Abstracts from the 55th European Society of Human Genetics (ESHG) Conference: Hybrid Posters

Volume 31 | Supplement S1

Vienna, Austria

June 11-14, 2022

Sponsorship: Publication of this supplement was sponsored by the European Society of Human Genetics. All content was reviewed and approved by the ESHG Scientific Programme Committee, which held full responsibility for the abstract selections.

Disclosure Information: In order to help readers, form their own judgments of potential bias in published abstracts, authors are asked to declare any competing financial interests.

Contributions of up to EUR 10 000.- (Ten thousand Euros, or equivalent value in kind) per year per company are considered “Modest”. Contributions above EUR 10 000.- per year are considered “Significant”.

Presenting author names are bold in the contributor lists.

P01 Reproductive Genetics

P01.001A An unusual number of high mutations expand in the male germline in tyrosine kinase receptors

Irene Tiemann-Boege ¹, Ingrid Hartl¹, Sofia Moura¹, Renato Salazar¹

¹Biophysics, Linz, Austria

Background/Objectives: The higher risk of older fathers having an affected offspring with early or late-onset rare disorders has been quite unsettling; but unfortunately, the methods have been limited to better characterize this phenomenon. So far, studies have focused on well-characterized mutations mainly identified in the receptor tyrosine kinase receptor (RTK) signalling pathway [1-3].

Methods: The establishment of duplex sequencing opened exciting new possibilities in ultra-rare variant detection with a very high accuracy for a sequencing-based method [4, 5]. This is the first study that has used this sequencing approach to explore this type of mutagenesis directly in sperm in the FGFR3 gene.

Results: We found mutations associated with congenital disorders at increased frequencies and identified new unreported selfish mutations expanding with age [6]. We further characterized the expansion of these in the male germline with droplet digital PCR and analysed the change in receptor signalling [7, 8].

Conclusion: Our work sheds light into different mutational mechanisms potentially affecting the receptor kinase activity.

References: 1 Arnheim, N. et al. Annu Rev Genomics Hum Genet 2016.

2 Goriely, A. et al. Am J Hum Genet 2012.

3 Shinde, D. N. et al. Hum Mol Genet 2013.

4 Kennedy, S. R. et al. PLoS Genet 2013.

5 Salk, J. J. et al. Nat Rev Genet 2018.

6 Salazar, R. et al. bioRxiv 2021.04.26.441422 2022.

7 Lanzerstorfer, P. et al. PLoS One 2014.

8 Motsch, V. et al. Sci Rep 2019.

Grants: FWFW1250, FWFP30867000, REGGEN ATCZ207.

Conflict of Interest: Irene Tiemann-Boege Johannes Kepler University Linz, principal investigator, Ingrid Hartl Johannes Kepler University Linz, Sofia Moura: None declared, Renato Salazar: None declared.

P01.002.B Using accurate duplex sequencing to explore the connection between elevated germline mutation rates, sperm selection, and male (sub)fertility

Jason Kunisaki ¹, Suchita Lulla¹, Xichen Nie², Joemy Ramsay³, Yixuan Guo², Joshua Horns³, Jim Hotaling³, Kenneth Aston³, Aaron Quinlan^1;4

¹University of Utah School of Medicine, Human Genetics, Salt Lake City, United States; ²University of Utah School of Medicine, Oncological Sciences, Salt Lake City, United States; ³University of Utah School of Medicine, Surgery, Salt Lake City, United States; ⁴University of Utah School of Medicine, Biomedical Informatics, Salt Lake City, United States

Background/Objectives: Elevated germline de novo mutation rates can impact health and fertility, especially in the context of male subfertility. In 2020, we associated elevated paternal germline mutation rates with reduced lifespans, mirroring the somatic theory of aging. Similarly, studies of subfertile men report elevated individual and familial cancer risks compared to age-matched fertile controls. We will examine the mechanistic connection between germline instability, somatic mutation burden, subfertility, and poor health.

Methods: Analysis of germline mutation rates frequently involves the use of whole-genome sequencing from pedigrees. However, this strategy suffers from high sequencing error rates (10^-3) and the indirect determination of mutations in sperm. To account for this, we use TwinStrand Duplex Sequencing (TDS) on bulk sperm and blood samples to accurately detect true mutations in individual gametes as those occurring on complementary DNA strands, reducing our per-nucleotide error rate to <10^-9.

Results: Studying a normozoospermic individual’s bulk sperm and blood, we obtained high (≤50,000X) coverage across our sequencing panel. We estimate sperm and blood mutation rates of ~4e-7 (95% CI: 3.2-4.8e-7) and ~2.3e-7 (95% CI: 2.1-2.6e-7), respectively.

Conclusion: We find mutation rates in sperm to be elevated compared to those inferred from pedigree studies, and in line with expectations based on per-cell-division error rates. This supports a hypothesis that sperm with elevated mutation rates are selected against during capacitation and/or fertilization which has significant implications for IVF and ICSI procedures that bypass these processes. We will share results from additional samples to explore the link between mutation rates, male fertility, and health.

References:.

Grants:.

Conflict of Interest: Jason Kunisaki: None declared, Suchita Lulla: None declared, Xichen Nie: None declared, Joemy Ramsay: None declared, Yixuan Guo: None declared, Joshua Horns: None declared, Jim Hotaling NIH R01 Grant - Fertility Status as a Marker for Overall Health, Kenneth Aston NIH R01 Grant - Fertility Status as a Marker for Overall Health, Aaron Quinlan NIH R01 Grant - Fertility Status as a Marker for Overall Health.

P01.003.C Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility

Ann-Kristin Dicke ¹, Margot Julia Wyrwoll¹, Jakob Christian Albrethsen², Alexander Busch², D. Fietz³, Adrian Pilatz⁴, Anders Juul², Sabine Kliesch⁵, Jörg Gromoll⁵, Birgit Stallmeyer¹, Frank Tüttelmann¹

¹Institute of Reproductive Genetics, University of Münster, Münster, Germany; ²Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; ³Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Gießen, Gießen, Germany; ⁴Clinic for Urology, Paediatric Urology and Andrology, Justus Liebig University Gießen, Gießen, Germany; ⁵Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany

Background/Objectives: Impaired testicular descent is a common birth defect, leads to cryptorchidism and predisposes to infertility. In mice, the hormone insulin-like factor 3 (INSL3) and its receptor relaxin family peptide receptor 2 (RXFP2) are essential for inguinal canal opening and gubernaculum dilatation pulling the testes into the scrotum. Heterozygous variants in INSL3 and RXFP2 were also proposed as associated with cryptorchidism but those variants are frequently found in unaffected controls.

Methods: We screened exome sequencing data of >1600 infertile men for variants in INSL3 and RXFP2. Clinical, semen, and testicular phenotypes were evaluated and INSL3 serum level measurements in patients with identified rare (MAF <0.01) LoF variants are ongoing.

Results: Two patients with homozygous LoF variants in either INSL3 or RXFP2 were identified. The INSL3 variant c.143dup p.(Arg50Profs*33) is located in the first exon and the RXFP2 variant c.1406del p.(Phe469Serfs*8) affects the transmembrane domain, thus encoding a non-functional protein if not degraded. Both patients showed bilateral cryptorchidism, azoospermia, elevated FSH and variably impaired spermatogenesis. In a multiply affected family, segregation indicates no association of heterozygous LoF variants with cryptorchidism.

Conclusion: Aside from a family in which a homozygous missense variant in RXFP2 segregated with cryptorchidism in boys, this is the first report of homozygous LoF variants in INSL3 and RXFP2 in adults. Our findings show that for both genes, bi-allelic variants are a more convincing cause of bilateral cryptorchidism than a previously assumed autosomal dominant inheritance.

References:.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‘Male Germ Cells’.

Conflict of Interest: None declared.

P01.004.D Associations between epigenetic biomarkers of aging and infertility

Yunsung Lee ¹, Jon Bohlin¹, Christian Magnus Page^1;2, Haakon Egdetveit Nustad^1;3, Jennifer Ruth Harris¹, Per Magnus¹, Astanand Jugessur¹, Maria Christine Magnus¹, Siri Håberg¹, Hans Ivar Hanevik^1;4

¹Norwegian Institute of Public Health, Centre for Fertility and Health, Oslo, Norway; ²University of Oslo, Department of Mathematics, Oslo, Norway; ³Deepinsight, Oslo, Norway; ⁴Telemark Hospital Trust, Fertility Department Soer, Porsgrunn, Norway

Background/Objectives: As more individuals in developed countries postpone parenthood, more experience infertility and use assisted reproductive technologies (ART). While the effect of increased chronological age on the risk of infertility is well known, the relationships between epigenetic biomarkers of aging and male or female infertility are understudied.

Methods: Using 1,945 mother-father pairs from the Norwegian Mother, Father and Child Cohort Study, we compared five epigenetic biomarkers of aging between 1,000 couples who conceived by coitus and 894 couples who conceived by ART – in vitro-fertilization (IVF, n = 525) and intracytoplasmic sperm injection (ICSI, n = 369).

Results: We found a significant difference in one epigenetic biomarker of a pace of aging, the standardized Dunedin Pace of Aging methylation (DunedinPoAm), between non-ART and IVF mothers (0.208, P-value = 0.0004) after adjustment for chronological age and potential confounders. Further, we detected elevated DunedinPoAm in mothers with tubal factor infertility (0.326, P-value = 0.0001), ovulation factor (0.248, P-value = 0.0075) and unexplained infertility (0.245, P-value 0.0014) compared to non-ART mothers. No differences in epigenetic biomarkers of aging between non-ART and ICSI fathers were found. DunedinPoAm also showed stronger associations with smoking, education, and parity than other epigenetic biomarkers of aging.

Conclusion: In conclusion, DunedinPoAm captured a difference in the pace of epigenetic aging between non-ART and IVF mothers, indicating an association between female epigenetic aging and infertility.

References:.

Grants: Research Council of Norway (Women’s fertility, project no. 320656 and Centres of Excellence Funding Scheme, project no. 262700) and the European Research Council under the European Union’s Horizon 2020 research, grant no. 947684.

Conflict of Interest: Yunsung Lee: None declared, Jon Bohlin: None declared, Christian Magnus Page: None declared, Haakon Egdetveit Nustad: None declared, Jennifer Ruth Harris: None declared, Per Magnus Research Council of Norway. Centres of Excellence Funding Scheme (project no. 262700), Astanand Jugessur: None declared, Maria Christine Magnus European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 947684)., Siri Håberg Research Council of Norway. Women’s fertility (project no. 320656) and Centres of Excellence Funding Scheme (project no. 262700)., Hans Ivar Hanevik Research Council of Norway. Women’s fertility (project no. 320656).

P01.005.A Aftermath of long-term cigarette smoking on telomere length and mitochondrial DNA copy number in human cumulus cells prior to In Vitro Fertilization

Fani Konstantinidou ^1;2, Maria Cristina Budani³, Francesca Gonnella^1;2, Gian Mario Tiboni³, Liborio Stuppia^1;2, Valentina Gatta^1;2

¹“G. d’Annunzio” University of Chieti-Pescara, School of Medicine and Health Sciences, Chieti, Italy; ²“G. d’Annunzio” University of Chieti-Pescara, Unit of Molecular Genetics, Center for Advanced Studies and Technology (CAST), Chieti, Italy; ³“G. d’Annunzio” University of Chieti-Pescara, Department of Medical, Oral and Biotechnological Sciences, Chieti, Italy

Background/Objectives: The female reproductive system represents a sensitive target of the harmful effects of cigarette smoke, with folliculogenesis as mostly affected by this exposure. Some of the main causes of ovarian injury can be represented by cigarette smoke-induced oxidative stress (OS), abnormal crosstalk between oocyte and granulosa-cells and increased cell death [1]. Studies have shown that smoking can also result in telomere shortening and mitochondrial dysfunction in human reproduction [2]. Based on these premises, we estimated the effect of tobacco smoking on telomere length and mitochondrial DNA (mtDNA) copy number in cumulus cells (CCs) of healthy smoking and non-smoking women (<35 years of age) enrolled from Assisted Reproductive Technology protocols.

Methods: DNA was manually extracted from CCs of all subjects and subsequently quantified. 96-well plates were prepared in order to perform dual quantification qPCR assays using telomere, mtDNA and a single copy reference primer sets, as well as a reference genomic DNA sample. Results were deemed significant when showing a fold change >1.4 or <0.7 and a P-value < 0.05 (two-tailed T-test).

Results: Statistical analysis indicated a significantly lower relative telomere length and mtDNA copy number (p-value ~ 0,014/0,012) of the target sample to the reference sample in CCs of smokers compared to corresponding controls.

Conclusion: A significant correlation between smoke exposure and telomere length, as well as mtDNA copy number has been evidenced in cumulus cells of smokers. Further epigenetic and proteomic investigations could be useful to better elucidate the underlying mechanisms.

References: [1] Konstantinidou, F et al. (2021).

[2] Thilagavathi, J et al. (2013).

Grants:.

Conflict of Interest: None declared.

P01.006.B BMP15 and FSHR genetic polymorphisms in Bulgarian patients with premature ovarian insufficiency

Ivelina Oprova ¹, Petya Andreeva¹, Kalina Mihova², Martin Georgiev², Radka Kaneva², Ivanka Dimova³

¹Medical complex “Dr Shterev”, Sofia, Bulgaria; ²Medical University Sofia, Molecular Medicine Center, Sofia, Bulgaria; ³Medical University Sofia, Sofia, Bulgaria

Background/Objectives: Premature ovarian insufficiency (POI) is the early depletion of the ovarian reserve affecting 1–2% of women < 40 years of age and 0.1% of women < 30 years. Genetic factors have been identified in approximately 20–25% of cases. Among the genes associated with POI, BMP15 and FSHR have been proposed to be incorporated as diagnostic biomarkers. The aim of our study was to determine the frequency of genetic polymorphisms in both genes in Bulgarian women with POI in comparison to non-selected Bulgarian individuals (controls), in order to find an association of these genetic factors with the disease.

Methods: Eighteen patients with POI (median age of 30.6 years) were subjected to genetic sequencing of all BMP15 exons and FSHR exon 10, containing SNPs rs6165 (c.919G>A, p.Ala307Thr) and rs6166 (c.2039G>A, p.Ser680Asn). The sequencing data were compared to data from clinical exome sequencing in 485 controls.

Results: We didn’t find any difference for the analysed FSHR variants between groups. Statistically significant difference was discovered for BMP15 rs41308602 (c.308A>G, p.Asn103Ser) heterozygotes frequency, as it was 4.8 times higher in POI patients compared to controls.

	Allele frequency		Heterozygous frequency		Homozygous frequency
	POI	Controls	POI	Controls	POI	Controls
BMP15 rs3810682	33.3%	23.2%	22.2%	19.6%	22.2%	13.4%
BMP15 rs41308602	13.9% p < 0.06	6.2%	27.8% p < 0.0002	5.8%	0%	3.3%
FSHR rs6165	47.2%	49.5%	38.9%	45.8%	27.8%	26.6%
FSHR rs6166	52.8%	49.4%	61.1%	46.4%	22.2%	26.2%

Conclusion: We discovered an association between BMP15 rs41308602 polymorphism and POI in Bulgarian patients and lack of association for FSHR polymorphisms with this condition.

References:.

Grants:.

Conflict of Interest: None declared.

P01.007.C A GWAS of parental allelic interactions associated with subfertility

Siri Naerland Skodvin ¹, Miriam Gjerdevik¹, Julia Romanowska^1;2, Christian Magnus Page^1;3;4, Yunsung Lee¹, Astanand Jugessur^1;2, Haakon Gjessing^1;2

¹Norwegian Institute of Public Health, Centre for Fertility and Health, Oslo, Norway; ²University of Bergen, Department of Global Public Health and Primary Care, Bergen, Norway; ³University of Oslo, Section for Statistics and Data science, Department of Mathematics, Faculty of Mathematics and Natural Sciences, Oslo, Norway; ⁴University of Oslo, PharmaTox Strategic Initiative, Faculty of Mathematics and Natural Sciences, Oslo, Norway

Background/Objectives: Subfertility is a heterogeneous phenotype, the causes of which are often unclear. One hypothesis is that the joint contribution of risk alleles in the parents may partly explain subfertility. We, therefore, performed a GWAS to study parental allelic interactions.

Methods: In the 29,199 pregnancies available for analysis in the Norwegian Mother, Father and Child Cohort Study (MoBa), 609 newborns were conceived using assisted reproductive technology (ART). We used ART as proxy for subfertility and analyzed interaction effects using logistic regression and a multiplicative dose-response model, adjusting for principal components, age, and BMI.

Results: Our results revealed interaction effects between maternal and paternal SNPs and use of ART. Entries in various public gene databases indicated that several of the identified genes are highly expressed in tissues relevant for the phenotype studied here. For example, the gene for coiled-coil domain containing 171 (CCDC171, 9p22.3) is expressed in both testis and endometrium. Two other genes, DENN domain containing 6B (DENND6B, 22q13.33) and karyopherin subunit beta 1 (KPNB1, 17q21.32), are also expressed in testis.

Conclusion: Our results pointed to several significant interaction effects between maternal and paternal SNPs in genes that are highly relevant to subfertility. Further replications are necessary to confirm these findings.

References: Magnus et al. Cohort profile update: the Norwegian Mother and Child Cohort study (MoBa). Int J Epidemiol. 2016.

Gjessing & Lie. Case-parent Triads: Estimating Single-and Double-dose Effects of Fetal and Maternal Disease Gene Haplotypes. Annals of human genetics. 2006.

Grants: Norwegian Research Council (grant 262700).

Conflict of Interest: None declared.

P01.008.D Establishment of the niPGT-A programme in two IVF centres

Antonio Urbano ^1;2;3, Marina Sánchez^1;3, Isabel Ochando^1;2;3, Mireia Poveda^3;4, Juan Iñiguez^3;5, Estefanía Montoya^1;3, Rosario Vazquez^1;3, Juan Manuel Moreno^3;4, Jose Jesús López-Gálvez^3;4, JOAQUIN RUEDA^1;2;3

¹Unidad de Genética. Hospital HLA Vistahermosa, Alacant, Spain; ²Universidad Miguel Hernández (UMH), Departamento de Histología y Anatomía, UMH, Alicante, Spain, San Juan de Alicante, Spain; ³Cátedra de Biomedicina Reproductiva Vistahermosa, UMH, Alicante, Spain, Alacant, Spain; ⁴UR Vistahernosa, UR Grupo Internacional de Reproducción Asistida, Alacant, Spain; ⁵UR IMED Valencia, UR Grupo Internacional de Reproducción Asistida, Burjasot, Spain

Background/Objectives: Preimplantation genetic testing for aneuploidy (PGT-A) selects euploid embryos, improving the results in in vitro fertilisation (IVF) cycles. The evolution of next-generation sequencing (NGS) has made it possible to reduce the amount of genetic material needed to obtain reliable results. NGS allows analysis of spent culture medium (SMC) to determine embryo ploidy, with even better concordance with embryo ploidy than trophectoderm (TE) biopsy (1). Non-invasive PGT-A (niPGT-A) promises to be one of the main tools for embryo selection, as it provides insight into embryo ploidy without altering embryo development at all. Our objective is to determine the best protocol for niPGT-A.

Methods: We established a niPGT-A programme in 2 IVF centres. We determined concordance, FPR and FNR.

Centre A stablish standard protocol for remove granulosa cells from oocyte and Centre B stablish a strict protocol for remove granulosa cells.

Results: Control: 47 SMC from vitrified embryos. Concordance 95.74% SMC vs TE, FNR of 2.13% and FPR 2.13%.

Centre A: 34 fresh SMC, 52,94% concordance, 38,24% FNR and 8,82% FPR.

Centre B: 16 fresh SMC. 100% concordance and 0% FPR and FNR.

Conclusion: niPGT-A is one of the best techniques for embryo selection, but it is necessary to establish a strict protocol for the removal of granulosa cells from the oocyte and/or embryo to ensure consistent results.

References: 1. Huang L, Bogale B, Tang Y, Lu S, Sunney X, Racowsky C. Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy. 2019:1-8. https://doi.org/10.1073/pnas.1907472116.

Grants: UMH-Citolab1.19A.

Conflict of Interest: None declared.

P01.009.A Experimental validation of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos

Barış Yaşar ^1;2, Nina Boskovic^2;3, Eeva-Mari Jouhilahti⁴, Piibe Vill¹, Thomas Bürglin⁵, Shintaro Katayama^2;4;6, Tõnis Org^1;7, Juha Kere^2;4;6, Ants Kurg¹

¹Institute of Molecular and Cell Biology, University of Tartu, Department of Biotechnology, Tartu, Estonia; ²Karolinska Institutet, Department of Biosciences and Nutrition, Huddinge, Sweden; ³Helsinki University Hospital, University of Helsinki, Department of Obstetrics and Gynecology, Helsinki, Finland; ⁴Stem cells and Metabolism and Biomedicum Stem Cell Centre, Faculty of Medicine, University of Helsinki, Research Programs Unit, Helsinki, Finland; ⁵University of Basel, Department of Biomedicine, Basel, Switzerland; ⁶Folkhälsan Research Center, Helsinki, Finland; ⁷Estonian Genome Centre, University of Tartu, Institute of Genomics, Tartu, Estonia

Background/Objectives: Identification of genes involved in embryonic genome activation (EGA) in humans revealed a role of PRD-like homeobox genes during early embryo development. In this study we investigate bovine (Bos taurus) oocytes and preimplantation embryos in order to clarify the possible roles of PRD-like homeobox genes in EGA.

Methods: Bovine oocytes/embryos were used to prepare cDNA by STRT-N which was followed by PCR for subsequent TOPO cloning and Sanger sequencing.

Results: We analysed RNA-seq data derived from a bovine model system to investigate the transcriptome in germinal vesicle and metaphase II oocytes, and in embryos at the four-, eight-, 16-cell, and blastocyst stages. This revealed the expression of a similar set of early embryo PRD-like homeodomain transcription factors previously identified in humans. In addition, evolutionary comparisons of oocyte and early embryo transcription factors helped predict gene structures and the genomic location of these PRD-like homeobox genes. cDNA cloning allowed the validation of these genes in bovine as candidate igniters of EGA.

Conclusion: Orthologues of human PRD-like homeobox genes, ARGFX, DUXA, NOBOX, LEUTX, TPRX1, and TPRX2 were experimentally identified in bovine at the transcript level. A TPRX duplicate, TPRX3, which was earlier predicted but not confirmed, was also shown to be expressed in bovine.

References: Töhönen, V. et al. Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development. Nat Commun 6, 8207 (2015).

Grants: This work is funded from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813707.

Conflict of Interest: None declared.

P01.010.B SizeMatters: a novel algorithm for fetal-fraction estimation based on fragment-size distributions

Jesper Eisfeldt ^1;2, Camilla Holmgren², Malin Tirfing², Anna Lindstrand^1;2, Erik Iwarsson^1;2, Agne Lieden^1;2

¹Karolinska Institute, Molecular medicine and surgery, Solna, Sweden; ²Karolinska University hospital, Clinical genetics, Solna, Sweden

Background/Objectives: Non-invasive prenatal testing (NIPT) for trisomy 13,18, and 21 is performed on cell free DNA (cfDNA) extracted from peripheral blood from the pregnant woman. The cfDNA contains both foetal and maternal DNA and the foetal fraction (FF) is an important factor for NIPT test accuracy. In general, NIPT on cfDNA with an FF < 3% is considered unreliable.

In a male fetus, the FF is estimated from chromosome Y signals (FFY), however, no such patterns are available for female fetuses. Instead, the FFY is complemented by machine learning approaches, requiring large training sets and homogenous data. As such, there is a need to develop robust computational tools for the estimation of FF in both male and female fetuses. Herein, we present a novel algorithm (SM) based on a machine-learning consensus approach for FF estimation from DNA fragment size distribution in shallow genome sequencing data (sGS).

Methods: We develop the SM-algorithm, combining multi-layer perceptron regression, support-vector machine, and multiple linear regression in a machine-learning framework.

Results: We analysed 4000 pregnancies using the SM-algorithm for FF estimation based on sGS data and fragment size distribution and compared it to current leading methods for sGS FF estimation. The SM-algorithm was able to successfully estimate FF, providing high concordance with FFY (median absolute error (MAE) =0.9), significant correlation with trisomy 21 signal (r = 0.75), and final NIPT quality (fail < 1%).

Conclusion: Our novel algorithm for FF estimation provides a FF estimation in both male and female fetuses.

References: None.

Grants: None.

Conflict of Interest: None declared.

P01.011.C Shedding light on Endometriosis (EM) disease: Whole Exome Sequencing (WES) and new genes discovery in a fully clinical characterized Italian cohort

Aurora Santin ¹, Paola Tesolin¹, Beatrice Spedicati¹, Gabriella Zito², Federico Romano², Anna Morgan³, Maria Pina Concas³, Giuseppe Ricci^1;2, Giorgia Girotto^1;3

¹University of Trieste, Department of Medicine, Surgery and Health Sciences, Trieste, Italy; ²Institute for Maternal and Child Health – I.R.C.C.S. “Burlo Garofolo”, Department of Obstetrics and Gynecology, Trieste, Italy; ³Institute for Maternal and Child Health – I.R.C.C.S. “Burlo Garofolo”, Medical Genetics, Trieste, Italy

Background/Objectives: EM is a multifactorial disease, involving the proliferation of endometrial epithelium and stromal tissue outside the uterine cavity. Despite its prevalence, little is known about the genetic factors involved.

Methods: To date, 50 EM adult women with a deep clinical evaluation (i.e., second-level ultrasound echography) were recruited and WES was performed. WES focused on a first list of 285 known EM genes and later on new candidates. Likely pathogenic variants have been selected and their frequency checked in 1000 already sequenced healthy controls.

Results: Preliminary results allow the identification of 47 variants within 23 genes. In particular, two approaches have been conducted: a) the identification of recurrent genes shared among many patients (e.g., LAMA5, CSMD1, NEB), b) private variants within specific genes (e.g., IL18). In group a) 4/50 patients carry different heterozygous missense variants within LAMA5 gene, already known to be associated with EM and EM-related infertility; 3/50 patients carry pathogenic variants (i.e., two heterozygous missense/one intronic deletion) within CSMD1 gene, a negative complement regulator previously associated with infertility. Furthermore, 7/50 patients carry heterozygous missense variants within NEB gene, a new candidate encoding a structural sarcomere protein. In group b) one patient carries a heterozygous missense variant in IL18 gene, known to be involved in EM pathogenesis and in embryo implant regulation.

Conclusion: This study allows the identification of novel pathogenic variants in EM known genes as well as new candidates which can guide clinicians in defining diagnostic/prognostic markers and tailoring targeted therapies for patients and their families.

References:.

Grants:.

Conflict of Interest: None declared.

P01.012.D Design and validation of a next-generation sequencing workflow for simultaneous detection of aneuploidy, ploidy, and common pathogenic microdeletions within Preimplantation Genetic Testing (PGT)

Matteo Figliuzzi ¹, Silvia Caroselli¹, Francesco Cogo², Paola Zambon², Cristina Patassini², Dany Bakalova³, Federico Favero⁴, Attilio Anastasi⁵, Francesco Capodanno⁵, Andrea Gallinelli⁵, Danilo Cimadomo⁶, Laura Rienzi⁶, Filippo Maria Ubaldi⁶, Carmen Rubio⁷, Jose Miravet-Valenciano⁷, Jorge Jimenez Almazan⁷, David Blesa⁷, Carlos Simon⁷, Antonio Capalbo¹

¹Igenomix Italia, Reproductive Genetics, Rome, Italy; ²Igenomix Italia, Reproductive Genetics, Marostica, Italy; ³Igenomix UK, Reproductive Genetics, Guildford, United Kingdom; ⁴Arc-Ster, ART center, Mestre, Italy; ⁵Hospital “del Delta”, Physiopathology of Human Reproduction Center, Lagosanto, Italy; ⁶GeneraLife, ART center, Rome, Italy; ⁷Igenomix Spain, Reproductive Genetics, Valencia, Spain

Background/Objectives: Standard methodologies employed in preimplantation genetic testing allow to identify chromosomal aneuploidies but not to determine embryo ploidy status nor the presence of microdeletions (MD). Transferring embryos with these abnormalities can result in miscarriage, molar pregnancy, or multiple congenital anomalies, requiring later-stage invasive prenatal diagnosis. Our objective is the development of a novel NGS-based framework to resolve current limitations.

Methods: PGT-A products were reamplified and sequenced using a custom AmpliSeq panel targeting 357 genomic regions harbouring high frequency SNPs and/or associated to 8 pathogenic microdeletions (-4p = Wolf-Hirschhorn, -8q = Langer-Giedion, -1p = 1p36 deletion, -22q = DiGeorge, -5p = Cri-du-Chat, -15q = Prader-Willi/Angelman, -11q = Jacobsen, -17p = Smith-Magenis). Sequencing data were processed by a bioinformatic algorithm which accounts for sequencing noise through gaussian-mixture modeling of B-allelic ratios measured at each SNP locus and estimates the likelihood of different ploidy levels and of the presence of MD.

Results: Ploidy was correctly determined in 233/234 cases, with only one diploid sample misclassified as triploid (PPV = 94.1%, NPV = 100%). Microdeletions could be consistently detected with high reliability (PPV = 98.5%, NPV = 99.5%) in 6 out of 8 microdeletion regions. Detection in -1p and -4p was less reliable due to the presence of recurrent haplotype blocks in the population, as confirmed by the analysis of a dataset of 2504 whole genome sequencing from 1KGP database.

Conclusion: This study provides, for the first time, detection of common pathogenic microdeletions and ploidy status from a single trophectoderm biopsy, expanding PGT clinical validity. This new assay will also help elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of apparently euploid embryos.

References:.

Grants:.

Conflict of Interest: Matteo Figliuzzi Igenomix Italy, Silvia Caroselli Igenomix Italy, Francesco Cogo Igenomix Italy, Paola Zambon Igenomix Italy, Cristina Patassini Igenomix Italy, Dany Bakalova Igenomix UK, Federico Favero Arc-Ster, Attilio Anastasi: None declared, Francesco Capodanno: None declared, Andrea Gallinelli: None declared, Danilo Cimadomo GeneraLife, Laura Rienzi GeneraLife, Filippo Maria Ubaldi GeneraLife, Carmen Rubio Igenomix Spain, Jose Miravet-Valenciano Igenomix Spain, Jorge Jimenez Almazan Igenomix Spain, David Blesa Igenomix Spain, Carlos Simon Igenomix Spain, Antonio Capalbo Igenomix Italy.

P01.013.A Actionable secondary findings in infertile men and their clinical significance

Kristiina Lillepea ¹, Laura Kasak¹, Liina Nagirnaja², Kenneth Aston³, Don Conrad², Margus Punab^1;4;5, Maris Laan¹

¹University of Tartu, Institute of Biomedicine and Translational Medicine, Tartu, Estonia; ²Oregon Health & Science University, Oregon National Primate Research Center, Division of Genetics, Beaverton, OR, United States; ³University of Utah School of Medicine, Department of Surgery, Division of Urology, Andrology and IVF Laboratory, Salt Lake City, UT, United States; ⁴University of Tartu, Institute of Clinical Medicine, Tartu, Estonia; ⁵Tartu University Hospital, Andrology Center, Tartu, Estonia

Background/Objectives: Whole exome sequencing (WES) has been increasingly used in medicine for patients with suspected genetic disorders. WES also enables the identification of secondary findings (SFs) – genetic variants in disease-causing genes that are unrelated to the patient’s primary condition but are still medically actionable. The prevalence of SFs in the general population is 1–3%; data of SFs in specific clinical conditions is limited. This study aimed to describe SFs in non-obstructive azoospermia (NOA) patients.

Methods: GEMINI¹ represents a multi-center study aiming to map the genetic composition of NOA. We evaluated SFs across 85 genes in a cohort of 836 GEMINI participants from six countries. Recommendations from the American College of Genetics and Genomics (ACMG), the Geisinger Health System’s MyCode and the Clinical Genome Resource were used to compile the gene list.

Results: Based on the ACMG guidelines for the interpretation of genetic variants, we identified 27 SFs in 21 genes in 30 patients (3.6%, 30/836). The majority of these patients carried SFs in genes linked to familial cancer syndromes, followed by cardiac conditions. Moreover, 11 patients with SFs (37%, 11/30) carried variants in genes linked to spermatogenic failure in knockout mice. Clinico-molecular diagnoses were confirmed in multiple cases during a retrospective evaluation of patients with SFs.

Conclusion: Every 28th analyzed NOA patient carried a medically actionable SF. Disclosure of SFs is valuable in genetic disease prevention and intervention, and long-term health management of andrology patients.

References: 1. Nagirnaja et al. (2021) N Engl J Med; 385:707-719.

Grants: Estonian Research Council IUT34-12, PRG1021; NIH R01HD078641, P50HD096723.

Conflict of Interest: None declared.

P01.014.B Asymptomatic male carriers of long copy number variants are predisposed to spermatogenic failure

Triin Kikas ¹, Laura Kasak¹, Margus Punab^2;3, Maris Laan¹

¹Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia; ²Andrology Centre, Tartu University Hospital, Tartu, Estonia; ³Institute of Clinical Medicine, University of Tartu, Tartu, Estonia

Background/Objectives: Infertility affects 7-10% of all men. About 60% of these cases remain idiopathic. Among other genetic factors, a burden of copy number variants (CNVs) has been suggested to increase the risk for spermatogenetic failure. Only a limited number of genome-wide studies have been conducted on the subject.

Methods: All study participants were recruited at the Andrology Center of Tartu University Hospital (2000-2015). The study group consists of azoospermia (no sperms, n = 73), severe (sperm count per ejaculate <10 mln, n = 83) or moderate oligozoospermia (10-39 mln, n = 58) and normozoospermia (>39 mln, n = 63) cases. All individuals were genotyped using Illumina HumanOmniExpress-24-v1.0/v1.1 BeadChips. Calling of autosomal CNVs (>100bp) from the genotyping data was performed with three algorithms (GADA, QuantiSNP, and CNstream) (1). CNVs detected by at least two programs were included in the analysis.

Results: No difference was detected between the groups for the mean number (7.2-7.4) and length (101-120kb) of CNVs per subject. CNVs longer than >1Mb (n = 33) were located in pericentromeric regions and near telomeres. Three loci overlapped with known pathogenic CNV regions (1q21.1, 7p22.2, 14q32.33). A recurrent CNV within the CSMD1 gene (n = 4) was detected only in azoospermia and severe oligozoospermia cases, supporting its reported link to spermatogenic failure (2).

Conclusion: Large pericentromeric and subtelomeres CNVs may affect spermatogenesis through meiotic and mitotic chromosomal missegregation. CNVs located within genes showing enhanced testicular expression may compromise normal spermatogenesis.

References: 1. Kasak et al (2015) Sci Rep 5:8342.

2. Lee et al (2019) Nat Commun 10:4626.

Grants: PRG1021 (Estonian Research Council).

Conflict of Interest: None declared.

P01.015.C Novel STARD8 and STARD9 mutations identified in 46,XY gonadal dysgenesis patients lend support to these genes as DSD candidates

Ludmila Livshits¹, Dmytro Sirokha ¹, Alexandra Gorodna¹, Nataliya Zelinska², Jadwiga Jaruzelska³, Kamila Kusz-Zamelczyk³, Serge Nef⁴

¹Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Laboratory of Human Genomics, Kyiv, Ukraine; ²Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues, Ministry of Health of Ukraine, Kyiv, Ukraine; ³Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; ⁴Faculty of Medicine, University of Geneva, Department of Genetic Medicine and Development, Geneva, Switzerland

Background/Objectives: Investigating mutation in genes, affecting gonad development is essential for understanding the genetic mechanisms causing Disorders/Differences in Sex Development. The aim of the research was to identify novel DSD genetic variants using whole exome sequencing (WES).

Methods: The WES was performed for two unrelated 46,XY SRY positive patients with gonadal dysgenesis.

Results: In first patient the hemizygous missense mutation NM_001142503.2 c.2659C>T (p.Arg887Cys) (rs766188656) in STARD8 gene (MAF = 0.0000251) was identified and confirmed as pathogenic using bioinformatic tools. After analysis of second patient two deferent mutations in compound heterozygous state were identified in STARD9 gene: NM_020759.3 c.5585_5590del (p.Ser1862_Thr1863del) (rs528276071) – inframe deletion (MAF = 0.0019) combined with NM_020759.3 c.3514C>T (p.Arg1172Cys) (rs12594837) – missense mutation (MAF = 0.00837). The analysis of genetic background which were performed for both patients did not reveal any pathogenic variants implicated in DSD phenotype. All detected mutant variants were inherited from healthy parents – heterozygous carriers and were not previously implicated in the pathogenesis of any disease. Bioinformatic analysis revealed that mutant variant in STARD8 and both mutations in STARD9 genes located in positions that are conserved in primates.

Conclusion: Based on the results obtained in current study, previous reports of STARD gene family mutations in DSD patients, expression patterns of STADR8 and STARD9 genes and steroidogenic properties of those protein products we conclude that STADR8 and STARD9 considered as 46,XY DSD causing genes.

References:.

Grants: Study was funded by the Swiss National Science Foundation, grant number SCOPES IZ73Z0_152347/1 and National Academy of Sciences of Ukraine [0121U110054].

Conflict of Interest: None declared.

P01.016.D Using marginal analysis to optimise the number of tested genes for reproductive genetic carrier screening

Leslie Burnett ^1;2;3, Matthew Hobbs¹, Heather Gordon¹

¹Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia; ²University of NSW, Sydney, School of Clinical Medicine, St Vincent’s Campus, Darlinghurst, Sydney, Australia; ³University of Sydney, Northern Clinical School, Royal North Shore Hospital, St Leonards, Sydney, Australia

Background/Objectives: Genomic technologies now permit carrier testing using very large panels, or even the entire exome. Including more genes increases diagnostic sensitivity, but at higher cost. We have explored if there is an optimum number of genes for clinical utility and cost effectiveness.

Methods: We used a comprehensive list of 1,300 genes causative of autosomal recessive and X-linked recessive conditions relevant to carrier screening. From data in the Genome Aggregation Database (gnomAD), for both total population as well as major population subgroups, we extracted the allele frequency (AF) in these genes for all known single nucleotide variants (SNV) and indel variants that have been reported as Pathogenic or Likely Pathogenic in ClinVar. We also considered a notional population subgroup, in which the AF was set equal to the highest encountered AF in any other population subgroup. Using marginal analysis techniques, we calculated the number of genes needing to be incorporated into a carrier screening panel to achieve 90%, 95% or 99% of theoretical maximal diagnostic sensitivity.

Results: To achieve the stated fraction of theoretical maximal diagnostic sensitivity, the number of genes needing to be included in a screening panel was found to be:

Diagnostic sensitivity	90%	95%	99%
Couples	84	160	381
Individuals	383	546	842

Conclusion: To achieve generally accepted levels of diagnostic sensitivity, the number of genes needing inclusion in a screening panel is much smaller than the total number of genes potentially available for testing. This has significant policy implications for both cost and public health benefits of carrier screening.

References:.

Grants:.

Conflict of Interest: Leslie Burnett Garvan Institute of Medical Research, Takeda Pharmaceuticals USA Inc, Invitae Asia-Pacific, Honorary academic positions at University of NSW and University of Sydney, Matthew Hobbs Garvan Institute of Medical Research, Heather Gordon Garvan Institute of Medical Research.

P01.017.A Cytogenetic investigation of infertile patients in Hungary: a 10-year retrospective study

Szilvia Andó ¹, Katalin Koczok¹, Beáta Bessenyei¹, Istvan Balogh^1;2, Aniko Ujfalusi¹

¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Genetics, Debrecen, Hungary; ²University of Debrecen, Department of Human Genetics, Debrecen, Hungary

Background/Objectives: Infertility is a very common health problem that can caused by numerical or structural abnormalities of sex chromosomes or autosomes. The main purpose of this retrospective study was to determine the types and frequency of chromosomal aberrations detected in infertile patients and to compare the frequency of structural aberrations with control group.

Methods: Constitutional karyotyping was performed in 1489 men and 780 women diagnosed with infertility between 2010 and 2020. The control group was represented by 869 male and 1160 female patients having cytogenetic investigation with other referral reason then infertility. Karyotyping was performed on peripheral blood lymphocytes as standard protocol by using G-banding method.

Results: Thirty three (2,22%) of 1489 infertile men showed sex chromosomal aberrations [47,XXY (n = 28); 47,XXY/46,XY (n = 3); 47,XXY/48,XXXY (n = 1); 46,XX (n = 1)] and 89 (5,98%) had structural abnormalities [inversion (n = 21); balanced translocation (n = 16); single cell translocation (n = 52)]. Three (0,38%) of the 780 infertile women had sex chromosomal aberrations [X numerical mosaicism (n = 2); 46,XY (n = 1)] and 58 (7,44%) had structural abnormalities [inversion (n = 21); balanced translocation (n = 10); single cell translocation (n = 27)]. Structural chromosomal abnormalities were detected in 27/869 (3,11%) control male cases [inv(9) (n = 23); single cell translocations (n = 4)] and 39/1160 (3,36%) control female samples [inversions (n = 31); single cell translocations (n = 8)]. The prevalence of single cell translocations was higher in infertile patients (3,5% vs. 0,46% in men and 3,46% vs. 0,7% in women).

Conclusion: The type and frequency of chromosomal abnormalities of infertile patients was comparable to literature data. The frequency of less-studied single cell translocations was significantly higher in infertile patients.

References:.

Grants:.

Conflict of Interest: None declared.

P01.019.C Familial Mayer-Rokitansky-Küster-Hauser syndrome

Hagit Daum ¹, Ayala Frumkin¹, Vardiella Meiner², Iris Harel³, Dvora Bauman⁴

¹Hadassah Medical Organization and Faculty of Medicine, Hebrew University of Jerusalem, Israel, Genetics, Jerusalem, Israel; ¹Hadassah Medical Organization and Faculty of Medicine, Hebrew University of Jerusalem, Israel, Genetics, Jerusalem, Israel; ³Barzilai Medical Center, OBGYN, Ashkelon, Israel; ⁴Hadassah Medical Organization and Faculty of Medicine, Hebrew University of Jerusalem, Israel, OBGYN, Jerusalem, Israel

Background/Objectives: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital agenesis/aplasia of the uterus and part of the vagina in women with a normal 46,XX karyotype. Most cases are sporadic and do not have a molecular diagnosis, although individual case reports have been described in association with copy number variants or candidate genes (WNT9B, LHX1, GREB1L, 1q21.1 deletion, 22q11.21 duplication etc). We describe a dominantly inherited case of MRKH, in which a mother with MRKH had a biological daughter with the same syndrome through a surrogate pregnancy.

Methods: We used G-banded chromosomal analysis in order to confirm a 46,XX karyotype, and exome sequencing in order to investigate single nucleotide variants (SNV). Copy number variants were inferred from read depth analysis of the exome data.

Results: Chromosome analysis on the mother and daughter with MRKH revealed a female karyotype with a deletion of chr2q37 in both. The deletion was found to be de novo in the mother. Exome sequencing, undertaken in the daughter, confirmed the ~4Mbp deletion and did not demonstrate pathogenic or likely pathogenic variants. Similar deletions have been described in the literature in association with a spectrum of neuro-physical findings but never with MRKH.

Conclusion: Our data suggest that deletion of 2q37 may be associated with MRKH. The developing options of surrogacy and uterine transplantation stress the importance of genetic investigation for MRKH patients.

References:.

Grants: Internal fund of the Hebrew University of Jerusalem and Hadassah Medical Center.

Conflict of Interest: Hagit Daum Mentoring Grant of the Hebrew university and Hadassah Medical Center. PI: Hagit Daum, Ayala Frumkin: None declared, Vardiella Meiner: None declared, Iris Harel: None declared, Dvora Bauman: None declared.

P01.020.D A Novel Cause of Male Infertility and Hypergonadotropic Hypogonadism: Inhibin-A Deficiency Due to Loss of Function Variations of INHA

Esra Arslan Ateş ¹, Mehmet Eltan², Bahadır Şahin³, Busra Gurpinar Tosun², Tuba Seven Menevse², bilgen bilge geçkinli⁴, Andy Greenfield⁵, Serap Turan², Abdullah Bereket², Tulay Guran²

¹Marmara University Pendik Training and Research Hospital, Genetic Diseases Diagnostic Center, İstanbul, Turkey; ²Marmara University School of Medicine, Department of Pediatric Endocrinology and Diabetes, Istanbul, Turkey; ³Marmara University School of Medicine, Department of Urology, Istanbul, Turkey; ⁴Marmara University School of Medicine, Medical Genetics, Istanbul, Turkey; ⁵MRC Harwell Institute, Mammalian Genetics Unit, Oxfordshire, United Kingdom

Background/Objectives: The human INHA gene encodes the inhibin subunit alpha protein, which is common to both inhibin A and B. The functional importance of inhibins in male sex development, sexual function, and reproduction remain largely unknown. Todate no clinical manifestation were related to INHA gene variations. Here we report for the first time two male siblings sharing the same clinical features associated with homozygous INHA pathogenic variations.

Methods: The medical documents were examined for clinical, biochemical, and imaging data. Genetic analysis was performed using next-generation and Sanger sequencing methods.

Results: Two brothers were referred to us because of gynecomastia, testicular pain, and having a history of hypospadias. They were born to a consanguineous parents and no similar cases were reported in the family. Biochemistry revealed low serum testosterone, high gonadotropin and anti-Mullerian hormone, and very low/undetectable inhibin concentrations, where available. Both patients had azoospermia in spermiogram. We have identified a homozygous 2bp deletion (c.208_209delAG, R70Gfs*3), which leads to a truncated INHA protein in both patients, and confirmed heterozygosity in the parents. The external genital development, pubertal onset and progression, reproductive functions, serum gonadotropins, and sex hormones of mother and father, who were heterozygous carriers of the identified mutation were normal.

Conclusion: Homozygosity for pathogenic INHA variations causes decreased prenatal and postnatal testosterone production and infertility in males, while the heterozygous female and male carriers of INHA variations do not have any abnormality in sex development and reproduction.

References:.

Grants:.

Conflict of Interest: None declared.

P01.023.C Copy number variant (CNV) analysis in couples with recurrent miscarriages

Evelina Dagyte ¹, Algirdas Utkus¹

¹Vilnius University, Faculty of Medicine, Institute of Biomedical Sciences, Department of Human and Medical Genetics, Vilnius, Lithuania

Background/Objectives: Recurrent miscarriage (RM) is an important global health issue and carries psychological and financial burden for affected couples. Anatomy factors, antiphospholipid syndrome, endocrinological and chromosomal abnormalities are the most commonly recognized aetiologies for RM. Aetiology of RM remains idiopathic in 40-60 % of couples. The aim of this study was to evaluate copy-number variation (CNV) characteristics in couples with RM and compare to the Lithuanian population cohort.

Methods: Microarray analysis was performed for couples with unexplained RM (anatomy factors, antiphospholipid syndrome, chromosomal and endocrinological abnormalities were excluded) to detect CNVs. Genotyping was performed with Illumina HumanCytoSNP-12 v2.1 arrays using the standard Illumina Infinium HD Ultra Assay Protocol Guide.

Results: In total, 68 RM patients were tested and 72 CNVs were detected. The average number of CNVs per patient was 1,1 (1,67 in Lithuanian population cohort). No CNVs were identified in approximately 40 % of patients (30-40 % in Lithuanian population cohort). CNV regions (CNVRs) presented >1 individual (criteria met 13 CNVRs) were selected for further analysis. Three of 13 CNVRs – at 3q29, 5p15.33 and 19p12-q11 – were not reported in Lithuanian population cohort or in DGV database.

Conclusion: The number of CNV is similar in the RM group and Lithuanian population cohort. For more detailed results, the study continues to involve more patients.

References:.

Grants:.

Conflict of Interest: None declared.

P01.024.D DNA methylation in newborns conceived by assisted reproductive technology

Robert Lyle ^1;2, Christian Page^2;3, Yunsung Lee², Haakon Nustad², Maria Christine Magnus^2;4;5, Kristine Løkås Haftorn², Ellen Carlsen², William Denault^2;6, Jon Bohlin^2;7, Astanand Jugessur^2;8, Per Magnus², Haakon Gjessing^2;8, Siri Håberg²

¹Oslo University Hospital, Department of Medical Genetics, Oslo, Norway; ²Norwegian Institute of Public Health, Centre for Fertility and Health, Oslo, Norway; ³University of Oslo, Department of Mathematics, Oslo, Norway; ⁴University of Bristol, MRC Integrative Epidemiology Unit, Bristol, United Kingdom; ⁵Bristol Medical School, Population Health Sciences, Bristol, United Kingdom; ⁶University of Chicago, Department of Human Genetics, Chicago, United States; ⁷Norwegian Institute of Public Health, Department of Method Development and Analytics, Oslo, Norway; ⁸University of Bergen, Department of Global Public Health and Primary Care, Bergen, Norway

Background/Objectives: Assisted reproductive technology (ART) may affect fetal development through epigenetic mechanisms as the timing of ART procedures coincides with the extensive epigenetic remodeling occurring between fertilization and embryo implantation. However, it is unknown to what extent ART procedures alter the fetal epigenome. Underlying parental characteristics and subfertility may also play a role.

Methods: Here we identify differences in cord blood DNA methylation, measured using the Illumina EPIC platform, between 962 ART conceived and 983 naturally conceived singleton newborns.

Results: We show that ART conceived newborns display widespread differences in DNA methylation, and overall less methylation across the genome. There were 607 genome-wide differentially methylated CpGs. We find differences in 176 known genes, including genes related to growth, neurodevelopment, and other health outcomes that have been associated with ART. Both fresh and frozen embryo transfer show DNA methylation differences. Associations persist after controlling for parents’ DNA methylation, and are not explained by parental subfertility.

Conclusion: Our results support the hypothesis that ART procedures influence DNA methylation in fetal life. The epigenetic differences between ART and naturally conceived children were not explained by parental DNA methylation or parental subfertility. Longitudinal assessments of DNA methylation are necessary to establish whether ART-induced differences in DNA methylation persist and influence later health outcomes.

References:.

Grants:.

Conflict of Interest: None declared.

P01.025.A Genome sequencing reveals causative CHH structural and single nucleotide variants in consanguineous families

Yassine Zouaghi ^1;2, Saba Irshad³, Ambrin Fatima³, mariam shahid³, Nida Najmi⁴, Fernanda De Azevedo Correa^1;2, Ansar Muhammad⁵, Alexia Boizot², Michela Adamo², Federico Santoni^1;2;6, Jim Acierno², Nelly Pitteloud²

¹University of Lausanne, Lausanne, Switzerland; ²Lausanne University Hospital, Service of Endocrinology, Diabetology and Metabolism, Lausanne, Switzerland; ³Aga Khan University, Department of Biological and Biomedical Sciences, Karachi, Pakistan; ⁴The Aga Khan University Hospital (AKUH), Department of Obstetrics and Gynaecology, Karachi, Pakistan; ⁵Hospital Ophthalmic Jules-Gonin - Fondation Asile Des Aveugles, Ophthalmology Department of the University of Lausanne, Lausanne, Switzerland; ⁶Medigenome, Genève, Switzerland

Background/Objectives: Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder that causes incomplete or absent puberty leading to infertility due to defects in secretion or action of gonadotropin-releasing hormone. CHH is very heterogeneous phenotypically, and its genetics are complex. More than 25 genes have been associated with it explaining up to 50% of cases. Discovering new causative genes, or new variants in known genes may aid explaining the intricacies of CHH improving diagnosis and treatment. Here, we present 6 consanguineous families from Pakistan, each with at least one member affected by CHH. Using different approaches, we determined in each case the genetic origin for the disorder.

Methods: 18 genomes from 6 families were sequenced. They were analysed using Sentieon. Then single nucleotide variants (SNVs) were filtered based on frequency in gnomAD and/or potential for pathogenicity. Copy number variations (CNVs) were extracted using CoverageMaster.

Results: A genetic cause for CHH was identified in all families. 4 SNVs following an autosomal recessive mode of inheritance: GNRHR:p.P320Q, GNRHR:p.F309del, KISS1R:p.S262X, KISS1R:p.W108X in 4 of them and 2 CNVs following an X-linked mode of inheritance ANOS1:NC_000023.11:g.8,593,145-8,733,4053del, ANOS1:NC_000023.11:g.8,471,269-8,568,633del in the last two. Except for GNRHR:p.P320Q, all the variants were not known before.

Conclusion: Systematic analysis of CNVs on top of SNVs can heavily increase the success rate of finding a genetic cause for CHH in patients. Performing such analysis in consanguineous families can uncover new causative variants leading to better understanding the function of genes involved in CHH.

References: Boehm, U., Bouloux, PM., Dattani, M. et al. Nat Rev Endocrinol 2015.

Grants:.

Conflict of Interest: None declared.

P01.026.B Autosomal recessive disorders - A common cause of early pregnancy losses?

Gjorgji Bozhinovski ¹, Katerina Kubelka-Sabit², Marija Terzikj¹, Dijana Plaseska-Karanfilska¹

¹Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Macedonia; ²Private Hospital “Acibadem-Sistina”, Skopje, Macedonia

Background/Objectives: Miscarriage or early pregnancy loss (EPL) represents one of the most common pathologies in the reproductive medicine occurring in about 15% of couples trying to conceive. About half of the miscarriages are caused by numerical chromosomal abnormalities. Up to 5% of couples experience recurrent miscarriage, defined as two or more consecutive pregnancy losses. Increasing number of case reports suggest Mendelian causes of recurrent miscarriages. Whole exome sequencing (WES) may help uncover the genetic etiology, but so far it has been rarely used to study EPL.

Methods: We have performed WES on seven euploid miscarried tissues from couples with recurrent EPL.

Results: Compound heterozygous pathogenic variants in CPLANE1 (c.1819delT and c.5820+3_5820+6delAAGT) and DHCR7 (c.964-1G>C and c.452G>A) genes were detected in two miscarriages. The variants were confirmed with Sanger sequencing and the partners in both families were heterozygous carriers. The compound heterozigosity in CPLANE1 was also confirmed in a second miscarriage from the same couple. Pathogenic variants in CPLANE1 and DHCR7 genes cause Joubert syndrome 17 and Smith-Lemli-Opitz syndrome, respectively. Both are multi-systemic disorders associated with variable clinical severity and, besides in patients, have been confirmed in pregnancy losses and/or fetuses with ultrasound abnormalities.

Conclusion: In conclusion, our study shows a high percentage of autosomal recessive disorders (2/7 or 28.5%) as a cause of EPL. Increased number of WES analysis in recurrent euploid miscarriages could improve our understanding of the etiology of EPL and relevant biological processes. The knowledge of genetic causes responsible for EPL allows for genetic counselling and gives hope to the families.

References:.

Grants:.

Conflict of Interest: None declared.

P01.028.D Genetic variation in genes linked to telomere length in Estonian non-obstructive azoospermia patients

Anna-Grete Juchnewitsch ¹, Laura Kasak¹, Liina Nagirnaja², Kenneth Aston³, Don Conrad², Margus Punab^1;4;5, Maris Laan¹

¹University of Tartu, Institute of Biomedicine and Translational Medicine, Tartu, Estonia; ²Oregon Health & Science University, Division of Genetics, Oregon National Primate Research Center, Beaverton, United States; ³University of Utah School of Medicine, Andrology and IVF Laboratory, Department of Surgery (Urology), Salt Lake City, United States; ⁴Tartu University Hospital, Andrology Center, Tartu, Estonia; ⁵University of Tartu, Institute of Clinical Medicine, Tartu, Estonia

Background/Objectives: Intact telomeric structures are important in protecting chromosomes from damage. Association between telomere length and risk to chronic diseases has become an engrossing topic. Sperm quality has been correlated with shorter telomeres¹ and rare variants in genes implicated in meiotic telomere complex have been linked to non-obstructive azoospermia (NOA)². We investigated the suggested potential causative link between rare variants in genes linked to telomere length and spermatogenic failure.

Methods: Exomes of 85 idiopathic NOA cases from Estonia were sequenced in collaboration with the GEMINI (GEnetics of Male INfertility Initiative) project. A list of 66 genes associated with telomere length in the scientific literature was compiled. Rare variants were identified and their possible pathogenicity was evaluated based on the ACMG guidelines.

Results: In our study group, no likely pathogenic/pathogenic rare variants in 66 candidate genes were identified. However, two autosomal dominant genes (pLI > 0.8) PELI2 and TERF1 carried two heterozygous missense variants of uncertain significance (VUS). Among these TERF1 is also implicated in the male reproductive system.

Conclusion: None of 85 Estonian NOA cases could be explained by causative variants in the analyzed genes modulating telomere length. The study may have been limited by an insufficient number of patients given a high heterogeneity of monogenic NOA and uncertainties in evaluating the effect of variants.

References: 1. Darmishonnejad et al Andrologia 52, e13546 (2020).

2. Salas-Huetos et al Human Genetics 140:217-227(2021).

Grants: IUT34-12, PRG1021; NIH R01HD078641, P50HD096723.

Conflict of Interest: None declared.

P01.029.A Role of progesterone receptor genetic variants rs4754732 and rs1942836 in spontaneous premature birth

Jasenka Wagner ¹, Mirta Kadivnik², Andrijana Muller², Kristina Kralik³, Nena Arvaj¹, Siniša Šijanović²

¹Faculty of Medicine, J.J. Strossmayer University Osijek, Department of Medical Biology and Genetics, Osijek, Croatia; ²Clinic of Obstetrics and Gynecology, University Hospital Center Osijek, Osijek, Croatia; ³Faculty of Medicine, J.J. Strossmayer University Osijek, Department of Medical Statistics and Informatics, Osijek, Croatia

Background/Objectives: To evaluate the roles of two selected genetic variations in fetal and maternal progesterone receptor gene (PGR) and to identify women who may have higher or lower odds for spontaneous premature birth compared to the general population.

Methods: A preliminary case–control study with two groups of pregnant women (with term and premature delivery, 219 in total) and two groups of newborns (term and preterm, 219 in total) was performed. Two single nucleotide polymorphisms (SNPs) of the progesterone receptor gene (rs4754732 and rs1932836) were genotyped.

Results: Our results suggest that women who gave birth to male newborns with CC or CT rs4745732 genotype have 2.5 higher chance for preterm birth (OR = 2.5; 95%CI 1.09 – 5.6; P = 0.03; Fisher’s Exact Test). We also found that women older than 35 whose newborns have CC or CT rs1942836 genotype have 5.3 higher chance for preterm birth (OR = 5.3; 95%CI 1.05 – 26.2; P = 0.04; Fisher’s Exact Test).

Conclusion: Our study suggests that patients with selected genetic variants of the progesterone receptor gene could have greater odds for premature birth compared to general population. Replication studies with a larger population of different ethnicity are needed in order to confirm these findings.

References:.

Grants: ‘Role of PROGINS Mutations in Progesterone Receptors as Modulators of Risk for Premature Birth’(VIF2017-MEFOS-3), Faculty of Medicine in Osijek, Croatia.

Conflict of Interest: None declared.

P01.030.B Does aneuploidy discordance between trophectoderm and spent culture medium suggest embryo mechanism of self-correction?

Anna Strychalska ¹, Wojciech Sierka¹, Joanna Smolen-Dzirba¹, Anna Kokot¹, Natalia Jodłowiec-Lubańska¹, Klaudia Simka-Lampa¹, Urszula Wroblewska¹, Patrycja Piotrowska¹, Adam Pudełko¹, Emilia Morawiec¹, Anna Bednarska-Czerwińska¹, Simone Palini^1;2

¹Gyncentrum Genetic Laboratory, Sosnowiec, Poland; ²‘Cervesi’’ Hospital Cattolica, IFV unit, Cattolica, Italy

Background/Objectives: Investigating the utility of spent culture media (SCM) and mixture of SCM and blastocoel fluid (BF) in non invasive preimplantation genetic testing for aneuploidy (PGT-A).

Methods: Embryo was cultured until day 4, then washed and transferred to fresh medium. Trophectoderm (TE) biopsied from embryo day 6 has been tested for PGT-A in our routine procedure. SCM and mix of SCM + BF were collected prior to TE biopsy. Testing was performed with the use of Illumina VeriSeq NGS platform and MiSeq system.

Results: Comparison of chromosome 8 changes is presented in table.

Material	Start cyto	End cyto	Type of change	Copy#
Trophectoderm	pter	q24.13	Gain	2.66
Spent coulter medium	q24.13	qter	Gain	6
SCM+BF	pter	q24.13	Loss	1
	q24.13	qter	Gain	4

TE analysis showed partial gain of chromosome 8 (pter->q24.13). In SCM we found gain of remaining part of chr8 (q24.13->qter). Sample of mix (SCM+BF) showed DNA gain of chr8 (q24.13->qter) and loss of chr8 (pter->q24.13).

Conclusion: Human embryo ability of self-correction has been discussed widely. Repairing mechanisms may result from increased aneuploid cells death and release of fragmented DNA into culture media. We tested cell-free embryo DNA released from the blastocyst to SCM and mix of SCM+BF during cell cleavage (day 4 to day 6). We have observed chromosomal status after breakage occurred in mitotic cell division of chromosome 8 in embryo. Discordance between results in collected samples may be evidence for embryo undergoing self-correction mechanism.

References:.

Grants: WND-RPSL.01.02.00-24-0477/19-007.

Conflict of Interest: Anna Strychalska Gyncentrum Genetic Laboratory, Wojciech Sierka Gyncentrum Genetic Laboratory, Joanna Smolen-Dzirba Gyncentrum Genetic Laboratory, Anna Kokot Gyncentrum Genetic Laboratory, Natalia Jodłowiec-Lubańska Gyncentrum Genetic Laboratory, Klaudia Simka-Lampa Gyncentrum Genetic Laboratory, Urszula Wroblewska Gyncentrum Genetic Laboratory, Patrycja Piotrowska Gyncentrum Genetic Laboratory, Adam Pudełko Gyncentrum Genetic Laboratory, Emilia Morawiec Gyncentrum Genetic Laboratory, Anna Bednarska-Czerwińska Gyncentrum Genetic Laboratory, Simone Palini Gyncentrum Genetic Laboratory.

P01.032.D Novel variant curation strategies and preclinical technology to improve diagnosis of differences of sex development

Emmanuèle Délot ^1;2, Surajit Bhattacharya², Hayk Barseghyan^1;2;3, Eric Vilain^1;2

¹George Washington University, Genomics and Precision Medicine, Washington, D.C., United States; ²Children’s National Research Institute, Center for Genetic Medicine Research, Washington, D.C., United States; ³Bionano Genomics Inc, San Diego, United States

Background/Objectives: Despite being collectively among the most frequent congenital developmental conditions, differences of sex development (DSD) lack recognition and research funding. As a result, what constitutes optimal management remains uncertain. Identification of the individual conditions is challenging and molecular diagnosis frequently not achieved, which may have psychosocial and health-related repercussions for individuals and their families. While research genome sequencing of samples from the DSD-TRN repository contributed to the description of the new GUBS syndrome (OMIM 618820) and diagnosed previously unsolved cases, network-wide diagnosis rate remains under 50%.

Methods: New pre-clinical genomic approaches have the potential to increase diagnostic yield through ascertainment of under-recognized etiology such as mosaic, structural, non-coding, or epigenetic variants. We undertook a study to determine the usefulness of Optical Genome Mapping for clinical DSD diagnosis and etiology discovery. As interpretation of now widespread exome sequencing requires expert interpretation rarely available in clinical settings, we also examined variants in ClinVar for 32 DSD genes.

Results: OGM identified 7 common DSD so far. We showed that curation of DSD gene variants in clinical databases used by algorithms to classify variants is currently insufficient even for the best validated DSD genes.

Conclusion: To address this gap, a special-interest group on the Franklin.genoox.com platform, opened to interested curators worldwide, integrates curation of DSD-specific variants into the genetic data analysis platform, with variant classification available through the free Franklin interface. Analysis of DSD-TRN registry data also shows that practice needs to evolve to prioritize early genetic testing.

References: PMID33513338.

Grants: NICHD RO1HD093450.

Conflict of Interest: Emmanuèle Délot Estranged spouse has stock options in Bionano Genomics, current value $12,000 USD., Surajit Bhattacharya: None declared, Hayk Barseghyan Bionano Genomics Inc, part-time employee, Stock/stock options, Eric Vilain Stock options, Bionano Genomics. These are current theoritical value $13,000, acquired over 4 years, they are non vested (non-sellable)., Scientific consultancy, Bionano Genomics.

P01.033.A Polygenic risk score for recurrent pregnancy loss in LUCAR study, Ukraine

Yevheniya Sharhorodska ^1;2, Zhanna Balkhiyarova^2;3, Anna Ulrich^2;3, Vincent Pascat⁴, Liliia Chorna¹, Ivanna Shymanska¹, Harmen M Draisma³, Danuta Zastawna¹, Oleg-Roman Gnateyko¹, Halyna Makukh¹, Marika Kaakinen^2;3, Inga Prokopenko^2;4

¹Institute of hereditary pathology, National Academy of Medical Sciences, Lviv, Ukraine; ²University of Surrey, Department of Clinical and Experimental Medicine, Guildford, United Kingdom; ³Imperial College London, Department of Clinical and Experimental Medicine, London, United Kingdom; ⁴University of Lille, Lille, France

Background/Objectives: The loss of 2+ sequential pregnancies before 24 weeks of gestation is defined as recurrent pregnancy loss (RPL). In RPL etiology, endocrine, immunological, anatomical and other factors could drive individual susceptibility, still ~50% of RPL cases remain idiopathic. We assessed the predictive ability of a polygenic risk score (PRS) for RPL in the LUCAR study (Lviv Ukrainian Cohort for Advancing Reproductive Health) from the Western Ukraine.

Methods: LUCAR includes 350 idiopathic RPL cases/458 controls with at least one healthy child, all having the genome-wide association study (GWAS) data imputed to TopMED and RPL association analysis performed using log-additive model. We used summary statistics from recurrent miscarriage (RM) GWAS meta-analysis (Laisk et al., PMID:33239672) and PRS software PRSice-2.

Results: In LUCAR, the unweighted common variant PRS_RM, with independent SNP sets after LD clumping, did not increase the RPL risk (Table). The lead variants from the Laisk et al., rs146350366 and rs7859844, did not affect RPL susceptibility.

Laisk et al.	PRSRM in LUCAR
Association test Significance Threshold	Total SNPs	R2	Beta(SE)	P-value
5 × 10-8	50	0.0021	0.082(0.075)	0.27
10-3	336	0.0054	0.13(0.076)	0.075
0.01	767	0.0013	0.065(0.075)	0.39
0.5	9889	0.0022	0.084(0.075)	0.26
0.99	21013	0.015	0.23(0.076)	0.0028

Conclusion: Despite the previous meta-analysis showing good predictive ability of the PRS for RPL, we were unable to replicate this in dataset from Western Ukraine. We believe this highlights the commonly observed problem of low portability of PRS derived in one population to another, and an urgent need for well-powered GWAS for RPL in world-wide populations.

References:.

Grants:.

Conflict of Interest: None declared.

P02 Prental Genetics

P02.001.B Exome sequencing for structurally normal fetuses - yields and dilemmas

Hagar Mor-Shaked ¹, Hagit Daum¹, Tamar Harel¹, Avital Eilat¹, Duha Fahham¹, Shiri Gershon-Naamat¹, Chaggai Rosenbluh¹, Orly Elpeleg¹, Vardiella Meiner¹

¹Hadassah-Hebrew University Medical Center, Human Genetics, Jerusalem, Israel

Background/Objectives: There is a growing body of literature regarding the yield of chromosomal microarray analysis (CMA) in structurally normal fetuses (1-3). We aimed to assess the empirical data as to the yield of exome sequencing (ES) in this population.

Methods: From February 2017 to January 2022, 1,298 fetuses were subjected to ES; 374 of them were structurally normal (28.8%). Only pathogenic and likely pathogenic (P/LP) variants, per the American College of Medical Genetics and Genomics (ACMG) classification, were reported. Additionally, ACMG secondary findings relevant to childhood were reported.

Results: Five fetuses (5/374; ~1.33%) had a P/LP variants indicating a moderate to severe disease: Wilson disease, Enhanced S-cone syndrome, Legius and Muenke syndromes (upon affected genes ATP7B, NR2E3, SPRED1 and FGFR3 respectively). One fetus had a paternally inherited pathogenic variant in RET.

Conclusion: Our data suggest that offering only CMA for structurally normal fetuses may provide false reassurance. Prenatal ES mandates restrictive analysis, and careful management combined with pre and post-test genetic counselling.

References: 1. Stern S, Hacohen N, Meiner V, et al. Universal chromosomal microarray analysis reveals high proportion of copy number variants in low risk pregnancies. Ultrasound Obstet Gynecol. 2020.

2. Sagi-Dain L, Cohen Vig L, Kahana S, et al. Chromosomal microarray vs. NIPS: analysis of 5541 low-risk pregnancies. Genet Med. 2019.

3. Moshonov R, Hod K, Azaria B, Abadi-Korek I, Berger R, Shohat M. Benefit versus risk of chromosomal microarray analysis performed in pregnancies with normal and positive prenatal screening results: A retrospective study. PLoS One. 021;16(4):e0250734.

Grants:.

Conflict of Interest: None declared.

P02.002.C DES-NIPT study: Global approach to detect inherited paternal variants in NIPT samples using deep exome sequencing

Ieva Miceikaite ¹, Charlotte Brasch-Andersen^1;2, Qin Hao², Christina Fagerberg^1;2, Britta Schlott Kristiansen², Pernille Torring², Geske Sidsel Bak³, Lene Sperling³, Martin Larsen^1;2

¹University of Southern Denmark, Human Genetics, Department of Clinical Research, Faculty of Health Sciences, Odense, Denmark; ²Odense Universitetshospital, Department of Clinical Genetics, Odense, Denmark; ³Odense University Hospital, Fetal Medicine Unit, Department of Obstetrics and Gynecology, Odense, Denmark

Background/Objectives: Deep sequencing has now enabled detection of sequence variants and diagnosis of specific monogenic diseases by non-invasive prenatal testing (NIPT), using tailor-made assays for each pregnancy. However, a comprehensive non-invasive genome-wide diagnostic approach suitable for all cases and all known monogenic disorders is needed.

The aim of this study was to utilize high-throughput deep exome sequencing (DES) on cfDNA as a global approach to detect inherited paternal sequence variants, allowing the detection of known dominant monogenic disease variants and enabling paternal variant exclusion in recessive disease diagnostics.

Methods: For our pilot study, we included 16 pregnancies with high-risk criteria for genetic testing. The gestational age ranged 14-21 weeks. We obtained plasma, parental blood and invasive fetal samples (trio), and applied DES. We performed bioinformatic evaluation of the results from trio and DES-NIPT analysis. To estimate DES-NIPT sensitivity and precision, we compared paternal variants found in the overlapping exonic regions in both the NIPT and the invasive sample, where variants from invasive samples were the reference.

Results: We achieved a mean alignment target coverage of 537x. Using the paternal allele frequency in NIPT samples, we estimated fetal fraction, which ranged 5.13-18.77%. The sensitivity and precision of our approach to detect all inherited paternal variants from plasma samples were 98.19% and 99.41%, respectively.

Conclusion: Using DES-NIPT approach, we can detect and exclude inherited paternal variants from NIPT samples with high sensitivity and precision. This potentially allows the detection of the majority known inherited monogenic disorders early in the pregnancy and non-invasively.

References:.

Grants: RegionSyddanmarksForskningspulje(20/14085);SyddanskUniversitet;FondentilLægevidenskabensFremme(19-L-0281);AaseogEjnarDanielsensFond(19-10-0259).

Conflict of Interest: None declared.

P02.004.A Valuable insights after one year whole exome sequencing in a fetal/prenatal setting

Sarah Delbaere ¹, Machteld Baetens¹, Candy Kumps¹, Ellen Roets², Noortje Van Oostrum², Bert Callewaert¹, Sandra Janssens¹, Olivier Vanakker¹, Björn Menten¹

¹Center for Medical Genetics Ghent, Ghent University Hospital, Ghent University, Department of Biomolecular Medicine, Gent, Belgium; ²Women’s Clinic, Ghent University Hospital, Department of Obstetrics, Gent, Belgium

Background/Objectives: The aim of this study is to evaluate the implementation of whole exome sequencing (WES) for fetuses with congenital anomalies in a diagnostic workflow.

Methods: A fast-WES track for prenatal testing was implemented, with a maximal turn-around-time of 8 weeks. Depending on the fetus’s abnormalities, targeted gene panel or Mendeliome analysis was performed in-silico, the latter comprising all human disease-linked genes. The variant analysis strategy was optimized to efficiently identify the causal variant, by focusing on de novo, X-linked or biallelic inheritance. National Belgian guidelines have been established to standardize the testing and reporting strategy.

Results: Over the period of one year, 59 structurally abnormal fetuses with normal copy number variant (CNV) analysis were investigated via exome sequencing. The overall diagnostic yield was 16.9%; in 10 out of 59 fetuses, a class 4 or 5 variant could be identified. When comparing analysis in on-going versus terminated pregnancies or miscarriages, diagnostic yield was 17.9% (5/28) and 15.6% (5/32) respectively. Diagnostic yield for targeted gene panels was 13% (3/23), which is less compared to 18.4% (7/38) for Mendeliome analysis. Additional CNV analysis on the WES data using the ExomeDepth algorithm revealed a small intragenic ASCC1 homozygous deletion in a fetus with heterozygous carrier parents. Furthermore, two inherited pathogenic incidental findings were reported (APOB and BRIP1).

Conclusion: These results proof an important contribution of WES within prenatal and fetal context to obtain a higher number of molecular diagnoses and can broaden the understanding of aberrant fetal phenotypes and their underlying genetic cause.

References:.

Grants:.

Conflict of Interest: None declared.

P02.005.B C-terminal cFLIP disruption in humans causes intrauterine multisystem anomalies due to aberrant cell death response switch

Ivana Lessel¹, Leonie Piehl¹, Kira Kirchner¹, Thilo Diehl², Christian Kubisch¹, Davor Lessel ¹

¹University Medical Center Hamburg-Eppendorf, Institute of Human Genetics, Hamburg, Germany; ²University Medical Center Hamburg-Eppendorf, Department of Neonatology and Pediatric Intensive Care Medicine, Hamburg, Germany

Background/Objectives: Pregnancy losses are mostly attributable to chromosomal abnormalities although recent studies also identified selected Mendelian disorders responsible for stillbirth. However, in the majority of cases the underlying cause of foetal death remains elusive. Here, we studied a consanguineous family with history of recurrent early miscarriages and two foetuses who presented with severe intrauterine multi-system anomalies, resulting in early demise.

Methods: Next-generation sequencing and segregation analyses were used to identify candidate variants. Functional characterization was performed in a dual-cellular model utilizing cell viability analyses, immunoblotting, immunoprecipitation, caspase activity assays, RNA-sequencing and methylation profiling.

Results: We identified a homozygous frameshift variant in CFLAR resulting in C-terminally truncated cFLIP, a master regulator of multiple cell death pathways. In-vitro analyses of the identified variant revealed enhanced binding to unprocessed procaspase-8, which correlated with abrogated caspase-8 activity, thus disclosing the inability to execute CASP8-mediated extrinsic apoptosis. Furthermore, we observed enhanced binding to RIPK1 and phosphorylated-RIPK1, which correlated with an enhanced aberrant execution of the RIPK1-RIPK3-MLKL phosphorylation cascade upon apoptotic stimuli. Thus, aberrant necroptosis induction concomitant with impaired ability to execute apoptosis during embryonic development underlies the cFLIP-deficiency. Moreover, integrative transcriptome and epigenome analysis of patient-derived fibroblasts revealed compensatory, pro-survival silencing of RIPK3 and CASP10.

Conclusion: Above identifying a novel Mendelian cause for foetal death and linking for the first time a pathogenic CFLAR variant to a human disease, we further expand the knowledge underlying the molecular switch between apoptosis and necroptosis, and provide the first direct evidence for the importance of necroptosis inhibition for proper human embryonic development.

References:.

Grants:.

Conflict of Interest: Ivana Lessel Full time, Leonie Piehl Full time, Kira Kirchner Full time, Thilo Diehl Full time, Christian Kubisch Full time, Davor Lessel Full time, 1. Deciphering molecular mechanisms of CHAMP1 deficiency-associated developmental delay, German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). 2. The role of RNA interference in human neurodevelopment, Werner-Otto Foundation. 3. Identification and characterization of highly penetrant risk genes in testicular germ cell tumors, German Cancer Aid (Deutsche Krebshilfe). 4. Elucidating the role of stress granules associated translational control in aging and aging-associated diseases, Fritz Thyssen Foundation. 5. The RNA helicase DHX30: Physiological function and role in a neurodevelopmental disorder, German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). 6. Genomic context analysis of clinically-relevant disease-associated variants in zinc finger proteins (mis-ZFs), German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD), The Champ1 Research Foundation, Member of the Scientific Advisory Board, https://champ1foundation.org/scientific-advisory-board/ AGO2 Association, Member of the Scientific Advisory Board, https://ago2.org/en.

P02.006.C Increased nuchal translucency (NT). Can we do more? Prenatal trio exome sequencing of >150 cases revealed unexpected findings in fetuses with increased NT

Heinz Gabriel ¹, Björn Schulte¹, Martin Ritthaler¹, Saskia Biskup¹

¹Praxis fuer Humangenetik/CeGaT Tuebingen, Tübingen, Germany

Background/Objectives: Ultrasound diagnosis allows for the detection of half of all major structural anomalies. As a soft marker, nuchal translucency (NT) is associated with a spectrum of structural anomalies. Mostly, NT measurement is offered as part of screening for chromosomal abnormalities. About 20% of fetuses with increased NT will have a chromosomal abnormality. Noonan syndrome is considered the most frequently reported syndrome associated with increased NT. Prenatal panel testing for Noonan syndrome genes is nowadays widely offered in cases with increased NT. Aside from these Noonan syndrome genes, there are only a limited number of gene for which increased NT is documented.

Methods: In this study, we performed prenatal trio exome sequencing in cases with increased NT.

A cohort of 158 fetuses with increased NT were analysed by trio exome sequencing. The mean turn-around-time (TAT) was 12 days.

Results: We detected (likely) pathogenic variants in 46 cases (29%). Noonan syndrome genes were found in 11 cases. In 22 cases pathogenic variants were found in genes which are not to be known to be associated with increased NT so far.

Conclusion: Our data demonstrate that prenatal testing should not be limited to chromosomal aberrations, or RASopathies/Noonan syndrome, in cases with increased NT. Increased NT can be found in a wide range of syndromic conditions and might be one of the earliest detectable phenotypic features in the first trimester. We have shown the value of trio exome sequencing for the prenatal genetic diagnosis of fetuses with increased NT.

References:.

Grants:.

Conflict of Interest: Heinz Gabriel full time, Björn Schulte full-time, Martin Ritthaler full-time, Saskia Biskup full-time.

P02.007.D Prenatal WES for rapid detection of copy number variants and single gene disorders in uncultured samples

Vincenzo Cirigliano ¹, Elisabet Lloveras², Elena Ordonez¹, Isabel Castilla¹, Mireia Lechuga¹, Mafalda Almeida¹, Cristina Perez², Miriam Leon^1;1;1

¹Veritas Intercontinental, Barcelona, Spain; ²Synlab Diagnosticos Globales, Esplugues de Llobregat, Spain

Background/Objectives: aCGH is currently the first line test in prenatal diagnosis. However, most cases with normal results require additional tests targeting specific indications or whole exome sequencing. We evaluated an optimized WES capture assay to detect both CNVs and SNVs in a single assay aiming to assess its performance for genomewide detection of CNVs in uncultured prenatal samples.

Methods: DNA extracted from uncultured amniotic fluids (17) and CVSs (8) were selected with pathogenic CNVs of different sizes reported by aCGH. Samples were sequenced using an improved whole exome capture of 51 Mb designed to allow genomewide CNV detection and optimised for coverage of a wide subset of genes related with fetal structural abnormalities and developmental delay.

Results: All samples passed sequencing QC metrics, full concordance between WES and aCGH results was observed with an expectable slight difference in CNVs breakpoints locations. CNVs ranged between 13.8Kb and 52.9Mb in size: 17 cases with a unique event, 4 cases with 1 gain and 1 loss, two more samples had 2 and 3 losses. One rare case of chromothripsis, with 8 duplications along different regions of the same chromosome was also correctly classified.

Conclusion: WES can be used to identify pathogenic CNVs in uncultured prenatal samples. CNVs testing as first analysis step on sequencing data has the advantage of allowing rapid inclusion of SNVs/indels detection in multiple genes if required. While reducing time/costs for the analyses, this single test approach has the potential to increase the overall diagnostic yield in prenatal diagnosis.

References:.

Grants:.

Conflict of Interest: None declared.

P02.008.A A positive nonivasive prenatal testing result for sex chromosome aneuploidies. What could we expect?

Radostina Raynova ¹, Stoyan Bichev², Katerina Vladimirova², Kameliya Kercheva¹, Violeta Dimitrova³, Irena Bradinova², Ivanka Dimova⁴, Alexey Savov¹

¹National Genetic Laboratory, University Hospital of Obstetrics and Gynecology, Medical University, Sofia, Bulgaria; ²National Genetic Laboratory, University Hospital of Obstetrics and Gynecology, Medical University, Sofia, Bulgaria, Sofia, Bulgaria; ³University Hospital of Obstetrics and Gynecology, Medical University, Sofia, Bulgaria, Sofia, Bulgaria; ⁴SAGBAL “Dr.Shterev”, Sofia, Bulgaria

Background/Objectives: Non-invasive prenatal testing (NIPT) for detection of fetal aneuploidy risk has been widely adopted in clinical practice due to its high positive predictive value (PPV) for common aneuploidies, involving chromosomes 21, 18, 13. However, PPV for sex chromosome aneuploidies (SCA) is as lower as 57.6 %. NIPT remains classified as a screening, non-diagnostic test with standard recommendations that any positive NIPT result be followed by confirmatory diagnostic testing.

Methods: We present three cases referred to our Laboratory for confirmation of positive results for SCA from NIPT: 1) for Jacobs syndrome (47, XYY), 2) Turner syndrome (45, X) and 3) a deletion of 9.71 Mb on Xp11.4-Xp11.23. Amniocenteses were performed and DNA was extracted from uncultured amniocytes. Commercial QF-PCR kit and MLPA probe mix were used and samples were analyzed on ABI 3130. Karyotyping was performed on cultured amniocytes using standard protocol.

Results: For the first case, QF-PCR showed a XXY profile instead of a XYY, and the karyotype was 46, XY[12]/47,XXY[18]. Monosomy X was excluded for the second case, karyotype was 46, X, del(Y)(q12). The Xp deletion of the third case wasn’t confirmed by MLPA probe mix P034; the QF-PCR result was consistent with Turner syndrome.

Conclusion: The confirmation of a positive NIPT result for SCA demands an integral approach for the elucidation of the case and thus facilitating the counseling and patients’ decision making.

References: None.

Grants: None.

Conflict of Interest: None declared.

P02.009.B Exome sequencing in prenatal diagnosis: results from 156 cases

Emmanuelle Ranza ¹, Xavier Blanc¹, Federico Santoni¹, Katayoun Afshar², Guerry Frederic³, Bernard Conrad⁴, Isabelle Eperon⁵, Cécile Tissot⁶, Cecile Deluen⁷, Tanguy Araud⁷, Loïc Baerlocher⁷, Yasmine Sayegh-Martin⁸, Anne-Claude Müller-Brochut⁹, Christian Bisch⁵, Wawrzyniec Rieder⁵, Joel Fluss¹⁰, Jean-Marie Pellegrinelli¹¹, Marta Carrasco¹², Thibaud Quibel¹³, Sylvie Lacroix¹³, Philippe Extermann⁵, Graziano Pescia⁴, Romaine Robyr Susini¹³, Stylianos Antonarakis²

¹Medigenome, Swiss Institute of Genomic Medicine, Geneva, Switzerland; ¹Medigenome, Swiss Institute of Genomic Medicine, Geneva, Switzerland; ³Genesupport, Lausanne, Switzerland; ⁴Genesupport - Lausanne, Lausanne, Switzerland; ⁵Dianecho, Genève, Switzerland; ⁶Hirslanden Clinique des Grangettes, Chêne-Bougeries, Switzerland; ⁷Genesupport - Genève, Plan-les-Ouates, Switzerland; ⁸Centre Jean Violette, Geneva, Switzerland; ⁹GynEcho, Fribourg, Switzerland; ¹⁰Hôpitaux Universitaires de Genève (HUG), Pediatric Neurology, Genève, Switzerland; ¹¹Hôpitaux Universitaires de Genève (HUG), Maternity, Genève, Switzerland; ¹²Dre. Marta Carrasco Gynécologue, Nyon, Switzerland; ¹³Echofemme, Genève, Switzerland

Background/Objectives: Exome sequencing (ES) has become a standard test for undiagnosed developmental disorders, now increasingly used in prenatal diagnosis.

Methods: We report here our experience with 156 consecutive prenatal cases through ES. The most common fetal signs were: increased nuchal translucency (25 %), congenital heart defect (21.8 %), skeletal malformations (18.6 %) and brain abnormalities (9.6 %). After performing a-CGH, 28 cases had solo and 128 trio ES.

Results: In 21.8 % of cases, we have identified a pathogenic or likely pathogenic variant (as per the ACMG guidelines) likely causative of the fetal phenotype. The diagnostic yield was 34.5 % for skeletal malformations, 15.4 % for nuchal translucency, 11.8 % for congenital heart defect, and 33.3 % for brain anomalies. In 29 of the solved cases, the pathogenic variants were SNVs, while 2 were pathogenic structural variants (SV). Furthermore, the trio ES has identified in three cases complete uniparental disomies UPD6, UPD16 and UPD17, all including isodisomic segments with likely causative recessive genes.

Conclusion: ES is a powerful method for the identification of causative variants in prenatal; trio sequencing reduces the turnaround time and increases the diagnostic yield. The VUS remain a challenge and guidelines are needed to assist in the interpretation and disclosure of the results.

The discovery of novel mendelian genes and the introduction of additional laboratory and computational methods such as long-read sequencing and improvement of SV detection may further increase the diagnostic yield.

References:.

Grants:.

Conflict of Interest: Emmanuelle Ranza Medigenome, Medigenome, Xavier Blanc Medigenome, Federico Santoni Medigenome, Katayoun Afshar Medigenome, Guerry Frederic Genesupport, Bernard Conrad Genesupport, Genesupport, Isabelle Eperon Dianecho, Cécile Tissot Clinique des Grangettes, Cecile Deluen Genesupport, Tanguy Araud Genesupport, Loïc Baerlocher Genesupport, Yasmine Sayegh-Martin Centre Jean-Violette, Centre Jean-Violette, Anne-Claude Müller-Brochut GynEcho, Christian Bisch Dianecho, Wawrzyniec Rieder Dianecho, Joel Fluss HUG, Jean-Marie Pellegrinelli HUG, Marta Carrasco: None declared, Thibaud Quibel Echofemme, Sylvie Lacroix Echofemme, Philippe Extermann Dianecho, Dianecho, Graziano Pescia Genesupport, Genesupport, Romaine Robyr Susini Echofemme, Echofemme, Stylianos Antonarakis Medigenome, Medigenome.

P02.010.C Frequency of cystic fibrosis in fetuses with echogenic bowel in Bulgarian population

Nadezhda Yaneva ¹, Guergana Petrova², Irena Bradinova¹, Kameliya Kercheva¹, Radostina Raynova¹, Alexey Savov¹

¹National Genetic Laboratory, University Hospital of Obstetrics and Gynecology “Maichin Dom”, Medical University, Sofia, Bulgaria, Obstetrics and Gynecology, Sofia, Bulgaria; ²Pediatric cilinic, UMHAT “Alexandrovska”, Sofia;, Medical University of Sofia, Pediatric, Sofia, Bulgaria

Background/Objectives: Cystic fibrosis (CF) is the most common life-limiting condition with autosomal recessive inheritance. The at birth prevalence of CF in Bulgaria was estimated to be 1:3,600 live births. According to prenatal ultrasound evaluations 0,5-9,9% of cases with fetal echogenic bowel (FEB) are affected with CF. Recent studies revealed that detected echogenic bowel is associated with CF, chromosome abnormalities, congenital CMV infections, intrauterine growth retardation, and even fetal or neonatal death. However, the relation between the presence of FEB and cystic fibrosis is still scarcely studied. The purpose of this study was to assess the frequency of occurrence of cystic fibrosis in cases with fetal echogenic bowel.

Methods: 101 cases with FEB, detected during a routine ultrasound examination were screened for transmembrane conductance regulator (CFTR) mutations with Sanger sequencing at National Genetic Laboratory.

Results: Of the 101 cases studied, in 9 cases only one parent was carrier for CFTR mutation. Additionally, in 7 cases both parents were carriers of CFTR mutation. In five of these cases the fetuses had CF. Only one pregnancy was terminated. Other four was confirmed postnatally. In the other two cases the fetuses were negative.

Conclusion: We observed relatively high frequency of occurrence of CF in fetuses with echogenic bowel (5%, 5/101). However, the presence of CFTR mutation in the parents is not mandatory associated with the presence of CFTR mutation and CF in their fetus. For this reason after screening for carrier status of the parents, we recommend prenatal diagnosis for fetal CF when FEB is detected.

References: None.

Grants: None.

Conflict of Interest: None declared.

P02.011.D Genetic variants identified in Slovenian families with apparent non-syndromic orofacial cleft phenotypes

Lara Slavec ^1;2, Ksenija Geršak^1;3, Andreja Eberlinc⁴, Nataša Karas Kuželički²

¹University Medical Centre Ljubljana, Department of Obstetrics and Gynaecology, Research Unit, Ljubljana, Slovenia; ²University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Ljubljana, Slovenia; ³University of Ljubljana, Faculty of Medicine, Department of Gynaecology and Obstetrics, Ljubljana, Slovenia; ⁴University Medical Centre Ljubljana, Department of Maxilofacial surgery, Ljubljana, Slovenia

Background/Objectives: Orofacial clefts (OFCs) are common congenital anomalies that manifest as cleft lip and/or cleft palate. Most OFCs are non-syndromic (nsOFCs), but can also occur in various syndromes (syOFCs). The majority of syOFCs have a known, monogenic cause, while nsOFCs usually have a complex aetiology. In some syOFCs, the presence of additional anomalies is not obvious, thus they often remain undiagnosed as such. Examples of such syOFCs are Van der Woude syndrome (VWS; IRF6, GRHL3 mutations), and X-linked cleft palate (CPX; TBX22 mutations). Moreover, there is increasing evidence that mutations in single genes may be implicated in some families with nsOFCs.

Methods: We recruited 35 Slovenian multiplex families. Using whole-exome sequencing (WES) and Sanger sequencing, we firstly screened the proband of each family for causal variants in IRF6, GRHL3, and TBX22. In probands without any relevant variants in these three genes, we filtered WES data for 72 additional genes. Family segregation was checked for each identified variant.

Results: We genetically confirmed the diagnosis of VWS in five families. We identified four (likely) pathogenic variants in IRF6: two stop-gain, one frameshift, and one missense variant. In one family, we identified the deletion of TBX22 and confirmed CPX. We have also found one nsOFC family with deletions in coding regions of ARHGAP29.

Conclusion: With a two-step approach in gene selection and analysis, we identified families with syOFCs, and found a monogenic mutation in a family with nsOFCs. We present the first genetic study performed on Slovenian patients with apparent nsOFCs.

References:.

Grants: Slovenian Research Agency (J3-8207, MR51882).

Conflict of Interest: None declared.

P02.012.A Is nuchal translucency of 3.0- 3.49 mm an indication for NIPT or microarray-still needs a debate

Magda Rybak-Krzyszkowska ^1;2, Anna Madetko-Talowska³, Katarzyna Szewczyk³, Miroslaw Bik-Multanowski³, Agata Sakowicz⁴, David Stejskal⁵, Marie Trková⁵, Dagmar Smetanova⁵, Silvia Serafim⁶, Hildeberto Correia⁶, Julián Nevado^7;8, Fe Amalia García Santiago^7;8, Maria Angeles Mori^7;8, Lena Rutkowska⁹, Agata Kucińska⁹, Agnieszka Gach⁹, Hubert Huras¹, Magdalena Kołak¹, Malgorzata Srebniak¹⁰

¹Department of Obstetrics and Perinatology University Hospital, Kraków, Poland; ²Hi-Gen Centrum Medyczne, Krakow, Poland; ³Department of Medical Genetics, Institute of Paediatrics, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland; ⁴Department of Medical Biotechnology, Medical University of Lodz, Łódź, Poland; ⁵Centre of Medical Genetics and Reproductive Medicine GENNET, Prague, Czech Republic; ⁶Unidade de Citogenética, Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisbon, Portugal; ⁷Instituto de Genética Médica y Molecular (INGEMM), Hospital Universitario La Paz- IdiPaz and Centro de Investigación Básica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; ⁸ITHACA, International Research Network in Rare Diseases. Hospital Universitario La Paz, Madrid, Spain; ⁹Department of Genetics, Polish Mother’s Memorial Hospital Research Institute, Łódź, Poland; ¹⁰Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands

Background/Objectives: Nowadays, fetal nuchal translucency (NT) values of >3.5 mm constitute an indication for invasive testing, optimally followed by chromosomal microarray. In our analysis, we wanted to evaluate the occurrence of clinically relevant sub-microscopic chromosomal aberrations in fetuses with the NT range from 3.0 to 3.49 mm, which would be missed by currently available non-invasive prenatal tests (NIPT).

Methods: A retrospective analysis of 271 fetuses with NT between 3.0 and 3.49 mm and increased risk after combined test in five cohorts of pregnant women referred for invasive testing and chromosomal microarray was performed: Czech Republic (n = 152), Poland (Cracow n = 48, Lodz n = 7) Portugal (n = 46) and Spain (n = 18).

Results: A chromosomal aberration was identified in 1:5 fetuses (19.19%; 52/271). In 15.13% (41/271) of cases trisomy 21, 18 or 13 was found. In 0.74% (2/271) sex chromosome aneuploidy was found. In 1.1% (3/271) of cases copy number variant (CNV) >10Mb was detected, which would potentially also be detected by NIPT. The residual risk for missing a clinically relevant (sub)microscopic chromosome aberration in the presented cohorts is 1:45 (2.21%; 6/271).

Conclusion: Our results indicate that a significant number of fetuses with increased risk after combined test and presenting NT of 3.0-3.49 mm carry a clinically relevant chromosomal abnormality other than common trisomy. Invasive testing should be offered and counselling on NIPT should include the resolution limitation that may result in NIPT false negative results in a substantial percentage of fetuses.

References:.

Grants:.

Conflict of Interest: None declared.

P02.013.B Sodium channel gene variants in fetuses with abnormal sonographic findings

Andrea Hadjipanteli ¹, athina theodosiou², ioannis papaevripidou², paola evangelidou², angelos alexandrou², nicole salameh², violetta anastasiadou³, Yiannis Kallikas⁴, LUDMILA KOUSOULIDOU², carolina sismani²

¹The Cyprus Institute on Neurology and Genetics, Cytogenetics and Genomics, Nicosia, Cyprus; ¹The Cyprus Institute on Neurology and Genetics, Cytogenetics and Genomics, Nicosia, Cyprus; ³Archibishop Makarios III hospital, Nicosia, Cyprus; ⁴AAK Ultrasound and Fetal Medicine Centre, Nicosia, Cyprus

Background/Objectives: Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSC may therefore cause epileptic encephalopathies and congenital myopathies resulting in severe phenotypes. This study presents two cases of sodium channelopathies in pregnancies with abnormal ultrasound findings; identified as part of a larger cohort study.

Methods: Fetal samples with abnormal sonographic findings accompanied by normal karyotype and array-CGH results were included in the study. Trio-based whole-exome sequencing was performed, followed by Sanger sequencing for the confirmation of pathogenic variants.

Results: Two unrelated fetuses presented pathogenic variants in genes encoding the alpha subunit of VGSCs. A known de novo heterozygous missense variant was identified in the gene SCN2A (c.751G>A; p.Val251Ile) in a fetus with intrauterine growth retardation, hand clenching, ventriculomegaly and other structural findings that neonatally also exhibited refractory epilepsy, spasms, and MRI abnormalities. The second fetus was compound heterozygous for two parentally inherited novel missense variants in the gene SCN4A (c.4340T>C; p.Phe1447Ser), (c.3798G>C; p.Glu1266Asp). The fetus presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and other. Both probands died soon after their birth.

Conclusion: Our results suggest a potentially crucial role of the sodium channel gene family in fetal development since non-functional VGSCs have been associated with severe fetal phenotypes and early lethality. New genotype-in utero phenotype associations related to this gene family may ultimately result in new additions to preconception and prenatal diagnostic panels.

References:.

Grants:.

Conflict of Interest: None declared.

P02.014.C Diagnostic yield and implications of exome sequencing analysis, including the use of copy number variant analysis pipeline, for pregnancy management in a series of 46 fetuses with structural anomalies

Sebastien Moutton^1;2, Ines Harzallah³, Laure Raymond ¹, Jérémie Mortreux¹, Nada Houcinat¹, Marine Dancer¹, Vanna Geromel¹, Radoslava Saraeva-Lamri¹, Angeline Preto¹, Luc Druart¹, Marc Nouchy¹, Fabienne Prieur³, Marine Lebrun³, Francis Ramond³, Rodolphe Dard⁴, Aude Tessier⁴, Benjamin Dauriat⁵, Valentin Marquet⁵, Constance Wells⁶, Caroline Deiller⁶, Elise Schaefer⁷, Leila Frigere⁸, Patrice Bouvagnet^1;8, Renaud Touraine³, Marie-Emmanuelle Naud-Barreyre¹

¹Laboratoire Eurofins Biomnis, Département de génétique, Lyon, France; ²Maison de Santé Protestante de Bordeaux Bagatelle, Centre Pluridisciplinaire de Diagnostic PréNatal, Talence, France; ³CHU de Saint-Etienne, Service de génétique médicale, Saint Etienne, France; ⁴CHI Poissy-Saint-Germain-en-Laye, Service de génétique médicale, Poissy, France; ⁵CHU de Limoges, Service de génétique médicale, Limoges, France; ⁶CHU de Montpellier, Service de génétique médicale et foetopathologie, Montpellier, France; ⁷CHRU de Strasbourg, Service de génétique médicale, Strasbourg, France; ⁸CHU de Martinique, Centre Pluridisciplinaire de Diagnostic PréNatal, Fort de France, France

Background/Objectives: During the past years, exome sequencing has been used more and more widely and contributed to increased diagnostic yield in many fields of human genetics including developmental disorders (25-40% according to the studies, the design, the limitation to diagnosis or extension to research). Its use has been progressively implemented in a prenatal setting, first to assess the yield in these particular patients, and second to assess its usefulness in pregnancy management.

Methods: Between June 2020 and November 2021, 46 exome analyses were asked for fetuses with structural anomalies (at the same time or after normal chromosomal microarray result), questioning the prognosis. Prescriptions came from several French multidisciplinary centers for prenatal diagnosis. Exome data were producted by Eurofins Biomnis laboratory and analyzed by 2 or 3 independant molecular geneticists, medical geneticists and/or scientists followed by meetings before reporting the results.

Results: In 11/46 = 24% of cases, a conclusive diagnosis could be made, with implications in pregnancy management in 5/11 = 45%, leading to opt for termination in 3 cases and to reassure in 2 cases (and 3 additional cases where the molecular diagnosis confirmed the suspicion by imaging and helped decision-making of termination). One diagnosis was an intragenic GPC3 duplication not seen by chromosomal microarray. In at least 16 until 29/35 unconclusive results, the negativity contributed to the continuation of the pregnancy.

Conclusion: Although the cohort remains quite small, this study suggests that exome sequencing could be useful in selected prenatal situations, as reported in still not so numerous other studies.

References: -.

Grants: -.

Conflict of Interest: None declared.

P02.015.D Designing a quantitative PCR assay for fetal RhD genotyping from maternal plasma derived cell-free fetal DNA (cffDNA)

Suvi Parviainen ¹, Ville Veikkolainen²

¹University of Turku, Life Technologies, Turku, Finland; ²PerkinElmer Wallac Oy, Turku, Finland

Background/Objectives: Hemolytic disease of the fetus and newborn (HDFN) is typically caused by maternal alloimmunization against antigens of RhD positive red blood cells. During pregnancy, these anti-RhD antibodies can induce the destruction of fetal RhD positive red blood cells and lead to HDFN. Maternal alloimmunization can be prevented with antenatal and postnatal anti-D prophylaxis treatment, which is often routinely performed for all RhD negative mothers. However, fetal RhD genotyping allows anti-D prophylaxis to be targeted only for women carrying RhD positive fetus. Therefore, unnecessary anti-D prophylaxis treatments can be avoided. The aim of the study was to develop a multiplexed, quantitative PCR (qPCR) assay for fetal RhD genotyping using maternal plasma derived cell-free fetal DNA (cffDNA) as sample material.

Methods: The fetal RhD genotyping assay was designed for simultaneous detection of RHD gene exons 5, 7 and 10, in addition to an internal control gene. Dry qPCR chemistry was utilized to enable simplified qPCR workflow. The assay was confirmed to be compatible with cfDNA samples extracted with PerkinElmer Vanadis Extract® system.

Results: The assay was able to distinguish RhD positive and negative cfDNA samples and showed similar performance with both liquid and dried qPCR chemistry. The presence of fetal DNA in the maternal cfDNA samples was demonstrated by amplification of male-specific sex-determining region Y (SRY) gene.

Conclusion: Dried qPCR chemistry enables a simple and time-saving workflow, which can be easily combined with other prenatal cffDNA screening. Further clinical testing is required to determine clinical sensitivity and specificity.

References:.

Grants: In collaboration with PerkinElmer Wallac Oy.

Conflict of Interest: Suvi Parviainen This Master’s Thesis study was done as collaboration with PerkinElmer Wallac Oy and University of Turku. I am currently working full-time at PerkinElmer Wallac Oy as Subject Matter Expert (customer support)., Ville Veikkolainen Employee at PerkinElmer (Senior R&D Scientist).

P02.016.A LARS1-related disorder presenting as recurrent severe early onset fetal growth restriction attributed to placental and immune disease

Dana Brabbing-Goldstein ¹, Sharon Perlman^2;3, lily bazak¹, Gabriel Arie Lidzbarsky¹, Yael Kilim¹, lina basel-salmon^1;2;4;5, Rivka Sukenik-Halevy^2;6

¹Raphael Recanati Genetic Institute, Rabin Medical Center-Beilinson Hospital, Petach Tikva, Israel; ²Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ³Ultrasound Unit, Helen Schneider Women’s Hospital, Rabin Medical Center, Petach Tikva, Israel; ⁴Paediatric Genetics Clinic, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel; ⁵Felsenstein Medical Research Center, Petach Tikva, Israel; ⁶Genetic Institute, Meir Medical Center, Kfar Saba, Israel

Background/Objectives: While the yield of chromosomal microarray in isolated fetal growth restriction (FGR) has been established, the added value of whole exome sequencing (WES) in such cases is unknown. We report recurrent severe early onset FGR in three of the five pregnancies in one family related to Infantile liver failure syndrome type 1 (OMIM 615438).

Methods:.

Results: Fetal autopsy showing hepatocyte hemosiderosis and placental pathology showing >50% necrosis of villi, multiple cysts and synechia, raised suspicion of both gestational alloimmune liver disease and thrombophilia. However, FGR fully reoccurred albeit IVIG and antithrombotic treatment. Fetal chromosomal microarray analysis was normal. Quatro WES revealed that two affected fetuses were compound heterozygotes for paternal truncating (c.1144G>T;p.Glu382Ter) and maternal missense (c.1511C>T;p.Ala504Val) variants in the LARS1 gene (NM_020117.11) which is related to autosomal recessive infantile liver failure syndrome type 1. The variants co-segregated in a third affected fetus but not in a healthy daughter.

Conclusion: Aminoacyl-tRNA synthetase deficiencies, including LARS1-related condition, cause multisystemic disorders mostly affecting growth and nervous system. Inefficient translation and consequential decreased cell proliferation may explain the FGR phenotype. The family’s genetic diagnosis will allow prenatal diagnosis or preimplantation genetic testing in future pregnancies and will help to avoid unnecessary treatments. This report highlights the importance of incorporating WES in the evaluation of early severe isolated FGR, as genetic diagnosis may completely change medical interventions and shed light on the expected outcome. Further studies are needed to evaluate the prevalence of single gene disorders in isolated FGR.

References:.

Grants:.

Conflict of Interest: None declared.

P02.017.B The role of a multidisciplinary team in managing variants of uncertain clinical significance in prenatal genetic diagnosis

Karin Diderich¹, Jasmijn Klapwijk¹, Myrthe van den Born¹, Martina Wilke¹, Marieke Joosten¹, Diane Van Opstal¹, Hennie Brüggenwirth¹, Malgorzata Srebniak ¹

¹Erasmus MC, Clinical Genetics, Rotterdam, Netherlands

Background/Objectives: The aim of this study was to evaluate the role of the multidisciplinary team (MDT), the elements that facilitate rapid management of variants of unknown clinical significance (VUS) in prenatal diagnosis, and which types of VUS were considered for reporting.

Methods: We reviewed the frequency of MDT meetings and factors contributing towards decision making on reporting VUS after prenatal exome sequencing.

Results: The crucial elements that facilitate rapid VUS management were regular meetings, appropriate expertise, professional connections with other experts (ad hoc present) and psychological team safety. The following VUS were considered for reporting: possibly matching the fetal phenotype, associated with severe disorders when a functional test is available (e.g. enzymatic test), possibly associated with a disorder where early post-partum diagnosis and treatment are crucial for a better prognosis.

Conclusion: In order to protect prospective parents from the burden of VUS, the professionals should limit reporting them. Apart from using software filters and building professional guidelines, this can be achieved by sharing professional experience and responsibility in a MDT setting. Moreover, we noted that emotional distress caused by communicating uncertain information to the prospective parents can only be borne when supported by psychological team safety.

References:.

Grants:.

Conflict of Interest: None declared.

P02.018.C CHRAB – a tool for identification and visualization of chromosomal abnormalities in preimplantation embryos using next-generation sequencing data

Timing Liu¹, Zainul Arifin¹, Vivienne Tan¹, Beatrice Pak¹, Arnold Tan¹, Simin Wong¹, Henry Yang¹, Caroline Lee¹, Mahesh Choolani¹, Peng-Cheang Wong¹, Samuel Chong ¹

¹National University of Singapore, Singapore, Singapore

Background/Objectives: Current commercial solutions for preimplantation genetic testing of aneuploidies (PGT-A) using next generation sequencing (NGS) data entail the use of specific proprietary analysis software, which preclude analysis of NGS data generated from other sources. We have developed and evaluated CHRAB, a tool for identifying CHRomosomal ABnormalities in preimplantation embryos using NGS data.

Methods: Single-cell and five-cell replicates from aneuploid and segmentally unbalanced cell lines were whole genome amplified using SurePlex and subjected to Nanopore single-molecule sequencing, followed by CHRAB analysis and visualization. An aliquot of each Sureplex-amplified sample was also sequenced using a leading commercial NGS-based PGT-A solution (Illumina VeriSeq PGS) and analyzed using its proprietary analysis software (BlueFuse Multi).

Results: Using Nanopore sequencing and CHRAB analysis and visualization, the expected cell-line specific aneuploidy was detected in 84 of 88 (95.45%) single- and multi-cells tested, and the expected segmental imbalance (≥10 Mb) was detected in 88 of 90 (97.78%) single- and multi-cells tested. The corresponding percentages obtained using the commercial PGT-A solution were 95.45% (84 of 88 single- and multi-cells with an aneuploidy) and 98.89% (89 of 90 single- and multi-cells with a segmental imbalance of ≥10 Mb). Detection sensitivity for smaller segmental imbalances of <10 Mb was significantly lower on both platforms.

Conclusion: We have developed a bioinformatics tool for aneuploidy and segmental imbalance detection from single- and multi-cell NGS data, with accuracy comparable to commercial platforms.

References:.

Grants: NMRC OF-IRG 20Nov-0060 (Ministry of Health, Singapore) and NUHSRO/2019/055/RO5+5/Seed-Mar/08 (National University Health System, Singapore).

Conflict of Interest: None declared.

P02.019.D Perlman Syndrome: A prenatal and genetic diagnostic challenge

Kim Van Berkel¹, Elise Vantroys ¹, Ileen Slegers¹, Astrid Leus², Stefanie Brock³, Boyan Dimitrov¹

¹UZ Brussel, Centre for Medical Genetics, Brussels, Belgium; ²UZ Brussel, Department of Radiology, Brussels, Belgium; ³UZ Brussel, Department of Pathology, Brussels, Belgium

Background/Objectives: Perlman syndrome (PS) is a very rare congenital overgrowth syndrome, with a prevalence estimated to be less than 1/1.000.000. Generally the diagnosis is based on neonatal clinical appearance and histologic findings. For a long time the genetic basis of PS was unknown with an assumed autosomal recessive inheritance. Since 2012 mutations in DIS3L2 gene have been found to be associated to PS. Some cases have been described prenatally, but precise diagnosis in a prenatal setting remains difficult to obtain due to overlap with other overgrowth syndromes. Both prenatal imaging and genetic diagnostic technologies have enormously evolved over the past decade and are being implemented in prenatal diagnosis today, enhancing diagnostic yield.

Methods: Here, we a present prenatal case of PS, with phenotyping by subsequent ultrasound and MRI examinations and molecular diagnosis by NGS, with an overview of existing literature. Accurate prenatal phenotyping of PS and comparison with Beckwith-Wiedemann and Simpson-Golabi-Behmel sydrome was made.

Results: Confirmed prenatal diagnosis of PS was obtained by subsequent high resolution imaging with next generation sequencing (NGS) on amniotic fluid sample, showing 2 pathogenic variants in the DIS3L2 gene, not yet described in existing literature.

Conclusion: Since knowledge of the concerned gene and use of new technologies, for imaging as well as genetic analysis, the diagnosis of PS can be made prenatally. This is of great value during prenatal counseling of pregnant couples, as PS is known to have a poor prognosis.

References: Astuti D et al Nature Genet. 2012.

Grants:.

Conflict of Interest: None declared.

P02.020.A Prenatal Trio-Exome-Analysis reveals not yet published prenatal manifestations of rare diseases

Uwe Ahting ¹, Melanie Isau², Bernt Schulze³, Markus Stumm², Konstanze Hoertnagel¹

¹MVZ-Martinsried GmbH, Human Genetics, Planegg, Germany; ²Medicover Genetics GmbH, Molekulargenetik, Berlin, Germany; ³Medicover Humangenetik Berlin Lichtenberg, Humangenetik Hannover, Hannover, Germany

Background/Objectives: Between 7-2021 and 12-2021 we performed 79 prenatal trio exome analyses with 23 positive results (29.5%) with a turnaround time of 12.05 days on average. In all cases, indication has been ultrasound abnormalities. Most frequent leading symptoms were increased nuchal translucency (n = 19), skeletal (n = 14), brain (n = 14), and heart (n = 13) abnormalities. Most pathogenic variants found in association with skeletal abnormalities were de novo and located in known disease genes. In some cases, pathogenic variants were associated with prenatal manifestations of rare diseases not published so far. Here we present two case reports.

Methods: Prenatal trio exome analysis.

Results: Case 1: Fetus with cerebella vermis hypoplasia, choroid plexus cyst and micrognathia in 19^th week of pregnancy. Trio exome analysis demonstrated a frameshift variant in CNOT3 (disease IDDSADF; OMIM #P: 618672), already published as pathogenic. Postnatal manifestations of IDDSADF are small chin, short stature, hypotonia, cerebellar abnormalities. Prenatal manifestations are not published yet.

Case2: Fetus with increased nuchal translucency and cystic hygroma in 13^th week of pregnancy. Trio exome analysis provided a probably pathogenic missense variant in KCNT2 (disease DEE57, OMIM #P: 617771). Postnatal manifestations of DEE57 are global developmental delay, hypotonia, intellectual disability and seizures. Hitherto only fetal hiccup has been mentioned in one case report.

Conclusion: Notwithstanding the possibility of spurious correlations, the two presented cases reveal so far unreported prenatal manifestations of rare diseases. More to be found. In addition prenatal trio exome analysis increases the diagnostic yield of genetic prenatal investigation, especially if increased nuchal translucency is present.

References:.

Grants:.

Conflict of Interest: None declared.

P02.021.B Expanded NIPS does not significantly increase the detection rate of abnormal genetic findings in pregnancies with normal ultrasound

Lena Sagi-Dain¹, Idit Maya ², Liat Sheelo Salzer², lina basel-salmon²

¹Carmel Medical Center, Haifa, Israel; ²Rabin Medical Center Golda Hasharon Campus, Petach Tikva, Israel

Background/Objectives: Non-invasive prenatal screening (NIPS) is currently widely used in many countries. The common NIPS technique is focused on trisomies 13, 18, 21 and sex-chromosomal aberrations (5-NIPS), while many laboratories offer expansion to include common microdeletions as well as genome-wide aberrations sized over 7Mb. We aimed to evaluate the theoretical yield of NIPS expansions compared to the commonly used 5-NIPS, based on chromosomal microarray analysis (CMA) results of pregnancies with normal ultrasound.

Methods: CMA results of pregnancies with normal ultrasound (including soft markers and abnormal maternal serum screening) were recorded. We have calculated the detection rate of 5-NIPS detectable aberrations and compared those to rates of findings detectable by 5-NIPS plus common microdeletions (1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.3-5p15.1, 15q11.2-15q13.1, and 22q11.2), and genome-wide NIPS-detectable findings (including variants >7Mb).

Results: Of the 8,605 pregnancies, 44 (0.51%) 5-NIPS detectable findings were noted, ranging from 1.56% in 642 pregnancies with abnormal maternal serum screening, 0.63% in 318 pregnancies with soft markers, 0.62% in 4,378 women aged over 35 years, to 0.15% in younger women. Three cases of common microdeletions were detected in the overall cohort (0.03%), as well as six genome-wide-NIPS detectable findings (0.07%), yielding a non-significant difference compared to 5-NIPS.

Conclusion: NIPS expansion to common microdeletions as well as to genome wide-findings does not significantly increase the detection rate compared to 5-NIPS. These results facilitate informed decisions of couples regarding prenatal genetic screening using NIPS technology in comparison to CMA from invasive fetal sample.

References:.

Grants:.

Conflict of Interest: None declared.

P02.022.C A rare case of fetoplacental discrepancy: a normal result in chorionic villi in contrast to de novo unbalanced translocation 46,XX,der(6)t(6;17) in amniotic fluid

Lucie Lišková ¹, Vlasta Cejnova¹, Monika Stará¹, Vjaceslav Harmas¹, Lilly Dejová¹, Lenka Vancová¹, Jana Lastuvkova¹

¹Masaryk Hospital in Ústí nad Labem, o.z., Krajská zdravotní, a.s., Department of Medical Genetics, Ústí nad Labem, Czech Republic

Background/Objectives: We present a gravidity with different results from invasive prenatal testing. Firstly, a normal analysis of chorionic villi and subsequently an abnormal karyotype of cultured amniocytes were detected. Combined first trimester screening was positive and the fetal ultrasound scan revealed hygroma colli cysticum.

Methods: Prenatal ultrasound, aCGH, karyotyping and FISH.

Results: Microarray analysis of native chorionic villi showed a normal female profile of the fetus. The amniocentesis (at 16 WG) was performed due to other fetal ultrasound findings: ascites, hyperdense lung and hyperechogenic fetal bowel. Cytological analysis of cultured amniocytes revealed an abnormal karyotype: 46,XX,der(6) which was confirmed by microarray: arr[GRCh37] 6q26q27(162799322_170884575)x1,17q21.32q25.3(47109888_81044553)x3. De novo unbalanced translocation was determinated in the fetus: 46,XX,der(6)t(6;17)(q26;q27.3), where the 8 Mb terminal deletion of long arm of chromosome 6 and 34 Mb terminal duplication of chromosome 17 were determined. Karyotypes of both parents were normal. The gravidity was terminated and the samples of placenta, amniotic fluid, cord blood and skin were analyzed. Cytological analysis of placenta confirmed a normal karyotype, whereas in the other types of tissues the normal cell line was detected together with abnormal cell line with der(6) in different levels of mosaicism. The mosaicism of cell line with der(6) was defined by FISH analysis: amniotic fluid, fetus tissue, cord blood; 90%, 66%, 17.5% of interphase nuclei, respectively.

Conclusion: The mosaicism of normal cell line 46,XX together with abnormal cell line with de novo unbalanced autosomal translocation is extremelly rare and needn´t be detected in CVS.

References:.

Grants:.

Conflict of Interest: None declared.

P02.023.D False positivity and false negativity as a standard part of noninvasive prenatal testing

Gabriel Minarik ^1;2, Michaela Hyblova^1;2, Martina Sekelska^1;2, Erika Tomkova³, Katarina Tothova³, Dagmar Landlova³, Peter Krizan^1;3

¹Medirex Group Academy n.p.o., Department of genomics, Nitra, Slovakia; ²Trisomy test Ltd., Nitra, Slovakia; ³Medirex Inc., Department of genetics, Bratislava, Slovakia

Background/Objectives: Nonivasive prenatal testing (NIPT) is becoming highly accepted as a routine prenatal screening tool in most of developed countries. With advanced versions of NIPT tests covering aneuploidies of all chromosomes and subchromosomal aberrations false positives and false negatives appear more frequently.

Methods: To get comprehensive information about the reason of NIPT result falseness methods as qPCR, aCGH, FISH, karyotyping in diagnostic settings were used additionally to original low coverage whole genome sequencing based NIPT.

Results: After 19159 NIPT performed in routine clinical laboratory 9 analyses were reported as false negative (2) and false positive (7), in statistics focused solely on chromosomes 21, 18 and 13 trisomies, representing less than 0.05% of cases. When focusing on all chromosomes and also subchromosomal aberrations the frequency of such false results is even higher. From the portfolio of potential biological reasons we detected and confirmed by molecular methods cases related to confined placental mosaicism, true fetal mosaicism, maternal aberration in full and mosaic state, precancerous maternal aberration, incorrect previous anamnestic data. As technical reasons grey zone result, insufficient coverage and syndrome specific information were detected.

Conclusion: False negative and false positive NIPT results are reported every day all over the world and need to be addressed with comprehensive supplementary diagnostic testing. Consensus about its routine usage is needed and international guidelines should reflect this need too.

References:.

Grants: This work was supported by the OPII programme as the project - Center for biomedical research – BIOMEDIRES – II. phase, ITMS 313011W428, co-financed by the ERDF.

Conflict of Interest: Gabriel Minarik part-time employee of Medirex Group Academy n.p.o and Trisomy test Ltd., principal investigator of running grants, Michaela Hyblova part-time employee of Medirex Group Academy n.p.o and Trisomy test Ltd., collaborator on several grants, Martina Sekelska part-time employee of Medirex Group Academy n.p.o and Trisomy test Ltd., collaborator on several running grants, Erika Tomkova full-time employee of Medirex Inc., Katarina Tothova full-time employee of Medirex Inc., Dagmar Landlova full-time employee of Medirex Inc., Peter Krizan part-time employee of Medirex Inc. and Medirex Group Academy n.p.o.

P02.024.A Clinical implementation of a custom oligonucleotide array-CGH. Experience in the largest cohort of Spanish prenatal samples (>5300 samples)

Olaya Villa Marcos¹, Marina Viñas-Jornet¹, Noemí Sousa¹, Nerea Alvarez¹, Neus Fornés¹, Manel García-Aragonés¹, Luis Pérez-Jurado², LLuís Armengol ¹

¹Quantitative Genomics Medicine Laboratories, qGenomics, Microarray, Esplugues del Llobregat, Spain; ²Universitat Pompeu Fabra, Hospital del Mar - IMIM, and CIBER de Enfermedades Raras (CIBERER), Genetics unit, Barcelona, Spain

Background/Objectives: The implementation of genomic array in prenatal routines, when accompanied by pre- and post-test genetic counseling, has demonstrated its utility by fulfilling the longstanding need for a diagnostic test with a higher resolution and higher diagnostic yield than its predecessor, the conventional karyotype.

Methods: Array CGH was performed in 5388 prenatal samples (3972 amniotic fluids, 1131 corions and 285 fetal samples), using a custom 60K oligonucleotide-based microarray (qChip® CM) designed to maximize the detection of clinically relevant copy-number alterations, and minimize the detection of variants of unknown significance (VOUS). As a general rule, VOUS with unclear phenotypic effect according to current knowledge, and some susceptibility variants are not reported.

Results: We identified a total of 363 pathogenic or likely pathogenic alterations (detection rate: 6.73%) and 76 VOUS (1.41%). As expected, the greatest pathogenic detection rate (7.28%, 326/4477) was among fetuses with ultrasound anomalies, while detection rate was 4.06% (37/911) in normal ultrasound gestations. When both parental samples were available, the vast majority of the VOUS were inherited from a non-affected parent (90.24%) and could be reclassified as likely benign.

Conclusion: Our series reinforces the clinical utility of prenatal microarray testing: it nearly doubles the diagnostic yield of conventional karyotype (153/363 with variants <10Mb), with no significant increase in the frequency of VOUS that could interfere in decision making. In our experience, we highlight the importance of implementing aCGH in prenatal routines, for all gestations with an indication of invasive fetal sampling.

References:.

Grants:.

Conflict of Interest: Olaya Villa Marcos qGenomics, Marina Viñas-Jornet qGenomics, Noemí Sousa qGenomics, Nerea Alvarez qGenomics, Neus Fornés qGenomics, Manel García-Aragonés qGenomics, Luis Pérez-Jurado qGenomics, LLuís Armengol qGenomics.

P02.026.C The diagnostic yield of prenatal exome sequencing in fetuses with ultrasound anomalies

Karin Diderich ¹, Florentine Thurik¹, Martina Wilke¹, Marieke Joosten¹, Myrthe van den Born¹, Robert-Jan Galjaard¹, Marjolein Weerts¹, Marjon van Slegtenhorst¹, Frank Sleutels¹, Sander Galjaard², Maarten Knapen², Attie Go², Lies Hoefsloot¹, Malgorzata Srebniak¹, Hennie Brüggenwirth¹

¹Erasmus MC, Clinical Genetics, Rotterdam, Netherlands; ²Erasmus MC, Obstetrics and Gynecology, Rotterdam, Netherlands

Background/Objectives: The aim of the study was to evaluate the diagnostic yield of routine exome sequencing (ES) in fetuses with ultrasound anomalies detected in the first two trimesters that had a normal molecular karyotype by chromosomal microarray.

Methods: A retrospective analysis of 138 fetuses with multiple or isolated ultrasound anomalies that were referred for ES in May 2019- March 2021. Some specific isolated abnormalities were excluded from ES: e.g. neural tube defect, ventricular septal defect and echogenic bowel. Variant analysis was limited to a broad gene panel consisting of genes involved in multiple congenital anomalies and/or intellectual disability as published before. We used trio analysis and filtering for de novo variants, compound heterozygous variants, homozygous variants, X-linked variants, variants in imprinted genes and for known pathogenic variants.

Results: Pathogenic and likely pathogenic variants (class 5 and 4 respectively) that explained the fetal phenotype were identified in 13% (18/138) fetuses. Potentially actionable class 3 variants (matching the phenotype) were reported in few cases after extensive discussion in a multidisciplinary team. The risk for a single gene disorder in the presented cohort was ~ 1:8. The time between the invasive procedure and genetic diagnosis was 13-18 days.

Conclusion: Our results indicate that a significant number of fetuses with ultrasound anomalies and normal molecular karyotype carry a clinically relevant (likely) pathogenic variant that can be diagnosed through prenatal exome sequencing. In our opinion not only microarray testing, but also exome sequencing should be offered in case of a fetus with ultrasound anomalies.

References:.

Grants:.

Conflict of Interest: None declared.

P02.027.D Use of prenatal exome sequencing in fetuses with ultrasound anomalies

Maria Segura-Puimedon¹, Marta Carreño¹, Raquel Garcia¹, Lidia Carreño¹, Hector San Nicolás¹, César Arjona¹, Cèlia Sintas¹, Olaya Villa Marcos¹, Marina Viñas-Jornet¹, Mònica Vall¹, Nina Bosch ¹, LLuís Armengol¹

¹qGenomics, NGS Clinical Department, Esplugues de Llobregat, Spain

Background/Objectives: Whole exome sequencing (WES) is an established diagnostic tool in postnatal settings for individuals with a suspected genetic condition. Recently, it is increasingly used as a diagnostic tool in prenatal settings as well. We present here our experience using this technology in fetuses with ultrasound anomalies.

Methods: WES was performed in 350 fetal samples with ultrasound anomalies. 248 samples were from evolutive pregnancies and 102 were from legal interruptions or stillbirths. Segregation studies were performed in cases with a candidate variant when possible.

Results: Common reasons for referral were skeletal anomalies, polymalformated fetuses, cerebral anomalies or specific syndrome suspicion. Pathogenic or likely pathogenic variants were identified in n = 56 (16%) of samples. In n = 64 (18.2%) cases, variants of unknown significance were identified. In more than half of the cases (58%) no candidate variant was identified. Diagnostic yield was higher in fetuses with skeletal anomalies, where pathogenic or likely pathogenic variants were identified in (n = 19) 33% of cases, and in fetuses with increased nuchal translucency or hydrops (Noonan syndrome suspicion), where a pathogenic variant was found in 23.5% (n = 13) of the samples.

Conclusion: Exome sequencing is a valuable diagnostic tool in fetuses with ultrasound anomalies, especially when skeletal anomalies are present or when Noonan syndrome is suspected.

References:.

Grants:.

Conflict of Interest: Maria Segura-Puimedon qGenomics, Marta Carreño qGenomics, Raquel Garcia qGenomics, Lidia Carreño qGenomics, Hector San Nicolás qGenomics, César Arjona qGenomics, Cèlia Sintas qGenomics, Olaya Villa Marcos qGenomics, Marina Viñas-Jornet qGenomics, Mònica Vall qGenomics, Nina Bosch: None declared, LLuís Armengol qGenomics.

P02.028.A Two novel variants in the POR gene causing Antley-Bixler syndrome type 2 detected prenatally in a foetus with abnormal ultrasound findings

Jana Lastuvkova ¹, Miroslav Brestak², Pavel Votypka³, Petra Peldova³, Vlasta Cejnova¹, Lucie Liskova¹, Vjaceslav Harmas¹

¹Masaryk Hospital, o.z., Krajska zdravotni, a.s., Department od Medical Genetics, Usti nad Labem, Czech Republic; ²Prague Prenatal Diagnosis Centre, ProfiG2, Prague, Czech Republic; ³2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Department of Biology and Medical Genetics, Prague, Czech Republic

Background/Objectives: Antley-Bixler syndrome (ABS) is a rare severe form of syndromic craniostenosis.

Type 1 ABS is autosomal dominant due to heterozygous pathogenic variants in the FGFR2 gene, type 2 ABS is autosomal recessive due to biallelic pathogenic variants in the POR gene. Phenotypic features include craniostenosis, characteristic facial features, proptosis, skeletal anomalies and wide range of organ malformations.

We present a case of ABS type 2 detected on the basis of prenatal ultrasound examination.

Methods: Prenatal ultrasound, aCGH, targeted NGS panel of 89 genes associated with craniostenosis.

Results: Atypical head shape of foetus was seen on ultrasound in the 21st week of pregnancy. Combined screening of the 1st trimester was negative but level of PAPP-A was extremely high - 41.112 MoM. Specialized ultrasound examination confirmed turicephaly, deformity of the cerebral hemispheres, hypertelorism, low set ears and talipes varus. Craniostenosis was suspected. Parents decided to terminate pregnancy.

Autopsy of the foetus showed turicephaly, frontal bones agenesis, hypertelorism, proptosis, low set ears, lack of thumbs and arachnodactyly forefingers on both feet. Examination of the targeted NGS gene panel revealed 2 novel heterozygous probably pathogenic variants in the POR gene - c.1898 + 1del/c.450>A p.(Asp150Glu). Each variant is present in one of the parents.

Conclusion: This case demonstrates the importance of collaboration between precision ultrasound diagnostics and modern laboratory methods (NGS) in elucidating the causes of rare serious foetal disorders. We believe this is the first case of prenatal detection of ABS type 2 in the Czech Republic.

References:.

Grants:.

Conflict of Interest: None declared.

P03 Sensory Disorders (Eye, Ear, Pain)

P03.001.B Accurate clinical evaluation and high throughput technologies for the molecular characterization of hereditary hearing loss in a large cohort of Italian patients

Anna Morgan ¹, Stefania Lenarduzzi¹, Beatrice Spedicati², Paola Tesolin², Aurora Santin², Elisa Rubinato¹, Giorgia Girotto^1;2

¹Institute for Maternal and Child Health – IRCCS “Burlo Garofolo”, Trieste, Italy; ²University of Trieste, Department of Medicine, Surgery and Health Sciences, Trieste, Italy

Background/Objectives: HL is the most prevalent sensory disorder, affecting ~6% of the world’s population. From a genetic perspective, HL is a challenging disease because of its genetic heterogeneity and clinical variability with different onset of symptoms complicating the molecular diagnosis.

Methods: We applied a multi-step approach for testing 290 syndromic and non-syndromic HL families (negative to GJB2 mutation), which included:

• 1. an accurate clinical evaluation,.

• 2. the assessment of STRC-CATSPER2 and OTOA Copy Number Variants (CNV) via Multiplex Ligation-dependent Probe Amplification (MLPA),.

• 3. Whole Exome Sequencing (WES) in patients negative to steps 2-3.

• 4. Whole Genome Sequencing (WGS) in a subset of families negative to 2-3.

.

Results: MLPA and WES led to the characterization of ~41% of cases. In particular, data analysis allowed to 1) confirm the relevant role of CNVs in the STRC gene, with ~5.9% of patients carrying a pathogenic deletion, 2) discover six new disease genes (e.g. PSIP1, TBL1Y, SPATC1L, PLS1, SLC12A2, MYO5C), and 3) shed the light on unexpected scenarios, such as the presence of dual molecular diagnosis, further exploring the complexity of HL. In negative cases to steps 2-3 we proceeded with WGS, searching for new possible causative alleles, including deep intronic and structural variants, starting from patients carriers of one pathogenic variant in recessive HL genes (e.g. SLC26A4).

Conclusion: Our approach led to an overall detection rate of ~41% in the Italian population. Furthermore, we expect WGS to unveil other possible disease mechanisms, deepening the knowledge of the biological mechanisms of HL.

References: NA.

Grants: NA.

Conflict of Interest: None declared.

P03.002.C A comprehensive WGS-based pipeline for the identification of new candidate genes in inherited retinal dystrophies

Elena Fernández-Suárez ^1;2, María González-del Pozo^1;2, Nereida Bravo-Gil^1;2, Cristina Méndez-Vidal^1;2, Marta Martín-Sánchez¹, Marcela Mena^1;2, Enrique Rodríguez-de la Rúa^3;4, Manuel Ramos-Jiménez⁵, Maria Jose Morillo Sanchez³, Salud Borrego^1;2, Guillermo Antiñolo^1;2

¹Virgen del Rocío University Hospital/CSIC/University of Seville, Department of Maternofetal Medicine, Genetics and Reproduction/Institute of Biomedicine of Seville, Seville, Spain; ²Center for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain; ³University Hospital Virgen Macarena, Department of Ophthalmology, Seville, Spain; ⁴Institute of Health Carlos III, Retics Patologia Ocular, OFTARED, Madrid, Spain; ⁵University Hospital Virgen Macarena, Department of Clinical Neurophysiology, Seville, Spain

Background/Objectives: To enhance the use of Whole Genome Sequencing (WGS) in clinical practice, it is still necessary to standardize data analysis pipelines. Herein, we aimed to define a WGS-based algorithm for the accurate variant interpretation in inherited retinal dystrophies (IRD).

Methods: This study comprised 429 phenotyped individuals divided into three cohorts: i) the training cohort with 209 genetically diagnosed IRD individuals was used to perform a statistical comparison of 14 pathogenicity predictors, redefine cutoffs and design the algorithm; ii) the validation cohort, consisting of 50 additional IRD individuals, 109 hereditary cancer patients and 47 with neurological diseases, allowed to select the optimal combination of predictors and to evaluate its translational value and iii) the discovery cohort including WGS data of 14 individuals from 7 genetically undiagnosed IRD families that were analysed applying our optimised workflow.

Results: The statistical analysis disclosed the most effective combination of predictors for non-splicing (CADDv1.6, MAPP, Grantham, and SIFT) and splicing (SpliceAI and NNS) variants. The pipeline validation showed high sensitivity, detecting 90% of causal variants in all the pathologies tested. This approach allowed identifying variants in candidate genes for IRD, such as CFAP20, in the discovery cohort families. Moreover, our results showed a 90% reduction of variants to be manually reviewed applying the customized cutoffs instead of the literature values.

Conclusion: Given the lack of consensus on the use of prediction tools, we offer a translational strategy for accurate WGS data prioritization in the clinical setting.

References: PMIDs: 24618965; 20238084.

Grants: ISCIII-ERDF/ESF (PI18/00612; PI21/00244; FI19/00091), Andalusian Government (PEER-0501-2019), F.Isabel Gemio/F.Cajasol [FGEMIO-2019-01].

Conflict of Interest: None declared.

P03.003.D Evaluation of CFAP20 as a candidate gene for autosomal recessive non-syndromic retinitis pigmentosa

María González-del Pozo ^1;2, Elena Fernández-Suárez^1;2, Nereida Bravo-Gil^1;2, Cristina Méndez-Vidal^1;2, Marta Martín-Sánchez¹, Marcela Mena^1;2, Enrique Rodríguez-de la Rúa^3;4, Manuel Ramos-Jiménez⁵, Maria Jose Morillo Sanchez³, Salud Borrego^1;2, Guillermo Antiñolo^1;2

¹University Hospital Virgen del Rocío/CSIC/University of Seville, Department of Maternofetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville, Seville, Spain; ²Center for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain; ³University Hospital Virgen Macarena, Department of Ophthalmology, Seville, Spain; ⁴Instituto de Salud Carlos III, Retics Patologia Ocular. OFTARED., Madrid, Spain; ⁵University Hospital Virgen Macarena, Department of Clinical Neurophysiology, Seville, Spain

Background/Objectives: The cilia and flagella-associated protein 20, CFAP20, encodes for a highly conserved protein that plays a critical role in cilia formation and morphology. Previous studies have demonstrated that CFAP20 (Bug22) depletion causes a ciliary phenotype in both in-vitro and in-vivo models, supporting its likely implication in human ciliopathies. On the other hand, retinitis pigmentosa (RP) is a highly penetrant phenotype among a vast majority of ciliopathies. Here, we aimed to evaluate the association between CFAP20 variants and autosomal recessive RP.

Methods: Two members of one consanguineous non-syndromic RP family underwent WGS using Illumina’s HiSeqX platform. Segregation analysis was conducted by Sanger sequencing. The retinal expression and localization of human CFAP20 were evaluated by real-time-qPCR and immunohistochemistry using retinal sections from healthy donors. The CFAP20 clinical impact was further investigated using 3D-modeling and protein-protein interaction networks.

Results: An in-house data analysis pipeline allowed the identification of one homozygous variant in CFAP20 (c.337C>T; p.Arg113Trp) segregating with the disease. Comparison of the relative mRNA levels between different tissues showed that CFAP20 mRNA was highest in the retina. Specific immunolabeling was observed in the photoreceptors layer. Our in-silico mutagenesis experiments also predicted conformational changes secondary to hydrogen-bonds loss. Additionally, CFAP20 is known to interact with the RP-associated protein, ARL2BP.

Conclusion: Although additional studies are needed, this work led us to propose CFAP20 as a candidate gene for non-syndromic RP and is expectedly to expand the mutational landscape of ciliary genes associated with human diseases.

References: PMIDs:24414207;20118210;24574454;31763178;23849777.

Grants: ISCIII-ERDF/ESF (PI18-00612;PI21-00244;FI19/00091), Andalusian Government (PEER _0501_2019), F.Isabel Gemio/F.Cajasol (2019-01).

Conflict of Interest: None declared.

P03.005.B Genetic heterogeneity of prelingual severe to profound hearing impairment in patients from North African countries and Jordan

Zied Riahi¹, Sophie Boucher², Fabienne Wong Jun Tai¹, Amrit Estivalet¹, Magali Niasme-Grare³, Yosra Bouyacoub¹, Samia Abdi⁴, Malika Dahmani⁵, Sonia Talbi⁵, Rhama mkouar⁶, Ghita Amalou⁷, Soukaina Elrharchi⁷, Mirna Mustafa⁸, Malak Salame⁹, Ely Cheikh Moctar⁹, Mouna Hadrami⁹, cherine charfeddine⁶, Marrakchi Jihene¹⁰, Rim Zainine¹⁰, Houda Chahed¹⁰, Besbes Ghazi¹⁰, Mediha Trabelsi¹¹, Ridha Mrad¹¹, Ichraf Kraoua¹², Ilhem Turki¹², Ahmed Houmeida⁹, Abdelhamid Barakat⁷, Fatima Ammar-Khodja⁵, Akila Zenati¹³, Mohamed Makrelouf¹³, Sonia Abdelhak⁶, Christine Petit¹, Crystel Bonnet ¹

¹Institut de l’audition, Institut Pasteur, INSERM, Paris, France; ²Service d’ORL, Centre Hospitalier Universitaire (CHU) d’Angers, Faculté de Médecine, Angers, France; ³Service de Biochimie et Biologie Moléculaire, Hôpital d’Enfants Armand-Trousseau, AP-HP, Paris, France; ⁴Laboratoire de biochimie génétique, Service de biologie - CHU de Bab El Oued, Université d’Alger 1, Laboratoire central de biologie, CHU Frantz Fanon, Faculté de médecine, Université Saad Dahleb, Blida, Algiers, Algeria; ⁵Equipe de Génétique, Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumédiène (USTHB), El Alia, Bab-Ezzouar, Algiers, Algeria; ⁶Institut Pasteur de Tunis, LR16IPT05, Biomedical Genomics and Oncogenetics Laboratory, Tunis, Tunisia; ⁷Human Molecular Genetics Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco; ⁸University of Sheffield, Department of Biomedical Science, Sheffield, United Kingdom; ⁹Unité de Recherche sur les Biomarqueurs dans la Population Mauritanienne, Université des Sciences, de Technologie, et de Médecine (USTM), Nouakchott, Mauritania; ¹⁰Hôpital La Rabta, Otorhinolaryngology Diseases, Tunis, Tunisia; ¹¹Hôpital Charles Nicolle, Congenital and Hereditary Diseases, Tunis, Tunisia; ¹²Institut National de Neurologie, Tunis, Tunisia; ¹³Laboratoire de biochimie génétique, Service de biologie - CHU de Bab El Oued, Université d’Alger 1, Algiers, Algeria

Background/Objectives: Prelingual hearing impairment (HI), one of the most frequent monogenic disorders, has a very high genetic heterogeneity. For genetic counseling as in the perspective of gene therapy, it is essential that all patients can benefit from molecular diagnosis.

Methods: We explored the variants responsible for deafness in a total of 450 unrelated patients affected by severe to profound early onset HI from North African (Algeria, Mauritania, Morocco, Tunisia) and Middle East (Jordan) countries. To this purpose, we developed a targeted exome high throughput sequencing of 156 known deafness genes (HearPanel-IdA), followed by qPCR analysis and only retained predicted pathogenic variants.

Results: This allowed us to ascertain molecular diagnosis for a total of 391/450 patients (86.9%). Remarkably, we found 63/450 patients (14%) with variants in Usher syndrome (USH) genes of whom 40 had been clinically diagnosed as Usher. In the other 23 patients, subsequent clinical exams established the existence of a retinitis pigmentosa. Moreover, a total of 213 predicted pathogenic variants were identified in 49 known deafness genes of which 84 (39.4%) never reported. This makes the HearPanel-IdA a powerful and cost-effective tool to identify rare mutations causing HI.

Conclusion: The use of HearPanel-IdA as a routine investigation should not only facilitate the detection of rare mutations in uncommon HI genes, but also contribute significantly to the early diagnosis of specific forms of syndromic HI such as Usher syndrome, for which early cochlear implantation is of utmost importance because of the secondary sight loss.

References:.

Grants: FPA-IDA05, ANR-10-LABX-65, ANR-11-IDEX-0004-02, ANR-11-BSV5-0011, ANR-16-CE13-0015-02.

Conflict of Interest: None declared.

P03.006.C Deciphering the genetic architecture of inherited retinal diseases in the Iranian population by integrated exome sequencing

Miriam Bauwens ¹, Kristof Van Schil², Marieke De Bruyne¹, Mattias Van Heetvelde¹, Quinten Mahieu¹, Toon Rosseel¹, Ebrahim Al Hajj³, Sarah Van Malderen¹, Reza Maroofian⁴, Fatemeh Suri⁵, Elfride De Baere¹

¹Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Ghent, Belgium; ²Center for Medical Genetics Antwerp, Universiteit Antwerpen, Antwerp, Belgium; ³Vrije Universiteit Brussel, Brussels, Belgium; ⁴Institute of Neurology, Department of Neuromuscular Diseases, University College London, London, United Kingdom; ⁵Ophthalmic Epidemiology Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Background/Objectives: To uncover the underlying molecular causes of inherited retinal disease (IRD) in 105 unrelated families of Iranian descent, an integrated approach consisting of whole exome sequencing (WES) and autozygosity mapping was used.

Methods: WES was performed in 105 Iranian IRD families, predominantly originating from a consanguineous background (77%). Data-analysis was performed using the in-house Seqplorer tool. Copy number variants (CNVs) were assessed via ExomeDepth. Variants were validated, classified (ACMG/ACGS guidelines) and segregation analysis was performed. AutoMap was used to determine runs of homozygosity (ROHs).

Results: By interrogating 290 known IRD genes using a WES-based analysis, we obtained a molecular diagnosis for 85% of the IRD cohort. In total, 103 (likely) disease-associated variants were identified in 42 genes, 58 of which are novel variants. ABCA4, EYS, AIPL1 and CRB1 were the four most implicated genes. In addition, the importance of structural variation (SV) in IRD was demonstrated, with CNVs identified in 8% of the cohort, including novel CNVs in CDHR1, CHM and RD3. Homozygous nonsense and missense deleterious variants were found in novel retina-expressed candidate IRD genes, including PFKFB2 and QRFPR.

Conclusion: This integrated study using WES and an in-depth analysis of the variants provides insight into the genetic architecture of IRD in Iran. We provided 85% of patients with a molecular diagnosis and expanded the molecular spectrum of IRD in Iran by the identification of novel variants in the majority of patients. Finally, autozygome-guided exome sequencing revealed several novel candidate genes for IRD in unsolved cases.

References:.

Grants: FWO1802220N.

Conflict of Interest: None declared.

P03.007.D Transient receptor potential ankyrin 1 (TRPA1) channel: rare variants in chronic neuropathic pain patients

Erika Salvi¹, Margherita Marchi¹, Elkadia Mehmeti¹, Mirna Andelic ^1;2, Daniele Cazzato³, Grazia Devigili⁴, Ilaria D’Amato¹, Raffaella Lombardi¹, Daniele Cartelli¹, Monique Gerrits⁵, Milena Ślęczkowska⁶, Rayaz Malik^7;8, Catharina Faber⁹, Giuseppe Lauria Pinter^1;10

¹Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neuroalgology Unit, Milano, Italy; ²School of Mental Health and Neuroscience, Maastricht University, Department of Neurology, Maastricht, Netherlands; ³Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neurophysiology Unit, Milano, Italy; ⁴Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Movement Disorders Unit, Milano, Italy; ⁵Maastricht University Medical Center, Department of Clinical Genetics, Maastricht, Netherlands; ⁶Maastricht University, Department of Toxicogenomics, Maastricht, Netherlands; ⁷University of Manchester and NIHR/Wellcome, Faculty of Biology, Medicine and Health, Manchester, United Kingdom; ⁸Weill CornellMedicine-Qatar, Qatar Foundation, Research Division, Doha, Qatar; ⁹Maastricht University Medical Centre, School of Mental Health andNeuroscience, Maastricht, Netherlands; ¹⁰University of Milan, Department of Medical Biotechnology and Translational Medicine, Milano, Italy

Background/Objectives: Chronic neuropathic pain is amongst the most common non-communicable disorders, with a significant impact on patients’ quality of life. The aim of this study is to assess the incidence of rare mutations in patients with neuropathic pain (NeuP).

Methods: We collected 480 patients complaining NeuP, and 216 healthy controls (HC) aged over 50. Subjects were classified according to symptoms distribution as “length-dependent”, “non-length-dependent” and “atypical” and analysed by target NGS of 107 genes involved in pain signalling. Rare genetic variants were selected and classified following ACGS recommendations.

Results: TRPA1 showed a significant overrepresentation of rare variants with functional or disrupting impact on the protein in NeuP patients compared to HC. We found that mutated samples with rare functional or disruptive variants were 2.92 time (p = 0.017) as prevalent in NeuP (7.7%) compared with HC (2.85%). TRPA1 variants are mainly clustered in the N-terminal domain containing the ankyrin repeats, involved in channel function and regulation. Among patients harboring TRPA1 mutations, 18 (69%) has non-length-dependent NeuP, 10 (38%) reported cold-induced or cold-accentuated pain, 12 (46%) neuropathic itch and 14 (48%) electric shock-like pain.

Conclusion: TRPA1 encodes for a polymodal Ca2+ channel, involved in pain and inflammation, activated by potentially noxious stimuli, chemicals, and cold. The only genetic disease associated with TRPA1 is Familial Episodic Pain Syndrome. High-throughput studies on SNPs frequency found no association with NeuP susceptibility. Focusing on rare high-impact mutations, our study suggests that TRPA1 could increase the risk for complex painful conditions.

References:.

Grants: GRANT AGREEMENT NO.602273 PROPANE STUDY-FP7-HEALTH-2013-INNOVATION-1.

Conflict of Interest: Erika Salvi: None declared, Margherita Marchi: None declared, Elkadia Mehmeti: None declared, Mirna Andelic: None declared, Daniele Cazzato: None declared, Grazia Devigili: None declared, Ilaria D’Amato: None declared, Raffaella Lombardi: None declared, Daniele Cartelli: None declared, Monique Gerrits: None declared, Milena Ślęczkowska: None declared, Rayaz Malik: None declared, Catharina Faber Dr. Faber reports grants from European Union’s Horizon 2020 research and innovation programme Marie Sklodowska-Curie grant for PAIN-Net, Molecule-to-man pain network (grant no. 721841), grants from Grifols and Lamepro for a trial on IVIg in small fibre neuropathy, grants from Prinses Beatrix Spierfonds., Steering committtees/advisory board for studies in small fibre neuropathy of Biogen/Convergence, Vertex, Lilly and OliPass, Giuseppe Lauria Pinter: None declared.

P03.008.A Molecular characterization of inherited retinal degenerations in a Swiss patient cohort

Karolina Kaminska ^1;2, Francesca Cancellieri^1;2, Quinodoz Mathieu^1;2;3, Giacomo Calzetti^1;2, Bence György^1;2, Lucas Janeschitz-Kriegl^1;2, Hendrik Scholl^1;2, Carlo Rivolta^1;2;3

¹Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland; ²University of Basel, Department of Ophthalmology, Basel, Switzerland; ³University of Leicester, Department of Genetics and Genome Biology, Leicester, United Kingdom

Background/Objectives: Inherited retinal degenerations (IRDs) are a group of disorders known for their genetic and clinical heterogeneity, which make molecular diagnosis a challenging process.

Methods: We collected approximately 200 DNA samples from more than 170 families with IRDs and other ocular disorders, ascertained by the Department of Ophthalmology at the University of Basel, and we developed a robust workflow based on the next generation sequencing (NGS) to identify the genetic determinants for these conditions. All patients underwent an ophthalmic examination performed by an IRD specialist, and their DNA was extracted from whole blood or saliva samples. Whole Exome Sequencing (WES) was performed for at least one affected individual from each family.

Results: Thus far, NGS analysis of 109 probands enabled the identification of causative variants in 37 known disease genes for 80 individuals, leading to an overall molecular characterization rate of 73%. Most identified mutations were single nucleotide variants (SNVs) and small deletion or insertions, whereas 6% of mutations were copy number variants (CNVs). Interestingly, mutations in intronic regions contributed to the diagnosis of 17 patients (>20% of all solved cases), with 3 deep intronic variants identified by an extensive analysis.

Conclusion: Integration of a research approach into analytical pipeline, together with the precise description of the clinical phenotype, lead to a high molecular diagnostic rate for patients with IRDs. Molecular diagnosis is crucial for the development of future gene therapies and proper genetic counseling of all patients.

References:.

Grants: The Swiss National Science Foundation, grant #204285.

Conflict of Interest: None declared.

P03.010.C Visual acuity GWAS in children identify novel genes and pathways

Krzysztof Marianski ¹, Judith Schmitz^1;2, Filippo Abbondanza¹, Michelle Luciano³, Silvia Paracchini¹

¹University of St Andrews, School of Medicine, St Andrews, United Kingdom; ²Georg-August-University Goettingen, Biological Personality Psychology, Goettingen, Germany; ³University of Edinburgh, Department of Psychology, Edinburgh, United Kingdom

Background/Objectives: Visual acuity (VA) is a measure of the eyes’ ability to distinguish between shapes and overall detail of objects. Reduction in VA is associated with myopia, reading disability (RD) and lower quality of life. Genetics of VA are not well understood due to most of the genome wide association studies (GWAS) related to vision, focusing on myopia or age-related macular degeneration, predominantly in adults.

Methods: We performed GWAS for VA on children aged 11,5 years old, from the ALSPAC data (N = 6,807), calculated polygenic risk scores and applied novel, non-standard post GWAS analyses tools - FINDOR and Downstreamer. FINDOR uses functional annotations to reweight GWAS summary statistics p-values and Downstreamer prioritises genes and pathways that would not normally be analysed for GWAS loci. Neither tool uses any phenotypic information.

Results: We report the first GWAS for VA in children. The top hit (NPLOC4 locus) was previously associated in GWAS for vision outcomes in adults and we find that having a higher polygenic risk for ADHD and RD is correlated with worse VA. Post-hoc analysis showed significant enrichments of genes and pathways related to vision. FINDOR re-weighted p-values resulted in six additional SNPs reaching statistical significance. Downstreamer prioritised two genes, namely PDE6G and RFSN, both important in sensory functions.

Conclusion: Our GWAS and post-GWAS analyses for VA resulted in significant associations despite a small sample size. This indicates the potential of VA as a useful phenotype for dissecting the genetics of vision.

References:.

Grants:.

Conflict of Interest: None declared.

P03.012.A Spectrum of ABCA4 variants in Slovenian patients with Stargardt disease

Jana Sajovic ¹, Andrej Meglič¹, Martina Jarc-Vidmar¹, Vlasta Hadalin¹, Zelia Corradi^2;3, Frans P.M. Cremers^2;3, Jana Zernant⁴, Rando Allikmets⁴, Damjan Glavac⁵, Aleš Maver⁶, Marija Volk⁶, Borut Peterlin⁶, Marko Hawlina¹, Ana Fakin¹

¹Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia; ²Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; ³Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, Netherlands; ⁴Department of Ophthalmology, Columbia University, New York, United States; ⁵Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; ⁶Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia

Background/Objectives: There are >1290 known pathogenic variants in ABCA4 causing heterogeneous phenotypes. This study aims to report the genetic and clinical characteristics of the Slovenian ABCA4 cohort.

Methods: Study included 75 patients (28 male; median age at the last exam 32 years, range 8-75). Genetic analysis was done with next-generation sequencing, deep intronic regions were included in 65/75. Phenotype analysis included age at disease onset, Snellen decimal best corrected visual acuity (VA), pattern and full-field electroretinography (ERG), and fundus autofluorescence (FAF) appearance.

Results: 63 (84%) patients had ≥2 identified variants, whereas 12 (16%) had 1 identified variant. Most frequent variants were p.(Asn1868Ile) (11% alleles), p.(Gly1961Glu) (11%), p.[(Leu541Pro;Ala1038Val)] (10%), p.(Arg681*) (7%) and c.5714+5G>A (7%). The latter was more frequent than in a multicentric study of 279 patients (2%) (1). 7/52 different variants were novel. Median age at onset was 15 years (range 5-60). Late-onset (>40 years) was present in 28%. Median VA was 0.16 (range counting fingers-1.0). According to ERG, generalized retinal dysfunction (ERG group 2 or 3) was present in 52%. Abnormalities on FAF extended beyond the vascular arcades in 60%. Patients with late-onset had a higher frequency of foveal sparing (44%) than other patients (9%). However, frequency of missense variants was similar between the two groups (72% vs 67%).

Conclusion: Slovenian ABCA4 cohort has a specific spectrum of ABCA4 variants. 13% of variants were novel, while c.5714+5G>A was relatively more frequent than in other cohorts. Around one-third of patients had late-onset disease, which may be misdiagnosed as age-related macular degeneration.

References: (1). PMID:29925512.

Grants:.

Conflict of Interest: Jana Sajovic Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia, Andrej Meglič Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia, Martina Jarc-Vidmar Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia, Vlasta Hadalin: None declared, Zelia Corradi Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands, Frans P.M. Cremers Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands, Jana Zernant Department of Ophthalmology, Columbia University, New York, New York, United States, Rando Allikmets Department of Ophthalmology, Columbia University, New York, New York, United States, Damjan Glavac Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, Aleš Maver Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia, Marija Volk Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia, Borut Peterlin Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia, Marko Hawlina Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia, Ana Fakin Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia.

P03.013.B Biallelic variants in KITLG cause Waardenburg syndrome type 2, albinism-deafness syndrome and oculocutaneous albinism

Katta Girisha ^1;2, Barbara Vona^3;4, Daniel Schwartzbaum⁵, Alejandro A. Rodriguez⁵, Solange S. Lewis¹, Mehran Beiraghi Toosi⁶, Periyasamy Radhakrishnan², Nazım Bozan⁷, Ramazan Akın⁷, Mohammad Doosti⁸, Ramachandran Manju⁹, Duygu Duman¹⁰, Claire J. Sineni⁵, Sheela Nampoothiri¹¹, Ehsan Ghayoor Karimiani^8;12;13, Henry Houlden¹⁴, Guney Bademci^5;15, Mustafa Tekin^5;15, Sofia Douzgou HOUGE^16;17, Reza Maroofian¹⁴

¹Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India, Manipal, India; ²Suma Genomics Private Limited, Manipal Universal Technology Business Incubator Manipal, Karnataka, India, Manipal, India; ³Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany, Göttingen, Germany; ⁴Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany, Göttingen, Germany; ⁵John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, Florida, USA, Miami, United States; ⁶Pediatric Neurology Department, Ghaem hospital, Mashhad University of Medical Sciences, Mashhad, Iran, Mashhad, Iran; ⁷Department of Otolaryngology, Yuzuncu Yıl University Faculty of Medicine, Van, Turkey, Van, Turkey; ⁸Department of Medical Genetics, Next Generation Genetic Polyclinic, Mashhad, Iran, Mashhad, Iran; ⁹Renai Medicity, Cochin, Kerala, India, Cochin, India; ¹⁰Department of Audiology, Ankara University Faculty of Health Sciences, Ankara, Turkey, Ankara, Turkey; ¹¹Department of Paediatric Genetics, Amrita Institute of Medical Sciences and Research Centre, Kochi, India, Kochi, India; ¹²Molecular and Clinical Sciences Institute, St. George’s, University of London, Cranmer Terrace, London SW17 0RE, UK, London, United Kingdom; ¹³Innovative medical research center, Mashhad branch, Islamic Azad University, Mashhad, Iran, Mashhad, Iran; ¹⁴Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology, London, UK, London, United Kingdom; ¹⁵Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL, USA, Miami, United States; ¹⁶Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway, Bergen, Norway; ¹⁷Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK, Manchester, United Kingdom

Background/Objectives: Pathogenic variants in KITLG, a key player in melanocyte proliferation and pigment production, cause autosomal dominant non-syndromic hearing loss, Waardenburg syndrome type 2 and familial progressive hyperpigmentation and familial progressive hyper- and hypopigmentation. We expand this spectrum to include a recessive disorder presenting with a hypomelanosis spectrum with or without hearing impairment.

Methods: We characterized the largest case series with biallelic KITLG variants identified through precision phenotyping, multiple sequencing approaches and extensive networking.

Results: We expand the KITLG-related hypomelanosis spectrum to include different patterns of distal depigmentation, partial depigmentation resembling Tietz albinism-deafness syndrome and complete depigmentation reminiscent of oculocutaneous albinism. We speculate that loss-of-function KITLG variants cause oculocutaneous albinism while those with possible residual function cause Waardenburg syndrome type 2 or albinism-deafness syndrome.

Conclusion: We expand the understanding of the mode of inheritance of Waardenburg syndrome type 2 to include autosomal recessive transmission. This work defines KITLG as a new molecular cause of autosomal recessive albinism-deafness syndrome and oculocutaneous albinism.

References:.

Grants: Funding was provided by the Bio-CARe Woman Scientist Award, Department of Biotechnology, Ministry of Science and Technology, Government of India (grant no. BT/Bio-CARe/07/ 9889/2013–14) (SSL and KMG), the John T. and Winifred M. Hayward Foundation (to MT), and the German Research Foundation Collaborative Research Center 889 (BV).

Conflict of Interest: Katta Girisha Founding director of Suma Genomics Pvt. Ltd., Barbara Vona: None declared, Daniel Schwartzbaum: None declared, Alejandro A. Rodriguez: None declared, Solange S. Lewis: None declared, Mehran Beiraghi Toosi: None declared, Periyasamy Radhakrishnan Scientist and Laboratory Director. Suma Genomics Private Limited, Nazım Bozan: None declared, Ramazan Akın: None declared, Mohammad Doosti: None declared, Ramachandran Manju: None declared, Duygu Duman: None declared, Claire J. Sineni: None declared, Sheela Nampoothiri: None declared, Ehsan Ghayoor Karimiani: None declared, Henry Houlden: None declared, Guney Bademci: None declared, Mustafa Tekin: None declared, Sofia Douzgou HOUGE: None declared, Reza Maroofian: None declared.

P03.014.C Dysregulated microRNA in skin epidermis contributes to small fiber neuropathy pathophysiology

Mirna Andelic^1;2, Erika Salvi ¹, Margherita Marchi¹, Daniele Cazzato³, Stefania Marcuzzo⁴, Daniele Cartelli⁵, Janneke Hoeijmakers², Catharina Faber², Raffaella Lombardi⁶, Giuseppe Lauria Pinter^5;7

¹Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neuroalgology Unit, Milano, Italy; ²School of Mental Health and Neuroscience, Maastricht University, Department of Neurology, Maastricht, Netherlands; ³Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neurophysiology Unit, Milano, Italy; ⁴Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neuroimmunology and Neuromuscular Diseases Unit, Milano, Italy; ¹Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neuroalgology Unit, Milano, Italy; ¹Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Neuroalgology Unit, Milano, Italy; ⁷Università degli Studi di Milano, Department of Medical Biotechnology and Translational Medicine, Milano, Italy

Background/Objectives: Small fiber neuropathy (SFN) is a multifactorial condition defined by impairment of peripheral nerve fibres often accompanied by pain. Lately, miRNAs are emerging as key fine-tuning regulators in complex neurodegenerative diseases, particularly associated with regulation of axon guidance and cross-talk. Our study aimed to investigate miRNA profiles of painful SFN patients and their possible correlation with underlying disease mechanisms.

Methods: Total RNA was isolated from skin epidermis. Unbiased miRNA expression quantification was performed using microfluidic array containing 754 miRNAs. Discovery and validation profiling involved 23 painful SFN patients and 15 healthy controls. We applied the relative threshold (Crt) method and NormFinder software was used to determine the optimal normalization gene. Results were expressed as relative quantification with healthy control tissue as reference group. Moreover, target and functional enrichment analyses were performed to explore possibly affected biological processes and molecular functions.

Results: We identified a miRNA as significantly downregulated in SFN patients respect to controls. The validation revealed an optimal diagnostic accuracy (AUC = 96.4%). Functional enrichment analysis showed involvement in biological processes and pathways that are closely related to nociception signaling cascade triggering various cellular responses and pain-related channel trafficking. Specifically, miRNA’s effect on target genes such as TRPV1, MAPK14, NTRK1 dysregulates inflammatory mediator regulation of TRP channels, cAMP and MAPK signaling pathways.

Conclusion: Our proof-of-concept study of miRNA profiling of human epidermis provided novel hints on epigenetic regulation of epidermal innervation and chronic neuropathic pain, while adding value to the dysfunction of axon-cell network in the contest of SFN.

References: None.

Grants: PAIN-Net (grant no. 721841).

Conflict of Interest: Mirna Andelic: None declared, Erika Salvi: None declared, Margherita Marchi: None declared, Daniele Cazzato: None declared, Stefania Marcuzzo: None declared, Daniele Cartelli: None declared, Janneke Hoeijmakers: None declared, Catharina Faber Grants from European Union’s Horizon 2020 research and innovation programme Marie Sklodowska-Curie grant for PAIN-Net, Molecule-to-man pain network (grant no. 721841), grants from Grifols and Lamepro for a trial on IVIg in small fibre neuropathy, Grants from Prinses Beatrix Spierfonds, Steering committtees/advisory board for studies in small fibre neuropathy of Biogen/Convergence, Vertex, Lilly and OliPass, outside the submitted work., Raffaella Lombardi: None declared, Giuseppe Lauria Pinter: None declared.

P03.016.A Achromatopsia: The curious case of triallelic inheritance

Ratna Tripathy ¹, Rainer König¹, Mojgan Drasdo¹, Nuha Al Zaabi²

¹Bioscientia Institute for Medical Diagnostics GmbH, Human Genetics, Ingelheim, Germany; ²College of Medicine and Health Science, United Arab Emirates University, Department of Pediatric, Al Ain, United Arab Emirates

Background/Objectives: Achromatopsia is a retinal disorder characterized by reduced or complete loss of color vision, reduced visual acuity, pendular nystagmus, and congenital photophobia (1). Affected patients show limited or complete lack of cone photoreceptor activity, caused in the majority of cases by biallelic variants in the CNGA3 or CNGB3 genes (2, 3), encoding CNG channel subunits that heterodimerize to mediate membrane hyperpolarization in response to light. A recent study by Burkard et al (2018) looked into the controversial p.R403Q hypomorphic variant in CNGB3, which in a biallelic state shows high variability, reduced penetrance, with high frequency in unaffected individuals. The authors showed that a trialleic co-occurrence of heterozygous pathogenic CNGA3 variant, in individuals with the homozygous CNGB3 p.R403Q variant, contributes to an exacerbated retinopathy (3).

Methods: We present a four-year-old boy with visual defects since age of six months, preferring dark or dimly lit rooms, showing nystagmus, and defective color discrimination. We performed whole exome sequencing to identify a possible genetic cause.

Results: The analysis lead to the identification of a new triallelic combination of the homozygous hypomorphic CNGB3 p.R403Q variant with the monoallelic known pathogenic CNGA3 variant p.A323P, which can explain the phenotype of the boy.

Conclusion: Along with broadening the list of triallelic variant combinations for achromatopsia, with this case, we aim to raise awareness in the clinical genetics community of the presence and importance of digenic-triallelic inheritance, which has implications for diagnosis, prognosis, and genetic counseling.

References: PMID: (1) 20301591, (2) 9662398, (3) 10958649, (4)30418171.

Grants:.

Conflict of Interest: None declared.

P03.017.B Retinitis pigmentosa caused by mutations in the ush2a gene: clinical trend comparison and genotype-phenotype correlations

Luigi Monti ^1;2, Nicole Balducci³, Antonio Ciardella³, Sara Taormina^1;2, Sofia Cesarini^1;2, Giulia Olivucci^1;2, Elena Luppi^1;2, Mina Grippa¹, Enrico Ambrosini¹, Giovanna Sartor¹, Francesca Montanari^1;2, Giulia Severi², Sara Miccoli², Claudio Graziano⁴, Cesare Rossi², Daniela Turchetti^1;2, Marco seri^1;2

¹Alma Mater Studiorum University of Bologna, Bologna, Italy, Department of Medical and Surgical Sciences, Bologna, Italy; ²IRCCS Azienda Ospedaliera Universitaria di Bologna, U.O. Genetica Medica, Bologna, Italy; ³Ophtalmology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Department of Medical and Surgical Sciences, Bologna, Italy; ⁴AUSL della Romagna, U.O. Genetica Medica, Cesena, Italy

Background/Objectives: Mutations in the USH2A gene are responsible for both isolated and syndromic (Usher syndrome type IIa) retinitis pigmentosa. We studied genotype-phenotype correlations and compared clinical prognosis between Usher syndrome type IIa and non-syndromic RP.

Methods: Clinical data, visual acuity (VA), visual field (VF), retinal imaging and electrophysiologic features were extracted from medical records of patients affected, with non-syndromic RP (n 6) and Usher syndrome type IIa (n 25). The patients were carriers of at least one pathogenetic USH2A mutation and they underwent to counseling in the medical genetics unit of University of Bologna (2003-2021). Statistical analysis has been performed (Student’s t-test, Chi-squared test, Fisher’s exact test).

Results: Participant groups had similar distributions of gender and similar ethnicity. Usher syndrome type IIa patients demonstrated earlier symptoms than non-syndromic RP (14 vs 27 years, P = 0,04), earlier clinical diagnosis (24 vs 43 years, P = 0,01), earlier molecular diagnosis (37 vs 58 years, P = 0,04) and earlier visual impairment (40 vs 56 years for VF, 45 vs 68 years for VA, P = 0,02). Truncating mutations are associated with earlier symptoms (13 vs 20) and with syndromic phenotype. We found novel variants (4 missense, 2 frameshift, 1 splicing) in both groups and suspected pathogenicity has been raised for other (n 3).

Conclusion: Patients with Usher syndrome type IIa have symptoms and severe visual impairment earlier than non-syndromic RP. One truncating mutation seems to predispose to worse clinical trend, two truncating mutations in USH2A is associated with syndromic phenotype.

References:.

Grants:.

Conflict of Interest: None declared.

P03.019.D Functional characterization of potential spliceogenics PAX6 variants in aniridia combining minigenes and long-reads sequencing

Carolina Ruiz-Sanchez¹, Alejandra Tamayo^1;2, Gonzalo Nuñez Moreno^1;2;3, Tudor Pastae¹, Carmen Ayuso^1;2, Pablo Mínguez^1;2;3, Marta Corton ^1;2

¹Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Genetics Department, Madrid, Spain; ²Center for Biomedical Network Research on Rare Diseases - CIBERER, Madrid, Spain; ³Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Bioinformatic Unit, Madrid, Spain

Background/Objectives: PAX6 is a highly-conserved transcription factor involved in ocular development. Several aniridia-associated exonic and intronic variants located in the hotspot exon 6 of PAX6 generate aberrant splicing patterns involving 5 cryptic donors. We aim to functionally study the potential spliceogenic effect of other variants affecting this exon.

Methods: Potential spliceogenic variants located in exon 6 were selected from our aniridia cohort or mutational databases based on in silico splicing analysis and further characterized in vitro. Then, a minigene was generated from the pSPL3 vector containing the full sequences between exons 5 to 7 of the PAX6 gene. Directed mutagenesis was used to introduce the selected variants in the PAX6_exon 5-7 minigene. Mutated and wild-type minigenes were transfected in HEK293 cells and total RNA was extracted. Finally, the splicing isoforms were analysed by RT-PCR using Oxford Nanopore long-read and Sanger sequencing.

Results: After an in silico analysis of 120 PAX6 variants, 6 of them showing potential splicing effects were analysed using in vitro minigenes. Four showed aberrant splicing patterns, including intronic retentions and partial or total exon skipping. Two of them, initially considered variants of uncertain significance, were reclassified as probably pathogenic.

Conclusion: In silico and in vitro minigenes assays are a good approach to test the involvement of different genetic variants in the splicing process. In addition, this functional characterization is useful to deepen into the pathogenicity of aniridia-causing variants.

References:.

Grants: Spanish Health Institute Carlos III (PI17_01164 and PI20_00851) and Spanish National Organization of the Blind (ONCE).

Conflict of Interest: None declared.

P03.020.A Rare missense variants and deletions in α-tectorin may drive a change in tectorial membrane stability in familial Meniere’s disease

Pablo Román-Naranjo^1;2;3;4, Alberto M. Parra-Perez^1;3;4, Alba Escalera-Balsera^1;2;3, Andres Soto-Varela^5;6, Alvaro Gallego-Martinez^1;2;3, Ismael Aran⁷, Nicolas Perez-Fernandez⁸, David Baechinger⁹, Andreas H. Eckhard⁹, Rocio Gonzalez-Aguado¹⁰, Lidia Frejo^1;2;3, Antonio Lopez-Escamez ^1;2;3;4

¹GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government, Otology & Neurotology Group CTS 495, Department of Genomic Medicine, Granada, Spain; ²Centro de Investigación Biomédica en Red en Enfermedades Raras, CIBERER, Sensorineural Pathology Programme, Madrid, Spain; ³Instituto de Investigación Biosanitaria ibs.Granada, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Department of Otolaryngology, Granada, Spain; ⁴University of Granada, Division of Otolaryngology, Department of Surgery, Granada, Spain; ⁵Complexo Hospitalario Universitario, Division of Otoneurology, Department of Otorhinolaryngology, Santiago de Compostela, Spain; ⁶Universidade de Santiago de Compostela, Department of Surgery and Medical-Surgical Specialities, Santiago de Compostela, Spain; ⁷Complexo Hospitalario de Pontevedra, Department of Otolaryngology, Pontevedra, Spain; ⁸Clinica Universidad de Navarra, Department of Otorhinolaryngology, Madrid, Spain; ⁹University Hospital Zurich, Department of Otorhinolaryngology, Head and Neck Surgery, Zurich, Switzerland; ¹⁰Hospital Universitario Marques de Valdecilla, Department of Otorhinolaryngology, Santander, Spain

Background/Objectives: Meniere’s disease (MD) is an inner ear disease characterized by low frequency sensorineural hearing loss associated with vertigo and aural fullness. Familial aggregation has been found in 9-10% of MD patients showing, mostly, an autosomal dominant inheritance with incomplete penetrance. Nevertheless, other inheritance patterns have been proposed, such as recessive and digenic inheritance involving rare variants in OTOG and MYO7A genes. This study aimed to search for relevant genes not previously linked to MD.

Methods: Exome sequencing was performed in 99 individuals diagnosed with MD. Candidate variants were classified according to the ACMG/AMP guidelines and their effects were evaluated by protein modeling. Audiometric evaluations were retrieved, and a case report was made of each family to assess genotype-phenotype correlations.

Results: TECTA gene was highlighted as candidate for four multicase MD families and two additional families with one MD patient and relatives with partial syndromes. Four rare missense variants and two frameshift deletions were found in heterozygous state segregating the MD phenotype. These variants could affect the stability of α-tectorin based on the predicted protein model.

Conclusion: Several MD families were identified carrying rare variants and deletions in the TECTA gene, which encodes one of the main proteins of the tectorial membrane (TM). The TM is an extracellular matrix localized over the sensory epithelium mediating the mechanical stimulation of cochlear hair cells. Modifications on the TM stability and the micromechanics involved in the sound-evoked motion of stereocilia may drive MD.

References:.

Grants: H2020-SC1-2019-848261 (UNITI), CECEU PY20-00303 (EPIMEN), European Regional Funds B-CTS-68-UGR20 (M3N-OMIC) and the Schmieder-Bohrisch Foundation.

Conflict of Interest: None declared.

P03.021.B Investigation of gene variations in inherited retinal dystrophies via next-generation sequencing

Şenol Demir ¹, esra arslan ateş², bilgen bilge geçkinli¹, ahmet arman¹

¹marmara university school of medicine, medical genetics, istanbul, Turkey; ²marmara university pendik training and research hospital, genetic diseases diagnosis center, istanbul, Turkey

Background/Objectives: Inherited Retinal dystrophies(IRD) are a group of progressive degenerative disorders of the retina with clinical and genetic heterogeneity. Common presentations include night blindness and peripheral vision abnormalities. Among retinal dystrophies most common sub-type is retinitis pigmentosa. In addition Leber congenital amaurosis, Usher syndrome, Stargardt disease are the other rare subtypes. This study aims to determine the effectiveness of the multigene panel testing in retinal dystrophies and to reveal genetic etiology of the IRD patients.

Methods: Thirty probands having retinal dystrophy with specific examination findings were included in this study. After evaluation of clinical and family histories, probands were screened using a custom designed retinal disorders panel including 140 genes via next-generation sequencing (NGS). The family members of the probands carrying pathogenic variations were screened via Sanger Sequencing.

Results: Among 30 cases, 14 were born to consanguineous parents and IRD family history was reported in 8 cases. Onset of the ages of the probands ranged between 3 months-48 years.We detected in 21 (70%) probands biallelic pathogenic variations, which of 5 were novel, in CYP4V2(n = 4), ABCA4(n = 4), USH2A (n = 3), RDH12 (n = 2), PROM1, ABHD12,TTC8, BBS1, RP1, CRB1, MAK, GRK1 genes.

Conclusion: The detection of 5 novel pathogenic variations has contributed to the expansion of the mutation spectrum. Also, the diagnosis of 70% of the patients supports that the NGS panel is an effective diagnostic method in IRD. Defining molecular etiology of IRD is important in terms of screening at risk family members and giving appropriate genetic counseling for preimplantation genetic diagnosis opportunities.

References:.

Grants:.

Conflict of Interest: None declared.

P03.022.C Cryptic chromosomal rearrangements disrupting the PAX6 locus identified in aniridia cases based on long-read whole-genome sequencing

Alejandra Damián ^1;2, Gonzalo Nuñez Moreno^1;2;3, Claire Jubin⁴, Alejandra Tamayo^1;2, Marta Rodriguez de Alba^1;2, Jean-François Deleuze⁴, Pablo Mínguez^1;2;3, Carmen Ayuso^1;2, Marta Corton^1;2

¹Fundación Jiménez Díaz University Hospital Health Research Institute (IIS-FJD)-Universidad Autónoma de Madrid (UAM), Department of Genetics & Genomics, Madrid, Spain; ²Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain; ³Fundación Jiménez Díaz University Hospital Health Research Institute (IIS-FJD)-Universidad Autónoma de Madrid (UAM), Bioinformatic Unit, Madrid, Spain; ⁴National Center of Human Genomics Research (CNRGH) Université Paris-Saclay, Evry, France

Background/Objectives: Haploinsufficiency of the transcription factor PAX6 is the main responsible for aniridia, a disorder mainly characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its 3’ regulatory regions were found in ~25% of aniridia patients. However, only few complex chromosomal rearrangements have been involved in aniridia which can be attributed to the inherent difficulty of detecting structural variants (SVs) involving repetitive/low-complexity regions. Long-read sequencing (LRS) techniques may overcome these limitations. Our objective was to assess the presence of cryptic SVs in our “PAX6-negative” cases suffering from aniridia using LRS approaches.

Methods: Long-read genome sequencing was conducted in two previously unsolved sporadic patients of our aniridia cohort after short-reads sequencing and CGH arrays. Libraries were prepared using high-weight molecular DNA following Oxford Nanopore Technologies protocols and sequenced at 30x on a PromethION. Detected SVs were validated using Sanger sequencing and/or FISH analysis.

Results: LRS analysis revealed a 4.9 Mb inversion in 11p13 involving the intron 7 of the canonical PAX6 isoform disrupting its linear structure. In a second patient, LRS revealed breakpoints for an apparently balanced translocation t(6;11) that affects the centromeric region on 6p11.1 and the downstream regulatory region (DRR) of PAX6 on the 11p13 region.

Conclusion: We first report two cases of cryptic balanced chromosomal SVs disrupting directly PAX6 or its regulatory elements. We demonstrate here how LRS can provide insight into hidden sources of variation.

References:.

Grants: H2020 EasiGenomics program (PID10291), PI20_00851 and Spanish National Organization of the Blind (ONCE).

Conflict of Interest: None declared.

P03.023.D Burden of rare coding variants in connexin genes involving Meniere Disease

Alba Escalera-Balsera ^1;2;3, Pablo Román-Naranjo^1;2;3;4, Marisa Flook^1;2;3, Paula Robles-Bolivar^1;3, Alberto M. Parra-Perez^1;3;4, Alvaro Gallego-Martinez^1;2;3, Jose Antonio Lopez-Escamez^1;2;3;4

¹Otology & Neurotology Group CTS495, GENYO, Centre for Genomics and Oncological Research, Pfizer University of Granada Andalusian Regional Government, Department of Genomic Medicine, Granada, Spain; ²Sensorineural Pathology Programme, Centro de Investigación Biomédica en Red en Enfermedades Raras, CIBERER, Madrid, Spain; ³Instituto de Investigación Biosanitaria ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Department of Otolaryngology, Granada, Spain; ⁴Division of Otolaryngology, University of Granada, Department of Surgery, Granada, Spain

Background/Objectives: Gap junctions consist of connexin proteins forming hemichannels in the sensory epithelia. Mutations in GJB2, GJB6 and GJB3 cause genetic deafness and hearing loss. Meniere Disease (MD) is an inner ear disorder characterized by episodic vertigo and associated with sensorineural hearing loss, tinnitus and/or aural fullness.

Methods: Whole Exome Sequencing (WES) was performed in 313 patients with MD. Gene Burden Analysis was done filtering the variants by a Minor Allele Frequency<0.05 and using gnomAD database as reference population. Connexin genes expressed in the mammalian inner ear were retrieved for further analyses. The pathogenicity of each variant was estimated and the protein stability changes were studied using PremPS and DynaMut2.

Results: An enrichment of rare missense variants was found in 2 genes expressed in the inner ear: GJB5 (OR = 29.53) and GJD2 (OR = 13.77). GJA10 presents an enrichment in rare stop-gain variants (OR = 2.28). GJB5 (OR = 103.28) and GJC2 are enriched in rare frameshift, inframe insertion and deletion variants (OR = 22.73). Functional analysis revealed biological processes involved in gap junctions, cell communication or transmembrane transport.

Conclusion: We have found a burden of rare missense variants, stop-gain variants and indels in patients with MD in 4 genes: GJB5 (CX31.1), GJD2 (CX36), GJA10 (CX62) and GJC2 (CX47). Further studies, including segregation analysis, are needed to confirm the role of these variants in MD.

References:.

Grants: European Union’s Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261.

Conflict of Interest: None declared.

P03.024.A PACS1-related syndrome: three cases with colobomas further delineating the phenotype

Simon Boussion ^1;2, Thomas Smol^2;3, Perrine Brunelle^2;3, Mélanie Rama³, Séverine Drunat⁴, Pierre Blanc⁵, Françoise Ernould⁶, Sabine Defoort⁷, Vasily Smirnov⁷, Jamal Ghoumid^1;2, Florence Petit^1;2, Catherine Vincent-Delorme¹

¹CHU Lille, Clinique de Génétique Médicale “Guy Fontaine”, Lille, France; ²Univ.Lille, RADEME, EA7364, Lille, France; ³CHU Lille, Institut de Génétique Médicale, Lille, France; ⁴Hôpital Robert Debré, AP-HP, Service de Génétique Médicale, Paris, France; ⁵Seqoia-FMG2025, Laboratoire de biologie médicale multisites, Paris, France; ⁶CHU Lille, Service d’Ophtalmologie, Lille, France; ⁷CHU Lille, Explorations de la Vision et de Neuro-Ophtalomologie, Lille, France

Background/Objectives: PACS1 gene (MIM #607492) is related to Schuurs-Hoeijmakers syndrome (MIM #615009), a condition characterized by the association of intellectual disability, recognizable facial appearance, and a wide range of congenital abnormalities. Here we report 3 patients with coloboma and developmental delay due to the recurrent PACS1 missense variant.

Methods: After clinical examination, NGS sequencing was performed (WGS for patient 1, gene panels for patients 2 and 3).

Results: The first proband is a 3 years old male presenting with congenital bilateral iris, chorioretinal and papillary coloboma, ectopia lentis, hypotonia, cryptorchidism. The second proband is a 9 years old female presenting with congenital bilateral chorioretinal coloboma, microphthalmia, right iris coloboma, hypotonia. The third proband is a 6 years old male presenting with congenital iris and chorioretinal coloboma, hypotonia, cryptorchidism. Cerebral MRI showed diffuse cortical atrophy, dysplastic corpus callosum, vermis hypoplasia. All 3 patients evolved towards global developmental delay. The same pathogenic de novo heterozygous variant in PACS1 (c.607C>G, p.(Arg203Trp)) has been identified in all 3 patients.

Conclusion: PACS1 encodes the Phosphofurin Acid Cluster Sorting Protein 1, a trans-golgi-membrane traffic regulator. So far, 61 patients with Schuurs-Hoeijmakers syndrome have been published, carrying the p.(Arg203Trp) heterozygous missense or rarely the p.(Arg203Gln) missense in PACS1. Intellectual disability is constant but congenital malformations are variable. Iris and chorioretinal colobomas are rare (9% and 13% respectively). Our patients present with coloboma at the forefront of the clinical description, further delineating the phenotype and highlighting the variable expressivity of this condition. PACS1 implication should be considered in patients with congenital coloboma.

References:.

Grants:.

Conflict of Interest: None declared.

P03.025.B Longitudinal follow-up of Slovenian patients with RPGR associated retinitis pigmentosa

Vlasta Hadalin ¹, Maša Marija Buscarino¹, Jana Sajovic¹, Marija Volk², Martina Jarc-Vidmar¹, Manca Tekavcic Pompe¹, Marko Hawlina¹, Ana Fakin¹

¹Eye Hospital University Medical Centre Ljubljana, Ljubljana, Slovenia; ²Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Slovenia, Ljubljana, Slovenia

Background/Objectives: To report the variant spectrum and longitudinal follow-up data of Slovenian RPGR-retinitis pigmentosa (RP) patients.

Methods: The study included 13 subjects (7 males) from 6 RPGR-RP families. Age at onset, logMAR best corrected visual acuity (BCVA), visual fields and fundus autofluorescence (FAF) were analysed. Eyes with milder disease were taken for the analysis to best reflect the functional impairment.

Results: Genetic analysis revealed two reported (c.1217dupT(p.Ser407fs), c.2236_2237delGA(p.Glu746ArgfsTer23)) and four novel variants (c.457G>A(p.Ala153Thr), c.1245+704_1415-2286del, c.G1978G>A(p.Glu660Ter), c.2340_2341delAG(p.Arg780SerfsTer54)). Median age at onset in males was 3 years (0-18). At first exam at median age of 16 years (0-61), median BCVA was 0.3 logMAR (0.15-2.0). All had constricted visual fields and hyperautofluorescent rings on FAF. At the last follow-up at median age of 39 years (4-71), median BCVA was 0.48 logMAR (0.0-2.30). FAF showed ring constriction, transitioning to patch in 2/7 cases. Median age at onset in females was 22.5 years (5-66). At first exam at median age of 40 years (6-66), median BCVA was 0.0 logMAR (0.0-0.30). One had normal/near-normal autofluorescence, 2 radial pattern, 1 focal pigmentary retinopathy and 2 male phenotype with partial AF ring. At the last follow-up at median age of 45 years (10-66), median BCVA was 0.10 logMAR (0.0-0.20). In 2 cases with male phenotype partial AF ring constricted.

Conclusion: RPGR-RP-causing variants in Slovenian cohort include 4/6 (67 %) novel variants, suggesting a distinct genetic background of the RPGR alleles in Slovenian population. Longitudinal follow-up of RPGR-RP patients showed disease progression in all males and 5/6 (83%) females.

References:.

Grants:.

Conflict of Interest: None declared.

P03.026.C Impaired bestrophin channel activity in a iPSC-RPE model of Best Vitelliform Macular dystrophy (BVMD) from an early onset patient carrying the P77S dominant mutation

Arnau Navinés-Ferrer ^1;2, Sheila Ruiz-Nogales^1;2, Pilar Mendez^1;2, Esther Pomares^1;2

¹Fundació de Recerca de l’Institut de Microcirurgia Ocular, Departament de Genetica, Barcelona, Spain; ²Institut de Microcirurgia Ocular, IMO Grupo Miranza, Barcelona, Spain

Background/Objectives: Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight.

Methods: In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this mutation and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and chloride ion entrance. The apoptotic profile is determined through several markers as well as TUNEL assay.

Results: Our cell model shows no differences in gene expression, protein expression/localization or phagocytosis capacity. There are no signs of apoptosis but the iPSC-RPE presents an increased chloride entrance that would explain the patient’s phenotype.

Conclusion: The molecular mechanisms underlying this dominant mutation from a BVMD patient widen the understanding of this pathology and open the door for a better diagnosis and prognosis of the disease.

References: Domingo-Prim et al., Generation of Best disease-derived induced pluripotent stem cell line (FRIMOi006-A) carrying a novel dominant mutation in BEST1 gene.

Grants:.

Conflict of Interest: None declared.

P03.030.C Targeted retinal dystrophy panel as a reliable tool for genetic diagnostics of retinal disorders in Polish patients

Ewa Matczyńska ^1;2, Anna Wąsowska^1;2, Marta Beć¹, Przemysław Łyszkiewicz¹, Robert Szymańczak¹, Ewa Suchecka¹, Monika Jurkowska¹, Maria Jędrzejowska¹, Sławomir Teper², Anna Boguszewska-Chachulska¹

¹Genomed S.A., Warsaw, Poland; ²Medical University of Silesia, Chair and Clinical Dept. of Ophthalmology, Zabrze, Poland

Background/Objectives: A developed targeted panel approach for genetic diagnosis of inherited retinal dystrophies (IRD) was successfully implemented in the routine diagnostics of Polish IRD patients. Here we present the results for the second part of Polish patients group subjected to IRD genetic diagnosis using the established method.

Methods: A group of Polish patients with clinical symptoms of retinal dystrophies (N = 182) were subjected to an NGS analysis using a custom panel capturing coding regions of 270 IRD genes (RetNet), developed with the Roche NimbleGen SeqCap EZ. Bioinformatic analysis was performed with the GATKv4 and CoNVaDING (for CNV analysis), using the GRCh38 reference genome. Variant analysis and interpretation were completed according to ACMG guidelines using the in-house variant interpretation tool - BroVar.

Results: The NGS data presented a satisfactory level of QC data metrics with mean target coverage in a range of 200-250x, heterozygote SNP calling sensitivity level above 0.997, and fraction of target bases covered >20x between 0.996 and 0.997). An IRD diagnosis was confirmed in 109 cases (59.8%). In several cases, the disease confirmation was enabled by CNVs or deep intronic variants calls. In further 29 patients only one pathogenic/likely pathogenic variant was identified in the context of AR disease.

Conclusion: The results of the application of the existing targeted retinal dystrophy panel are promising. Further increase in the method sensitivity is expected in the second version of the approach, which would broaden the targeted gene list, include more deep intronic clinvar variants and target the mtDNA using additional spike-in.

References:.

Grants:.

Conflict of Interest: Ewa Matczyńska Employed in Genomed S.A., Anna Wąsowska Employed in Genomed S.A., Marta Beć Employed in Genomed S.A., Przemysław Łyszkiewicz Employed in Genomed S.A., Robert Szymańczak Employed in Genomed S.A., Ewa Suchecka Employed in Genomed S.A., Monika Jurkowska Employed in Genomed S.A., Maria Jędrzejowska Employed in Genomed S.A., Sławomir Teper: None declared, Anna Boguszewska-Chachulska Genomed S.A. CEO.

P04 Internal Organs & Endocrinology (Lung, Kidney, Liver, Gastrointestinal)

P04.001.D Genetic polymorphisms and susceptibility to urinary tract infection among paediatric and adult populations: a systematic review and meta-analysis

Jiakun Yu ¹, Kristina Allen-Brady², Romana Cuffolo³, Felice Sorrentino⁴, John Chua⁵, Harmeet Sidhu⁶, Marianne Koch⁷, Philippe Violette⁸, Rufus Cartwright¹

¹Imperial College London, School of Medicine, London, United Kingdom; ²The Univeristy of Utah, Utah, United States; ³Stoke Mandeville Hospital, Aylesbury, United Kingdom; ⁴Department of Obstetrics & Gynecology-Policlinico Riuniti Foggia, Foggia, Italy; ⁵Frimley Health NHS Foundation Trust, Firmley, United Kingdom; ⁶LONDON NORTH WEST UNIVERSITY HEALTHCARE NHS TRUST, London, United Kingdom; ⁷Medical University of Vienna, Vienna, United Kingdom; ⁸McMaster University, Ontario, Canada

Background/Objectives: The lifetime risk of UTI is known to be highly heritable. Associations have also been observed across the life course from paediatric UTI to recurrent UTI in adulthood, suggesting lifelong susceptibility factors. We conducted a systematic review to identify all genetic polymorphisms tested for an association with UTI in children and adults, and to assess their strength, consistency, and risk of bias.

Methods: PubMed, HuGE Navigator and Embase were searched from 01/Jan/2005 to 01/Sep/2021, using a combination of genetic and phenotype key words. Fixed and random effects meta-analyses were conducted using co-dominant models of inheritance in metan. The interim Venice criteria were used to assess their credibility.

Results: 1283 studies were screened, with 42 included in the analysis (18 adult papers and 24 paediatric papers). All possible meta-analyses summarised in Table 1 (paediatric samples) and Table 2 (adult samples). These meta-analyses demonstrated significant pooled associations for paediatric UTI with variation in ACE, CXCR1, IL8, TGF, TLR4, VDR and VEGF. These meta-analyses also demonstrated a significant pooled association for adult UTI with variation in ACE and CXCR1. All significant pooled associations were graded as providing at most weak epidemiological credibility.

.

Conclusion: This systematic review provides a current synthesis of the known genetic architecture of UTI in childhood and adulthood, and should provide important information for researchers planning or analysing future genetic association studies. Although, overall, the credibility of pooled associations was weak, the consistency of findings for rs2234671(CXCR1) in both populations suggests a key role in UTI pathogenesis.

References:.

Grants:.

Conflict of Interest: None declared.

P04.002.A KIF12 variants and disturbed hepatocyte polarity in children with a phenotypic spectrum of cholestatic liver disease

Amelie Stalke ^1;2, Malte Sgodda³, Tobias Cantz³, Britta Skawran¹, Elke Lainka⁴, Björn Hartleben⁵, Ulrich Baumann², Eva-Doreen Pfister²

¹Hannover Medical School, Department of Human Genetics, Hannover, Germany; ²Hannover Medical School, Pediatric Gastroenterology and Hepatology, Hannover, Germany; ³Hannover Medical School, Translational Hepatology and Stem Cell Biology, Department of Gastroenterology, Hepatology and Endocrinology, REBIRTH-Center for Translational Regenerative Medicine, Hannover, Germany; ⁴University Children’s Hospital Essen, Department for Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Essen, Germany; ⁵Hannover Medical School, Institute of Pathology, Hannover, Germany

Background/Objectives: Recently, KIF12 has been identified as a cholestasis-associated candidate gene(1,2). As KIF12 is a member of a microtubule-associated motor protein family involved in intracellular transport, KIF12-associated cholestasis is assumed to be a result of disturbed cell polarity. We describe six cases with likely pathogenic KIF12 variants from four unrelated families, their different phenotypes and our investigations to study hepatocyte polarity.

Methods: Children with familial cholestasis and a likely pathogenic variant in the KIF12 gene were identified by exome sequencing. Segregation was analysed by testing parents and siblings. Immunofluorescence imaging of apical markers MRP2 und BSEP, basolateral marker OATP1B1, tight junction protein ZO-1 and KIF12 itself was performed on patient’s liver tissue sections.

Results: We detected two homozygous KIF12 variants in five patients ((NM_138424.1) 4 patients: c.655C>T p.(Arg219*); 1 patient: c.482-4_500del p.?). Segregation analyses confirmed autosomal recessive inheritance. The patients’ clinical manifestation ranged from neonatal cholestasis with complete clinical remission, or absent clinical symptoms to a progressive course ending in liver transplantation. Immunofluorescence imaging of liver sections of KIF12 patients revealed an ectopic cytoplasmic MRP2 staining. BSEP staining appeared in thickened long clustered structures, the latter was detected partly also for ZO-1. KIF12 and OATP1B1 staining was widely unremarkable.

Conclusion: Our results strongly support pathogenic KIF12 variants as cause for familial cholestatic liver disease and suggest that these variants result in functional cell polarity disturbance. Due to its wide clinical presentation with even asymptomatic cases, KIF12-associated cholestatic liver diseases are potentially underdiagnosed.

References: 1.PMID: 30250217; 2.PMID:30976738.

Grants: DFG:#433387263; BMBF (HiChol):#01GM1904B.

Conflict of Interest: None declared.

P04.003.B Elevated polygenic burden for brain haemorrhage related traits in kidney donors

Kane Collins ^1;2;3, Edmund Gilbert^1;2, Peter Conlon^1;4, Gianpiero Cavalleri^1;2, Elhussein Elhassan⁴, Graham Lord⁵

¹Royal College of Surgeons in Ireland, School of Pharmacy and Biomolecular Sciences, Dublin, Ireland; ²SFI FutureNeuro Research centre, Dublin, Ireland; ³SFI Centre for research training in Genomics Data Science, Galway, Ireland; ⁴Beaumont Hospital, Dublin, Ireland; ⁵The University of Manchester, Faculty of Biology, Medicine and Health, Manchester, United Kingdom

Background/Objectives: Brain haemorrhage (BH) is a common cause of death among kidney donors, but little research has been done to investigate polygenic risk scores (PRSs) for BH and related traits in kidney donors. A PRS estimates the cumulative effect of common genetic variation on an individual’s disease status.

Methods: We had 2,122 genotyped donor-recipient pairs from across the UK and Ireland and 5,519 ancestry matched controls. We calculated PRSs for stroke, Intracranial Aneurysm (IA) and hypertension. We compared PRSs between the donors who died of intracranial haemorrhage (DDICH) (1,303 individuals) and controls. We then used PRS to predict case/control status.

Results: PRS for stroke explained the greatest amount of variance in case/control status between DDICH and controls (6.7%, p: 8.1 x 10^-63), with still significant values for the other PRSs (IA: 5.3%, hypertension: 0.24%). A null model using just sex and principal components to predict case/control status achieved an AUC of 0.63, while models using just hypertension, IA and stroke PRSs achieved AUCs of 0.54, 0.61 and 0.67 respectively. A combined model with all the PRSs and covariates achieved an AUC of 0.74.

Conclusion: These observations support the hypothesis that DDICH carry an increased burden for traits related to BH. PRSs can play a part in being able to distinguish DDICH from the general population. These findings could have utility in testing relatives of DDICH to determine if they share the same risk for ICH.

References:.

Grants: Science Foundation Ireland grant number 18/CRT/6214.

Conflict of Interest: Kane Collins Full time, Science Foundation Ireland Grant number 18/CRT/6214.

UKIRTC was supported by grants awarded from the Wellcome Trust (090355/A/09/Z, 090355/B/09/Z and 088849/Z/09/Z, “WTCCC3”), Edmund Gilbert Full time, Peter Conlon Full time, Gianpiero Cavalleri Full time, Elhussein Elhassan Full time, Graham Lord Full time.

P04.005.D Genetic complexity of congenital hypopituitarism: oligogenic inheritance may explain the variable expression and incomplete penetrance of deleterious GLI2 variants in congenital hypopituitarism

Francisco Javier Rodríguez-Contreras¹, Purificación Ros Pérez², Fe Amalia García Santiago³, Elena Vallespín^4;5, Angela Del Pozo Maté^5;6, Mario Solís⁶, Isabel Gonzalez-Casado⁷, Karen E. Heath^5;8, Angel Campos-Barros ^5;9

¹Centro de Salud Galapagar, Pediatrics, Galapagar, Madrid, Spain; ²Hospital Universitario Puerta de Hierro, Pediatrics, Majadahonda, MADRID, Spain; ³Institute of Medical & Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, Prenatal Genetics & Fetal Medicine, Madrid, Spain; ⁴Institute of Medical &Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, Molecular Ophthalmogenetics, Madrid, Spain; ⁵Instituto de Salud Carlos III (ISCIII), CIBER de Enfermedades Raras (CIBERER) U753, Madrid, Spain; ⁶Institute of Medical & Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, Bioinformatics, Madrid, Spain; ⁷Hospital Universitaio La Paz, Pediatric Endocrinology, Madrid, Spain; ⁸Institute of Medical & Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, Madrid, Spain; ⁹Institute of Medical & Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, Molecular Endocrinology Division, Madrid, Spain

Background/Objectives: Pathogenic GLI2 variants are implicated in the etiology of the broad clinical spectrum of congenital hypopituitarism (CHYP), showing a highly variable expressivity and incomplete penetrance.

Aims: Genotype-phenotype correlations in a cohort of patients with CHYP and deleterious GLI2 variants.

Methods: Subjects: 114 patients with CHYP (n = 72), septo-optic dysplasia (SOD; n = 33) or isolated GH deficiency (n = 9).

Molecular analysis: Targeted NGS (HYPOPIT_V3 panel) of 310 genes implicated in the etiology of CHYP and hypothalamic-pituitary development.

Results: Deleterious GLI2 variants were identified in 11/114 (9.6%), all of them with CHYP. These included 4 truncating variants [p.(Arg1226*), p.(Leu709Trpfs*15), p.(Ser267*) and p.(Ser859Profs*53)] (ACMG: pathogenic). All had pituitary hypoplasia, 75% had ectopic neurohypophysis, and those with truncating variants had both conditions. 10/11 were GH and TSH deficient and 8/11 were also ACTH deficient. Other associated traits observed included postaxial polydactyly, ogival palate, bulbous nose, nephrourological dyspasia, and holoprosencephaly, with midfacial hypoplasia and hypotelorism being the most pervasive among the studied relatives (n = 25) of the 11 probands. All of them (11/11) also presented with a burden of additional deleterious variants in multiple genes involved in the regulation of pituitary development signaling pathways (SHH, BMP/TGFb, FGF, WNT, NOTCH, NODAL).

Conclusion: All probands with GLI2 variants presented deleterious variants in other genes involved in pituitary development and function, suggesting the possibility of a complex multifactorial genetic component.

This may explain the highly variable expressivity and incomplete penetrance observed, which seems determined by the burden of additional inherited variants in other relevant genes.

References:.

Grants: PI18/00402, ISCIII, to A.C.B.

Conflict of Interest: Francisco Javier Rodríguez-Contreras: None declared, Purificación Ros Pérez: None declared, Fe Amalia García Santiago: None declared, Elena Vallespín: None declared, Angela Del Pozo Maté: None declared, Mario Solís: None declared, Isabel Gonzalez-Casado: None declared, Karen E. Heath: None declared, Angel Campos-Barros Research grant as PI received from ISCIII (PI18/00402).

P04.006.A Clinical and genetic features in two patients carrying PAX2 variants

Deimante Brazdziunaite ¹, Ilona Rudminiene², Migle Gudynaite², Rasa Strupaite³, Gabija Mazur², Marius Miglinas⁴, Algirdas Utkus¹

¹Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ²Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; ³Center of Eye diseases, Clinic of Ear, Nose, Throat and Eye diseases, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ⁴Center of Nephrology, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Background/Objectives: Pathogenic variants in PAX2 have been associated with a spectrum of kidney and eye disorders, including isolated renal hypoplasia, papillorenal syndrome (renal coloboma syndrome) and focal segmental glomerulosclerosis.

Methods: We report clinical, pathology and genetic data of 2 unrelated adult male patients carrying previously reported heterozygous mutations in PAX2 gene.

Results: 27-years-old patient 1 referred to clinical geneticist with suspicion of hereditary cystic kidney disease. At 11 years, he was diagnosed with bilateral multicystic kidney dysplasia, now presenting with proteinuria and chronic kidney disease (CKD 3 stage). Patient’s father, grandmother had unspecified kidney disease. A kidney-focused NGS panel analysis identified heterozygous nonsense pathogenic c.685C>T, p.(Arg229*) variant in PAX2. Later ocular evaluation revealed bilateral optic disc dysplasia.

36-years-old patient 2 referred to clinical geneticist after his daughter was diagnosed with bilateral kidney hypoplasia, caused by heterozygous missense pathogenic c.250G>A, p.(Gly84Ser) variant in PAX2. Patient presented with persistent proteinuria, observed since 20 years of age, CKD 3 stage. Two kidney biopsies were performed since that time: the first one showing signs of IgA nephropathy, the second one – focal segmental glomerulosclerosis. Sanger sequencing analysis confirmed known familial mutation in PAX2 gene, ocular evaluation revealed optic disc pits.

Conclusion: Hereditary kidney diseases are rare and still underdiagnosed due to variable expressivity and wide range of clinical manifestations even within single families. Reported patients demonstrate this variability and expands knowledge about disorders, caused by PAX2 mutations, reported in literature. It also suggests that ocular evaluation should be performed for a better clinical diagnosis, when kidney pathology is present.

References:.

Grants:.

Conflict of Interest: None declared.

P04.007.B CD55-deficiency in Jews of Bukharan descent is caused by the Cromer Dr(a-) blood type variant

Alina Kurolap ¹, David Hagin^2;3, Tal Freund², Sigal Fishman^3;4, Noa Henig¹, Eli Brazowski⁵, Josepha Yeshaya⁶, Tova Naiman¹, elon pras^3;7, Jacob N. Ablin^3;8, Hagit Baris Feldman^1;3

¹Tel Aviv Sourasky Medical Center, The Genetics Institute and Genomics Center, Tel Aviv-Yafo, Israel; ²Tel Aviv Sourasky Medical Center, Allergy and Clinical Immunology Unit, Department of Medicine, Tel Aviv, Israel; ³Tel Aviv University, Sackler Faculty of Medicine, Tell Aviv, Israel; ⁴Tel Aviv Sourasky Medical Center, The Gastroenterology Institute, Tel Aviv, Israel; ⁵Tel Aviv Sourasky Medical Center, The Institute of Pathology, Tel Aviv, Israel; ⁶American Medical Genetics LaboratoryHerzliya, Herzliya, Israel; ⁷Sheba Medical Center, The Danek Gertner Institute of Human Genetics, Ramat Gan, Israel; ⁸Tel Aviv Sourasky Medical Center, Internal Medicine H, Tel Aviv, Israel

Background/Objectives: The complement system regulator CD55 was initially found to carry the Cromer blood group antigens, and its complete loss-of-function was recently revealed to cause a severe monogenic syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis.

Methods: Patient 1 was diagnosed using flow cytometry and targeted Sanger sequencing. Patient 2 was diagnosed by unrelated exome sequencing. Additional testing on Patient 1 samples included RNA analysis and B-cell immunophenotyping. A population screen was performed using enzymatic restriction method.

Results: Both patients are Bukharan Jewish (BJ) and presented with CD55-deficiency of variable severity; testing revealed homozygosity for the CD55:c.596C>T;p.Ser199Leu variant. RNA analysis confirmed that this missense variant causes aberrant splicing and deletion of 44bp in exon 5, leading to premature termination and low CD55 protein levels. Furthermore, Patient 1 exhibited a mildly abnormal B-cell immunophenotyping profile. The variant is highly prevalent in BJs (carrier frequency: 1:17).

Conclusion: The CD55 p.Ser199Leu variant is known as the Cromer Dr(a-) genotype, yet this is the first report in terms of CD55-deficiency. The phenotypic variability, ranging from abdominal pain on a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. The high frequency in the BJ population suggests that many similar patients are un- or mis-diagnosed. Establishing the diagnosis of CD55-deficiency in a timely manner may have critical effects on patient management and quality-of-life, since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations.

References:.

Grants:.

Conflict of Interest: Alina Kurolap Patent with Alexion Pharmaceuticals on eculizumab treatment protocol for CD55-deficiency (WO/2018/217638), which does not include any royalties., David Hagin: None declared, Tal Freund: None declared, Sigal Fishman: None declared, Noa Henig: None declared, Eli Brazowski: None declared, Josepha Yeshaya: None declared, Tova Naiman: None declared, elon pras: None declared, Jacob N. Ablin: None declared, Hagit Baris Feldman Patent with Alexion Pharmaceuticals on eculizumab treatment protocol for CD55-deficiency (WO/2018/217638), which does not include any royalties, Advisory board member for the C5-inhibitor Pozelimab clinical trial by Regeneron Pharmaceuticals.

P04.008.C Asthma exacerbations in the UK Biobank: a Genome-Wide Association Study

Ahmed Edris Mohamed ^1;2, Katherine Fawcett², Ian P. Hall³, Martin D Tobin², Lies Lahousse¹

¹Faculty of Pharmaceutical Sciences, Ghent University, Department of Bioanalysis, Ghent, Belgium; ²University of Leicester, Genetic Epidemiology Group, Department of Health Sciences, Leicester, United Kingdom; ³University of Nottingham, Faculty of Medicine & Health Sciences, Nottingham, United Kingdom

Background/Objectives: Asthma exacerbations affect morbidity and mortality of the disease, and increase the burden to both patient and healthcare system. We aimed to assess the effects of genetic variants on asthma exacerbation risk in UK Biobank participants with asthma.

Methods: Within the UK Biobank study, individuals with asthma were identified as those self-reporting doctor-diagnosed asthma, or with a code for asthma in hospital inpatient and/or General Practitioners (GP) records. Exacerbations were identified as either asthma–related hospitalization, GP record of asthma exacerbation, or an Oral Corticosteroid (OCS) burst prescription. A logistic regression model adjusted for age, sex, and smoking status was used to assess the association between genome-wide imputed SNPs and risk of an asthma exacerbation.

Results: We identified 11,604 individuals with asthma with at least one exacerbation (cases), and 37,890 individuals with asthma with no recorded exacerbations (controls). While no variants reached genome wide significance (p < 5x10^-8), two SNPs (rs1449836 (chr14, YWHAQP1/TUBBP3) and rs34643691 (chr15; intergenic variant)) were suggestively associated with asthma exacerbation risk (P < 1x10^-7). Rs1449836 has been previously associated with response to metformin, and is located in a genetic region previously associated with inflammatory auto-immune diseases.

Conclusion: We identified two SNPs suggestively associated with risk of asthma exacerbations. Further research and replication of these findings may be needed to ascertain genetic variants’ role in the severity of asthma and its clinical presentation.

References:.

Grants:.

Conflict of Interest: Ahmed Edris Mohamed: None declared, Katherine Fawcett: None declared, Ian P. Hall Research collaborations with GSK, Boehringer Ingelheim and Orion outside of submitted work., Martin D Tobin GSK and Orion for collaborative research projects outside of the submitted work., Lies Lahousse: None declared.

P04.009.D A screening of the genetic causes of Familial Pulmonary Fibrosis in patients from the Canary Islands (Spain)

Eva Tosco-Herrera ¹, Aitana Alonso-Gonzalez^1;2, Ibrahim Véliz-Flores³, Felipe Rodríguez de-Castro³, Rosario Perona⁴, Beatriz Fernández-Vara⁴, Desirée Alemán-Segura³, Angel Rodríguez-León³, Antonio Iñigo-Campos⁵, Luis A. Rubio-Rodríguez⁵, Alejandro Mendoza-Alvarez¹, JOSÉ MIGUEL LORENZO-SALAZAR⁵, Rafaela González-Montelongo⁵, Carlos Flores^1;5;6

¹Research Unit, Hospital Universitario Nuestra Señora de Candelaria, Universidad de La Laguna, Santa Cruz de Tenerife, Spain; ²Universidad de Santiago de Compostela, Santiago, Spain; ³Servicio de Neumología Hospital Universitario de Gran Canaria “Dr Negrín”, Las Palmas de Gran Canaria, Spain; ⁴Instituto de Investigaciones Biomédicas CSIC-UAM, Spain, Spain; ⁵Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain; ⁶CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain

Background/Objectives: Pulmonary Fibrosis (PF) is a rare progressive scarring lung disease with poor prognosis. A significant role of rare and common genetic variation underlying its etiology is known. We relied on whole-exome sequencing (WES) to screen the genetic causes of familial PF (FPF) from Canary Islands patients.

Methods: We recruited 54 subjects from 11 families. WES was obtained using a HiSeq4000 Illumina system and small germline variant identified with BWA-GATK v3.8 against the GRCh37/hg19 reference. Relative telomere length (TL) was measured by quantitative PCR and severe reduction denoted when length was <10^th percentile compared to age-matched controls. Rare (AF<1%) non-synonymous exonic and splicing variants from known PF or interstitial lung diseases genes were considered for manual review.

Results: Initial analysis excluded common genetic variants among affected individuals. A total of 22 rare variants from 15 genes were prioritized and classified for pathogenicity according to ACMG recommendations. Pathogenicity was supported only for a variant in NAF1 gene (NM_138386.3:c.1104T>G) found in one family and the TL of the index case was in the 1^st percentile. The variant was absent from gnomAD, local population controls (n = 920), and from healthy relatives (n = 2).

Conclusion: Our results evidence the complexity of this disease and the challenges of variant interpretation. Further studies are ongoing examining the remaining of the WES data and additional families.

References:.

Grants: Ministerio de Ciencia e Innovación (RTC-2017-6471-1; AEI/FEDER, UE); ITER agreement (OA17/008); Gobierno de Canarias & Fondo Social Europeo “Canarias Avanza con Europa” (TESIS2021010046), Ministerio de Universidades (modality Margarita Salas).

Conflict of Interest: None declared.

P04.010.A The value of genomic testing for the kidney patient and for pre-transplantation donor evaluation

Georgia Christopoulou¹, Stavroula Samara¹, Aikaterini Oikonomaki¹, Vassilis Filiopoulos², Stathis Tsiakas², Christina Melexopoulou², Ioannis Boletis², Pantelis Constantoulakis ¹

¹Genotypos M.S.A., Athens, Greece; ²National and Kapodistrian University of Athens, School of Medicine, “Laikon” University Hospital, Nephrology Department and Renal Transplant Unit, Athens, Greece

Background/Objectives: The spectrum of kidney genetic disease is extremely broad and phenotypes are variable and overlapping, involving extra-kidney manifestations in some disease groups. Elucidating potential genetic causes is challenging due to the number of genes involved and unknown disease mechanisms. Exome sequencing (ES) may unveil the genetic background in patients, allowing definitive diagnosis and/or timely interventions while it may provide valuable information for kidney-donor candidates.

Methods: Thirty-three individuals (kidney-patients/donor-candidates) were referred to our lab, by a single reference clinic for ES, performed on DNA extracted from peripheral blood: libraries preparation was performed with Clinical Exome (Sophia Genetics) or Whole Exome (Twist Bioscience) and sequenced on Illumina NextSeq-550 (Illumina). Bioinformatics analyses were conducted by SOPHiA DDM® bioinformatics pipelines.

Results: Pathogenic/likely pathogenic variants were detected in 19 patients: 10 in collagen-genes, 5 PKD1 and 1 INF2 confirming suspected diagnosis i.e., Alport syndrome, autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis, respectively. All cases were consistent with biopsy, imaging and urine/blood tests. Copy number variation analysis revealed a NPHP1 homozygous deletion (nephronophthisis) and a COL4A5 heterozygous deletion (Alport syndrome). In 9 cases results were negative either failing to elucidate genetic disease or clearing genetic burden for 5 kidney-donor candidates. In 5 cases, variants of unknown significance were detected giving eligibility to 1 donor, rejecting 2.

Conclusion: ES permitted timely, accurate diagnosis confirmation for kidney-patients allowing appropriate intervention, maintenance treatment or transplantation programming. Further, it provided valuable information for donor-candidates regarding their and the recipient’s best interest. Incorporating exome sequencing in Nephrology may lead to higher-quality healthcare.

References:.

Grants:.

Conflict of Interest: Georgia Christopoulou Full time employee: Genotypos M.S.A., Stavroula Samara Full time employee: Genotypos M.S.A., Aikaterini Oikonomaki Full time employee: Genotypos M.S.A., Vassilis Filiopoulos: None declared, Stathis Tsiakas: None declared, Christina Melexopoulou: None declared, Ioannis Boletis: None declared, Pantelis Constantoulakis Full time employee: Genotypos M.S.A.

P04.011.B Novel pathogenic ALG8 variants and evidence for somatic loss of heterozygosity in 542 autosomal dominant polycystic liver disease patients

Melissa M. Boerrigter ¹, Rene H.M. te Morsche¹, Anja Geerts², Anne Hoorens³, J.P.H. Drenth¹

¹Radboud University Medical Center, Department of Gastroenterology and Hepatology, Nijmegen, Netherlands; ²Ghent University Hospital, Department of Gastroenterology and Hepatology, Ghent, Belgium; ³Ghent University Hospital, Department of Pathology, Ghent, Belgium

Background/Objectives: Autosomal dominant polycystic liver disease (ADPLD) is caused by pathogenic variants in at least 6 different genes. ALG8 has been linked to ADPLD in 5 isolated cases. We aim to identify novel pathogenic variants in ALG8 by different next-generation sequencing techniques and to confirm loss of heterozygosity in liver cyst tissue in ADPLD patients.

Methods: We performed whole exome sequencing in a cohort of 60 genetically undiagnosed ADPLD patients and targeted sequencing of all ALG8 exons using MiniSeq in 482 genetically undiagnosed ADPLD patients. We performed expression analysis by immunohistochemistry on liver cyst tissue with a hepatocyte-specific, a cholangiocyte-specific, and an ALG8 antibody.

Results: We screened 542 ADPLD patients and identified 8 heterozygous variants in ALG8 (nonsense n = 4, frameshift n = 2, splice site n = 1, missense n = 1) in 16 ADPLD patients. A 19-member family included 4 patients with severe ADPLD who shared the novel ALG8 variant c.160C>T p.(Gln54*). This variant was also present in 7 asymptomatic family members with 0-2 liver cysts. The 7 other variants were found in 1 small family and 10 singletons. All patients had mild to severe ADPLD and 0-6 kidney cyst that did not affect renal functioning. We found evidence for somatic loss of heterozygosity, as ALG8 immunoreactivity was absent in the cyst lining and cyst bordering hepatocytes of a patient carrying the c.1501delG variant.

Conclusion: Our results are consistent with a pathogenic role of ALG8 in ADPLD. Loss of heterozygosity of ALG8 in liver cyst lining is a key event in the pathogenesis of ADPLD.

References: -.

Grants: -.

Conflict of Interest: None declared.

P04.012.C Clinical exome sequencing as a valuable diagnostic tool for glomerulopathies: a cohort study

Angelo Corso Faini ¹, Valeria Bracciamà¹, Silvia Kalantari¹, Giulia Margherita Brach del Prever¹, Martina Callegari¹, Licia Peruzzi², Roberta Fenoglio³, Dario Roccatello^3;4, Tiziana Vaisitti¹, Antonio Amoroso¹, Silvia Deaglio¹

¹Transplant Regional Center-Piedmont, Immunogenetics and Transplant Biology, AOU Città della Salute e della Scienza, Department of Medical Sciences, University of Turin, Turin, Italy; ²Nephrology Dialysis and Transplantation, Regina Margherita Children’s Hospital, Turin, Italy; ³SCU Nephrology and Dialysis (ERKnet member)-CMID, Center of Research of Immunopathology and Rare Diseases, San Giovanni Hospital, Turin, Italy; ⁴Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Background/Objectives: The diagnosis of glomerulopathies remains a hard task, as different diseases can present with similar characteristics and bioptic evidence is often non-specific. In this complex scenario, genetic analyses may play an important role.

Methods: Adult and pediatric patients were recruited from multiple centers of the Piedmont region (Italy), exploiting a pre-existing transplantation network. When referred to our center, patients had already undergone nephrological counselling and suspicion of glomerulopathy was raised. Clinical exome sequencing (CES) was performed, and analysis was restricted to a panel of genes associated with glomerulopathies.

Results: CES was performed on a diagnostic cohort of 166 patients. Alport syndrome scored the highest percentage of solved cases (59%), followed by glomerulonephritis (35%), non-nephrotic proteinuria (29%), FSGS (12%) and nephrotic proteinuria (19%). Of note, 54% of clinically undiagnosed glomerulopathies were solved. Overall, results show a diagnostic rate of 36% which is in line with published data¹.

Conclusion: Our data confirm genetic analysis is a valuable diagnostic resource when a precise glomerulopathy is suspected and – unexpectedly – also when the clinical suspicion is less defined. Analysis by CES i) shows a high diagnostic rate; ii) allows a precise diagnosis avoiding invasive procedures (biopsy); iii) prevents ineffective treatment; iv) speeds up the enrollment in transplantation awaiting lists and v) allows to determine recurrence risk, post-transplantation prognosis and living-donor eligibility. Taken together, our results strongly support genetic analysis as a first-line diagnostic tool in kidney disease.

References: 1. Connaughton et. al, Kidney Int., 2019,95(4):914-928.

Grants: Ministero dell’Istruzione, Progetto strategico di Eccellenza Dipartimentale #D15D18000410001.

Conflict of Interest: None declared.

P04.013.D Clinical exome sequencing (CES) as a tool for genetic diagnosis of Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Valeria Bracciamà ¹, Angelo Corso Faini¹, Silvia Kalantari¹, Giulia Margherita Brach del Prever¹, Martina Callegari¹, Licia Peruzzi², Roberta Fenoglio³, Dario Roccatello^3;4, Silvia Deaglio¹, Antonio Amoroso¹, Tiziana Vaisitti¹

¹Transplant Regional Center-Piedmont, Immunogenetics and Transplant Biology, AOU Città della Salute e della Scienza & Department of Medical Sciences, University of Turin, Turin, Italy; ²Nephrology Dialysis and Transplantation, Regina Margherita Children’s Hospital, Turin, Turin, Italy; ³SCU Nephrology and Dialysis (ERKnet member)-CMID, Center of Research of Immunopathology and Rare Diseases, San Giovanni Hospital, Turin, Turin, Italy; ⁴Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Background/Objectives: Autosomal dominant polycystic kidney disease (ADPKD #173900, #613095, #600666) is the most common inherited nephropathy and is predominantly caused by mutations in PKD1 and PKD2. Genetic testing of PKD1 is challenging for the presence of homologous pseudogenes with genetic and allelic heterogeneity. Due to phenocopies, diagnostic criteria are unable to provide a definitive diagnosis. We propose NGS-based strategy for genetic diagnosis of patients with ADPKD.

Methods: CES was performed in 121 subjects with clinical suspicion of PKD. Patients were recruited by the hospitals of the Piedmont region (Italy). PKD1 and PKD2 were analyzed and for negative cases additional cystogenes were investigated. Identified variants were segregated and validated by Sanger sequencing.

Results: Causative variants in PKD1 and PKD2 were identified in 78 patients, 56% of them presenting family history. 31 cases resulted negative or unconclusive. Most of the diagnostic genetic variants affected PKD1 (78%) and PKD2 (9%). 20 patients had more than one variant in the same gene or the 2 different cystogenes. Furthermore, we identified 12 possible alternative diagnoses among which 5 patients with biallelic mutations in PKHD1 and two patients with variants in TSC1 and DSTYK, respectively.

Conclusion: Identification of the specific molecular basis of a disease is essential to avoid genetic misdiagnosis, unnecessary and invasive therapies and for early detection of renal and extrarenal comorbidities. The broad phenotypic and genetic heterogeneity of cystic kidney diseases make CES a time- and cost-efficient diagnostic approach for these indications.

References: Cornec-Le Gall, et al., Lancet. 2019.

Grants: Ministero dell’Istruzione, Progetto strategico di Eccellenza Dipartimentale #D15D18000410001.

Conflict of Interest: None declared.

P04.014.A Transcriptomic landscape of pancreatic neuroendocrine tumours

Olesja Rogoza¹, Helvijs Niedra¹, Rihards Saksis¹, Aija Gerina-Berzina², Sofija Vilisova², Julie Earl¹, Ignacio Ruz Caracuel³, Bozena Smolkova⁴, Georgina Kolnikova⁵, Peter Dubovan⁶, Peter Makovicky⁴, Eythimios Koniaris⁷, Agapi Kataki⁸, Vita Rovite ¹

¹Latvian Biomedical Research and Study Centre, Research Group of Molecular and Functional Genomics, Riga, Latvia; ²Pauls Stradins Clinical University Hospital, Oncology Clinic, Riga, Latvia; ³Ramón y Cajal Health Research Institute (IRYCIS), Ramón y Cajal University Hopsital, Madrid, Spain; ⁴Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; ⁵National Cancer Institute, Department of Pathology, Bratislava, Slovakia; ⁶National Cancer Institute, Department of Surgical Oncology of Slovak Medical University, Bratislava, Slovakia; ⁷Hippokration General, Hospital of Athens, Department of Pathology, Athens, Greece; ⁸Hippokration General, Hospital of Athens, First Department of Propaedeutic Surgery, Athens, Greece

Background/Objectives: Pancreatic neuroendocrine tumours (PanNETs) arise from the neuroendocrine cells in pancreatic islets and account for approximately 12% of digestive system NETs, with an overall 5-year survival rate of 85%. PanNETs are heterogeneous lesions, classified according to cell proliferation rate and differentiation grade. In this study, we aimed to create a well-defined cohort of PanNETs and characterize the transcriptomic landscape on a molecular level to discover novel markers and therapeutic strategies for better tumour management options.

Methods: PanNET FFPE samples used in this study were obtained from different consortium members from Greece, Slovakia, and Spain. RNA was then extracted from FFPE samples and libraries prepared. Transcriptome sequencing was then carried out on DNBSEQ-G400 platform (MGI) to identify differentially expressed genes (DEGs) in tumour and non-tumour tissues.

Results: We have sequenced transcriptomes of 72 PanNET FFPE samples and detected different transcriptome profiles that distinguish tumour and non-tumour tissues of the pancreas. In total, we were able to find 265 DEGs. Among these were insulin regulation pathway genes and other markers related to tumour pathogenesis pathways, cell proliferation, and invasion, for example, ISL1, SYCN, KLK1, CUZD1 and others.

Conclusion: The results of the PanNET transcriptomic landscape highlight the heterogeneity of PanNETs, which is dependent on various tumour characteristics. Transcriptome analysis is valuable for understanding PanNET tumour biology, which will help to discover new markers and develop new therapeutic approaches for PanNETs.

References:.

Grants: European Regional Development Fund project “Establishing an algorithm for the early diagnosis and follow-up of patients with pancreatic neuroendocrine tumors (NExT)”, grant number 1.1.1.5/ERANET/20/03.

Conflict of Interest: None declared.

P04.015.B Four years’ experience in cystic fibrosis neonatal screening in Luxembourg: 2018-2021

Marizela Kulisic ¹, Dominique Bourgeois¹, Anna-Maria Charatsi^2;3, Meriem Mastouri^2;3, Caroline Eisele², Flore Nzuangue³, Isabel De La Fuente Garcia², Barbara KLINK¹, Patricia Borde⁴

¹Laboratoire national de santé (LNS), National Center of Genetics, Dudelange, Luxembourg; ²Centre Hospitalier de Luxembourg (CHL), Paediatric Department, Kannerklinik, Luxembourg, Luxembourg; ³Centre Hospitalier de Luxembourg (CHL), Pneumology Department, Luxembourg, Luxembourg; ⁴Laboratoire national de santé (LNS), Department of Medical Biology, Dudelange, Luxembourg

Background/Objectives: Cystic fibrosis (CF) newborn screening (NBS) was introduced in Luxembourg in January 2018. Biochemical and genetic testing is centralized at the Laboratoire national de santé (LNS) and we present the results of the first 4 years.

Methods: Immunoreactive Trypsinogen (IRT) is assessed for all newborns (app 7000/year) on day 3 of life (D3). In case of high IRT-D3 (≥60ng/mL), CFTR genetic testing using the CF-EU2v1 (Yourgene Health) kit to detect 50 frequent CFTR pathogenic variants is performed, and a second IRT test on day 21 (D21). Patients screened positive (one or two pathogenic variant detected and/or IRT-D21 ≥40ng/mL) are referred to the Paediatric National CF centre for sweat testing and further multi-disciplinary clinical follow up. In case of positive sweat test without confirmed molecular diagnosis, complete CFTR gene sequencing is performed.

Results: IRT-D3 level was above the threshold in 0.6% newborns in 2018, 0.97% in 2019, 1.38% in 2020 and 1.32% in 2021. CF was diagnosed in 10 infants (n = 3; n = 4; n = 2; n = 1 respectively). The positive predictive value (PPV) was 15%. All patients had at least one CFTR pathogenic variant detected by the kit (55% of them were F508del). In four cases, the second rare CF-causing variant was detected by sequencing (4382delA, E664X, I502T, S489L). The incidence of CF was 1/2454 live births.

Conclusion: CF NBS in Luxembourg is in line with current recommendations for standards of CF care. To improve the PPV, we will adapt the protocol and consider cases with no mutation and IRT-D3<100ng/ml as screened negative.

References:.

Grants:.

Conflict of Interest: None declared.

P04.017.D Genetic revision of the Hungarian cystic fibrosis registry

Anna Deák ¹, Katalin Koczok¹, Beáta Bessenyei¹, László Madar¹, Zsuzsanna Szűcs¹, Gabriella Csorba¹, Istvan Balogh^1;2

¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Genetics, Debrecen, Hungary; ²University of Debrecen, Department of Human Genetics, Debrecen, Hungary

Background/Objectives:.

In order to develop a diagnostic strategy for cystic fibrosis and to facilitate mutation-specific treatments, the genetic revision of the Hungarian cystic fibrosis Registry was performed.

Methods: 528 patients’ data and samples were used for the revision. First we reviewed the patients’ existing genetic findings. Wherever necessary, a comprehensive three-level genetic analysis of the CFTR gene was done.

Results: According to our study, of the 528 patients present in the Registry 395 (74.8%) had two pathogenic CFTR mutation. We completed and corrected 94 patients’ previously incomplete genetic status. 73 different pathogenic variants were detected, in which 6 aberrations were not previously reported. The five most common mutations were: F508del (68.3%); CFTRdele2,3 (3.7%); G542X (3.2%); 2184insA (2.7%); W1282X (2.3%). Based on genotype and age, 192 patients (36.4%) are eligible for the available lumacaftor-ivacaftor combination therapy. Soon ivacaftor-tezacafor-elexacaftor combination will be an option for 364 (68.9%) patients.

Conclusion: Due to the revision, we could identify the patients who can benefit from mutation-specific drugs instead of symptomatic therapy. In addition, the obtained data have been used to map the Hungarian distribution of mutations in the CFTR gene, which will help to develop a diagnostic strategy.

References:.

Grants:.

Conflict of Interest: None declared.

P04.018.A Gene expression profiling through Whole Transcriptome Sequencing predicts novel mechanisms in Hirschsprung Associated Enterocolitis

Francesca Rosamilia ¹, FRANCESCA CALCATERRA^2;3, Francesco Caroli⁴, Marta Rusmini⁵, Alice Grossi⁵, Alessio Pini Prato⁶, domenico mavilio^2;3, Isabella Ceccherini⁵, francesca lantieri¹

¹University of Genoa, Health Science Department (DISSAL), Biostatistics Unit, Genoa, Italy; ²University of Milan, Department of Medical Biotechnologies and Translational Medicine, Milan, Italy; ³IRCCS Humanitas Research Hospital, Laboratory of Clinical and Experimental Immunology, Milan, Italy; ⁴IRCCS Istituto Giannina Gaslini, University of Genoa, UOC Genetica Medica, Genoa, Italy; ⁵IRCCS Istituto Giannina Gaslini, UOSD, Laboratory of Genetics and Genomics of Rare Diseases, Genoa, Italy; ⁶Azienda Ospedaliera SS Antonio E Biagio E Cesare Arrigo, Umberto Bosio Center for Digestive Diseases, The Children Hospital, Alessandria, Italy

Background/Objectives: Hirschsprung (HSCR) associated Enterocolitis (HAEC) is a common life‐threatening complication in HSCR, likely due to a gut immune system impairment. We have recently identified a HAEC susceptibility variant in the Oncostatin‐M receptor gene, also implied in Inflammatory Bowel Diseases (IBDs), through Whole‐Exome Sequencing on HSCR and HAEC patients. This study also confirmed the immune system impairment in HAEC, however no data is available on possibly different gene expression in HAEC versus HSCR patients.

Methods: We have carried out a transcriptome analysis on Intraepithelial lymphocytes (IEL) derived from biopsies of the gut of 6 HAEC patients, 6 HSCR-only patients and 4 paediatric patients affected by neither Hirschsprung nor inflammatory related diseases. For the sequencing, the Ion AmpliSeq Transcriptome Human Gene Expression Kit was used.

Results: We found a clear clustering between the three different groups of patients. Several transcripts were nominally significantly over- and under-expressed in HAEC vs HSCR-only patients, among which several transcripts also involved in IBDs pathogenesis. Very preliminary results showed an enrichment in immune and inflammatory pathways in the HAEC group, although further analyses are still in progress.

Conclusion: We have identified several differentially expressed genes between HSCR patients that had developed HAEC vs those without HAEC occurrence, including genes or pathways already affected in several intestinal pathological conditions. This could lead to the identification of key factors in the HAEC pathogenesis, possibly shared with other intestinal inflammatory diseases.

References:.

Grants:.

Conflict of Interest: None declared.

P04.019.B Association between arterial hypertension and liver-related outcomes using polygenic risk scores – a population-based study

Fredrik Åberg¹, Katri Kantojärvi ², Ville Tapio Männistö³, Anna But⁴, Veikko Salomaa², Teemu Niiranen^2;5, Martti Färkkilä⁶, Panu Luukkonen^7;8;9, Satu Männistö², Annamari Lundqvist², Markus Perola², Antti Jula²

¹Helsinki University Hospital, Transplantation and Liver Surgery Clinic, Helsinki, Finland; ²Finnish Institute for Health and Welfare, Helsinki, Finland; ³University of Eastern Finland and Kuopio University Hospital, Department of Medicine, Kuopio, Finland; ⁴Helsinki University Hospital, Abdominal Center, Helsinki, Finland; ⁵Turku University Hospital and University of Turku, Department of Medicine, Turku, Finland; ⁶Helsinki University and Helsinki University Hospital, Abdominal Center, Helsinki, Finland; ⁷Minerva Foundation Institute for Medical Research, Helsinki, Finland; ⁸University of Helsinki and Helsinki University Hospital, Department of Medicine, Helsinki, Finland; ⁹Yale University, Department of Internal Medicine, New Haven, United States

Background/Objectives: Arterial hypertension (HTA) is associated with liver disease, but the causality remains unclear. We investigated whether genetic predisposition to HTA is associated with incident liver disease in the general population, and if antihypertensive medication modifies this association.

Methods: Participants of the Finnish health-examination surveys, FINRISK 1992-2012 and Health 2000, were followed for incident liver-related outcomes (ICD-10 codes: K70-K77, C22.0) and initiation of antihypertensive medication through the linkage with national registers. Polygenic risk scores (PRS) for systolic (SBP) and diastolic (DBP) blood pressure were derived from GWAS where over 1 million people were studied (Evangelou et al. 2018). PRSs were calculated with PRS-CS method. Cox regression analyses were adjusted for multiple confounders.

Results: In the fully-adjusted Cox regression models, both measured systolic blood pressure and clinically defined HTA were associated with increased risk of liver-related outcomes. Similarly, the PRSs for systolic and diastolic blood pressure were associated with liver-related outcomes. Recent initiation of antihypertensive medication was associated with reduced rates of liver-related outcomes in persons with high genetic HTA risk.

Conclusion: HTA and a genetic predisposition for HTA are associated with liver-related outcomes in Finnish population. New initiation of antihypertensive medication attenuates this association in persons with a high genetic risk for HTA.

References: Evangelou E. et al. 2018. https://doi.org/10.1038/s41588-018-0205-x.

Grants: Mary and Georg Ehrnrooth Foundation, Medicinska Understödsföreningen Liv och Hälsa, Finska Läkaresällskapet, Academy of Finland (#338544) and Sigrid Jusélius Foundation.

Conflict of Interest: None declared.

P04.020.C Whole Exome Analysis to identify related genes that predispose to the progression of Non-Alcoholic Fatty Liver (NAFL) to Non-Alcoholic Steatohepatitis (NASH)

Chiara Catalano ¹, Carla Debernardi¹, Gian Paolo Caviglia¹, Elisabetta Casalone¹, Chiara Rosso¹, Angelo Armandi¹, Alessandra Allione¹, Alessia Russo¹, Elisabetta Bugianesi¹, Giuseppe Matullo^1;2

¹University of Turin, Department of Medical Sciences, Turin, Italy; ²AOU Città della Salute e della Scienza, Medical Genetics Unit, Turin, Italy

Background/Objectives: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease that includes a wide range of liver damage ranging from simple steatosis (non-alcoholic fatty liver; NAFL) to non-alcoholic steatohepatitis (NASH)(1). The genetic factors underlying the progression from NAFL to NASH remain partially explored. We performed Whole Exome Sequencing (WES) analysis to better characterize the genetic landscape of NAFL vs NASH.

Methods: The study includes patients with biopsy-proven NAFLD enrolled at the Liver Unit of the Department of Medical Sciences, University of Turin. DNA samples from 157 subjects (111 NASH; 46 NAFL) were used for WES analysis (Illumina Novaseq6000).

Results: For variant analysis, we filtered based on the pathogenic/likely-pathogenic status according to ClinVar’s clinical annotation and Allele Frequency (Max AF) less than 5%. Preliminary results on a subset of 112 patients (76 NASH; 36 NAFL) revealed that NASH subjects have on average fewer mutations than NAFL subjects (Wilcox test p-value = 0.014). We identified 113 genes exclusively mutated in NASH patients. The majority of them is involved in pathways such as DNA repair, primary cilium formation and lipid metabolism, which are all linked to the pathophysiology of the progressive form of the disease.

Conclusion: WES analysis allows the detection of novel genetic alterations that could be useful to identify genes and pathways that may help in risk stratification in patients with NAFLD.

References: 1)Armandi, et al. Insulin Resistance across the Spectrum of Nonalcoholic Fatty Liver Disease. Metabolites 2021.

Grants: Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) Project “Dipartimenti di Eccellenza 2018–2022”.

Conflict of Interest: None declared.

P04.021.D Genetics of transient congenital hypothyroidism

Nikolina Zdraveska ¹, Violeta Anastasovska^1;2, Mirjana Kocova²

¹Ss. Cyril and Methodius University in Skopje (UKIM), University Children Hospital, Skopje, Macedonia; ²Ss. Cyril and Methodius University in Skopje (UKIM), Skopje, Macedonia

Background/Objectives: The etiology of transient congenital hypothyroidism (TCH) is heterogeneous and can result from maternal thyroid autoimmune disease, antithyroid medications, iodine deficiency or iodine excess iodine in the perinatal period or due to a genetic cause usually related to mild thyroid dyshormonogenesis.

Methods: The study cohort included 11 patients with TCH from unrelated families including 9 cases with normal sized eutopic thyroid gland and 2 cases with goiter. All patients had CH without extrathyroidal manifestations. Genomic DNA was extracted from peripheral blood leukocytes using standard techniques, and Sanger sequencing was used to screen for TSHR, DUOX2, and DUOXA2 genes were screened for mutations in all coding exons and exon/intron boundaries amplified by PCR specific primers. In cases for whom pertechnetate scan data were lacking, SLC5A5 (NIS) was also screened.

Results: Heterozygous DUOX2 variants were identified in 18% of the TCH cases. The p.E1546G mutation was previously reported to be associated with transient CH. An additional novel heterozygous DUOX2 variant p.D1440N was detected affecting a conserved amino acid within the NADPH-oxidase domain in which other pathogenic mutations have been identified, supporting the phenotype characteristics of mild, goitrous CH. Two novel monogenic TSHR mutations were identified p.L452P and p.E107D. TSHR variants were associated with mild CH and normal sized thyroid gland. In both cases TSH normalization was readily achieved after levothyroxine initiation without requiring supraphysiological FT4 levels.

Conclusion: Identification of genetic causes of TCH is important for delineation form permanent forms and possible earlier re-evaluation, as infants with TCH usually receive thyroid hormone replacement.

References:.

Grants:.

Conflict of Interest: None declared.

P04.022.A A comprehensive stepwise approach to study a large group of Egyptian referral patients with disorders of sex development (DSD)

Mona Mekkawy ¹, Inas Mazen², Alaa Kamel¹, Heba Hassan³, Mona Essawi³, Mohamad Abdelhamid³, Ola Eid¹, Amal Mahmoud¹, Aya Elaidy², Shereen Adel¹, Hala Soliman³, Ken McElreavey⁴, anu bashamboo⁵

¹National Research Centre, Human Cytogenetics, Cairo, Egypt; ²National Research Centre, Clinical genetics, Cairo, Egypt; ³National Research Centre, Medical Molecular genetics, Cairo, Egypt; ⁴Institut Pasteur, Developmental Genetics and Stem Cell Biology, Paris, France; ⁵Institut Pasteur, Developmental Genetics and Stem Cell Biology, Paris, France

Background/Objectives: Disorders of sexual development (DSD) include conditions affecting urogenital development, associated with atypical chromosomal, gonadal, or phenotypic sex. The phenotype variability result from a wide range of genetic alterations that may be chromosomal, CNVs, monogenic, digenic or polygenic (Mazen et al., 2021). The study aimed to improve the diagnostic strategy of DSD patients by applying a stepwise approach.

Methods: The study included 599 Egyptian DSD patients, over a period of 6 years. They underwent clinical examination, hormonal and imaging studies, cytogenetic and FISH analysis and Sanger sequencing. Detection of copy number variations (CNV) using MLPA was applied on 35 patients. Whole exome sequencing (WES) was applied on 18 patients and chromosomal microarray (CMA) was conducted for five patients with associated anomalies.

This study was approved by NRC Ethics Committee.

Results: Sex chromosomal abnormalities were found in 41%, while autosomal abnormalities were detected in 2.3%.

Sanger sequencing identified pathogenic variants in 33.7%. MLPA identified deletions of SOX9 in two patients. The detection rate of WES reached 66.7%, while CMA analysis revealed pathogenic copy number variations in two patients.

Conclusion: The study reports a large number of DSD patients from the same ethnic group with a wide cytogenetic spectrum and characteristic mutational profile with novel and rare variants.

References: Mazen I, Mekkawy M, Kamel A, et al. (2021) Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development. Am J Med Genet A. 185:1666-1677.

Grants: STDF–IRD Joint Innovative Projects Fund, Grant/Award Number: 4632.

In house NRC projects: 2013-2019.

Conflict of Interest: None declared.

P04.024.C NEK8-associated nephropathies: do autosomal dominant forms exist?

Cybel Mehawej ¹, Eliane Choueiry¹, ramy ghabril², sima tokajian³, Andre Megarbane^1;4

¹Lebanese American University, Department of Human Genetics, Gilbert and Rose-Marie Chagoury School of Medicine, Byblos, Lebanon; ²University of Balamand, Department of Clinical Pediatrics, Pediatric Nephrology Unit, Saint George University Hospital, Beirut, Lebanon; ³Lebanese American University, Department of Natural Sciences, School of Arts and Sciences, Byblos, Lebanon; ⁴Institut Jérôme Lejeune, Paris, Lebanon

Background/Objectives: Nephronophthisis (NPHP) is a group of autosomal recessive renal diseases characterized by a reduced ability of the kidneys to concentrate solutes, chronic tubulointerstitial nephritis and cystic kidney disease. It represents the most common genetic cause of childhood renal failure. To date, around 20 different genes, encoding primary cilia proteins, have been linked to NPHP. These contribute to one third of cases with NPHP while the majority of patients remain molecularly undiagnosed.

Methods: Whole exome sequencing (WES) was carried out on a two-year-old Lebanese boy with infantile NPHP characterized by multicystic kidney dysplasia, kidney insufficiency and enlarged kidneys in addition to chronic anemia. The candidate variant, detected by WES, was then tested in the patient and his parents by Sanger sequencing. Copy number variations analysis (CNV) was subsequently performed in the proband.

Results: Our studies enabled the detection of a heterozygous de-novo variant in NEK8 (NM_178170: pArg45Trp) in the proband. CNV analysis excluded the presence of big deletions or insertions in this gene.

Conclusion: In conclusion, here we report a de-novo heterozygous variant in the NEK8 gene in infantile NPHP. This variant was detected at a de-novo state in a patient presenting with the same clinical features as the proband (and reported in VarSome). This suggests that autosomal dominant forms of NEK8-linked nephropathies may exist. Reporting further patients is essential to confirm these findings and assess whether dominant forms of the disease are restricted to a specific mutational spot or are linked to variants scattered throughout the NEK8 gene.

References: NA.

Grants: NA.

Conflict of Interest: None declared.

P04.025.D Lipid metabolism is altered in alpha-1 antitrypsin deficiency derived-patients organoids

Sara Perez-Luz ¹, Jaanam Lalchandani¹, Nerea Matamala¹, Iago Justo², Alberto Marcacuzco², Cristina Garfia², Loreto Hierro Llanillo³, Gema Gomez-Mariano¹, Beatriz Martinez-Delgado¹

¹Instituto de Investigación de Enfermedades Raras, Molecular Genetics, Madrid, Spain; ²Hospital 12 de Octubre, Madrid, Spain; ³Hospital La Paz, Madrid, Spain

Background/Objectives: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder due to mutations in SERPINA1 gene presenting clinical manifestations in liver and lungs. More than 90% of severe deficiency patients are homozygous for PiZ (Glu342Lys) mutation located in exon 5, which produces an altered protein (AAT-Z) prone to accumulate in hepatocytes. The aggregated polymers avoid protein secretion into the circulation, resulting in plasma levels 10% to 15% of the levels of normal M homozygous while they related to different hepatic alterations such as fibrosis, hepatocarcinome or lipid alterations.

Methods: We have used hepatic organoids derived from patient homozygous for the Z mutation and HepG2 cells overexpressing Z-AAT to verify the relationship among AAT-Z protein and lipid accumulation by detecting neutral lipids using oil red O staining. In addition, we performed a lipidomic analysis to determine the lipidic species accumulated in our model as well as a transcriptomic study to reveal differential expressed genes, which could explain the aforementioned results.

Results: Our results show how Z-AAT protein accumulation triggered an increase in lipid content in hepatocytes and identified specifically three species presenting a more notably increment as measured by mass spectrography. Furthemore, the transcriptomic analysis expose several genes somehow related to lipid metabolism whose variation suggest involvement in such lipid deposits.

Conclusion: AAT polymers are associated to intracellular lipid accumulation mainly because the altered expression of lipogenic genes without ruling out the possibility that lipid accumulation could be cause, as well as consequence, and hence responsible of some clinical features associated with DAAT.

References:.

Grants: AESI/PI/307/20.

Conflict of Interest: None declared.

P04.027.B Benefits of whole exome sequencing to advance the genetic diagnosis in patients with differences (disorders) of sex development

Hannes Syryn ¹, Hannah Verdin¹, Julie Van De Velde¹, Marianne Becker², Cécile Brachet³, Marieke den Brinker⁴, Sylvia Depoorter⁵, Julie Fudvoye⁶, Daniel Klink⁷, Philippe Lysy^8;8, Guy Massa⁹, Nele Reynaert¹⁰, Anne Rochtus¹⁰, Willem Staels¹¹, Marlies Van Loocke¹⁰, Martine Cools¹², Elfride De Baere¹

¹Ghent University Hospital, Center for Medical Genetics, Department of Biomolecular Medicine, Ghent, Belgium; ²Centre Hospitalier de Luxembourg, Pediatric Endocrinology and Diabetology (DECCP), Luxembourg, Luxembourg; ³Hôpital Universitaire des Enfants Reine Fabiola, Paediatric Endocrinology Unit, Brussels, Belgium; ⁴Antwerp University Hospital, Department of Paediatric Endocrinology, Antwerp, Belgium; ⁵General Hospital Sint-Jan Bruges-Ostend, Department of Child Endocrinology, Bruges, Belgium; ⁶CHU de Liège, Department of Pediatrics, Liège, Belgium; ⁷ZNA Queen Paola Children’s Hospital, Division of Pediatric Endocrinology and Diabetes, Antwerp, Belgium; ⁸Cliniques universitaires Saint Luc, Pediatric Endocrinology, Brussels, Belgium; ⁹Jessa Hospital, Department of Pediatric Endocrinology and Diabetology, Hasselt, Belgium; ¹⁰University Hospitals Leuven, Department of Paediatric Endocrinology, Leuven, Belgium; ¹¹University Hospital of Brussels, Division of Pediatric Endocrinology, Jette, Belgium; ¹²Ghent University Hospital, Department of Internal Medicine and Pediatrics, Ghent, Belgium

Background/Objectives: Differences of sex development (DSD) are heterogeneous conditions affecting the development of chromosomal, gonadal or anatomical sex. Although over 75 genes have been associated with DSD, the diagnostic yield of whole exome sequencing (WES) studies is typically not higher than 35% in a clinical setting. Here, we investigated the benefits of WES for the genetic diagnosis in patients with DSD.

Methods: Between 2016 and 2022, 144 unrelated index patients with a clinical diagnosis of DSD or the broader DSD umbrella underwent WES-based panel testing interrogating the coding regions of 130 genes implicated in DSD, primary ovarian insufficiency and hypogonadotropic hypogonadism. Variants were extracted and classified according to the ACMG guidelines. Copy number variant (CNV) analysis was performed using the ExomeDepth algorithm.

Results: In 13% of patients, we identified a likely pathogenic (LP) or pathogenic (P) rare variant in 12 distinct DSD genes, including AR (6), NR5A1 (2), WT1 (2), ATRX, CYP21A2, DHX37, HSD3B2, HSD17B3, RXFP2, SRD5A2, SRY, and TXNRD2. The majority are sequence variants; four defects are CNVs identified using ExomeDepth. Interestingly, in two brothers displaying bilateral cryptorchidism and infertility an intragenic RXFP2 deletion was found to occur in trans with a heterozygous missense variant, corroborating its role in familial bilateral cryptorchidism.

Conclusion: We demonstrate the benefit of WES-based genetic testing of DSD in a clinical context. The low detection rate emphasizes the need for more stringent inclusion criteria on the one hand and for advanced genome analysis to solve missing heritability in this condition.

References:.

Grants: BESPEED, FWO1802220N, FWO1801018N.

Conflict of Interest: None declared.

P04.028.C Unique pipeline for the assessment of novel genetic variants leads to confirmation of PCD diagnosis

Nina Stevanovic ¹, Marina Andjelkovic¹, Anita Skakic¹, Vesna Spasovski¹, Maja Stojiljkovic¹, Marina Parezanovic¹, Milena Ugrin¹, Sonja Pavlovic¹

¹Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia

Background/Objectives: Primary ciliary dyskinesia (PCD) is a disease caused by impaired ciliary motility and mainly affects the lungs and reproductive organs. Inheritance is autosomal recessive and X-linked with more than 40 disease-causing genes, wherefore PCD patients have diverse clinical manifestations, thus making diagnosis difficult. The utility of next-generation sequencing (NGS) technology for diagnostic purposes allows a better understanding of the PCD genetic background. However, the identification of specific disease-causing variants is challenging. The objective of this study was to create a unique guideline that will enable the standardization of the assessment of novel variants within PCD associated genes.

Methods: The study included designing a pipeline for the classification of the rare genetic variants detected using NGS. The pipeline included in silico (translation, 3D-model, protein-protein interactions, sequence conservation, posttranslational modifications) and functional analysis (expressional analysis, Western Blot) of the variants.

Results: The designed pipeline consists of three steps: sequencing, detection, and identification of genes/variants; classification of variants according to their effect; and variant characterization using in silico structural and functional analysis. The pipeline was validated by the analysis of the variants detected in a disease-causing gene (DNAI1) and the novel candidate gene (SPAG16).

Conclusion: The application of the pipeline resulted in the identification of disease-causing variants, as well as pathogenicity validation, through the analysis on transcriptional, translational, and posttranslational levels.The application of created pipeline leads to the confirmation of PCD diagnosis and enables a shift from candidate to disease-causing gene.

References:.

Grants: This work was funded by MESTD, Republic of Serbia(451-03-68/2022-14/200042).

Conflict of Interest: None declared.

P04.029.D TSHB R75G is a founder variant and prevalent cause of low or undetectable TSH in Indian Jews

Marina Eskin-Schwartz ^1;2, David Shaki^3;4, Noam Hadar⁵, Emily Bosin⁶, Lior Carmon^2;3, Samuel Refetoff⁷, Eli Hershkovitz^2;3, Ohad Shmuel Birk^1;5, Alon Haim^2;8

¹Soroka Medical Center, Genetics Institute, Beer Sheva, Israel; ²Ben-Gurion University of the Negev, the Faculty of Health Sciences, Beer Sheva, Israel; ³Soroka Medical Center, Pediatric Endocrinology Unit, Beer Sheva, Israel; ⁴Ben-Gurion University of the Negev, the Faculty of Health Sciences, Beer Sheva, Israel; ⁵Ben-Gurion University of the Negev, the Morris Kahn Laboratory of Human Genetics, the Faculty of Health Sciences and National Institute for Biotechnology in the Negev, Beer Sheva, Israel; ⁶Soroka Medical Center, Endocrinology Laboratory, Beer Sheva, Israel; ⁷The University of Chicago, Departments of Medicine and Pediatrics and the Committee on Genetics, Chicago, Illinois, United States; ³Soroka Medical Center, Pediatric Endocrinology Unit, Beer Sheva, Israel

Background/Objectives: Bi-allelic loss-of-function mutations in TSHB, encoding the beta-subunit of TSH, cause congenital hypothyroidism. Homozygosity for the TSHB p.R75G variant, previously described in South Asian individuals, does not alter TSH function, but abrogates its detection by some immune-detection-based platforms, leading to erroneous diagnosis of hyperthyroidism. We set out to identify and determine carrier rate of the p.R75G variant among clinically euthyroid Bene Israel Indian Jews, to examine possible founder origin of this variant worldwide and to determine phenotypic effects of its heterozygosity.

Methods: Molecular genetic studies of Bene Israel Jews and comparative studies with South Asian cohort were performed. TSHB p.R75G variant was tested by Sanger sequencing and RFLP. Haplotype analysis in the vicinity of the TSHB gene was performed using SNP arrays.

Results: Clinically euthyroid individuals with low or undetectable TSH levels from three apparently unrelated Israeli Jewish families of Bene Israel ethnicity, originating from the Mumbai region of India, were found heterozygous or homozygous for the p.R75G TSHB variant. Extremely high carrier rate of p.R75G TSHB in Bene Israel Indian Jews (~4%) was observed. A haplotype block of 239.7kB in the vicinity of TSHB shared by Bene Israel and individuals of South Asian origin was detected.

Conclusion: Our findings highlight the high prevalence of the R75G TSHB variant in euthyroid Bene Israel Indian Jews, demonstrate that heterozygosity of this variant can cause erroneous detection of subnormal TSH levels, and show that R75G TSHB is an ancient founder variant, delineating shared ancestry of its carriers.

References:.

Grants:.

Conflict of Interest: None declared.

P04.030.A Genotype-phenotype correlation in patients with PHEX-related hypophosphatemia: identification of novel variants and a case of mosaicism

Irene Ambrosetti ^1;2, Giulia Olivucci^1;2, Claudio Graziano³, Giulia Lanzoni¹, Giulia Severi¹, Enrico Ambrosini¹, Sofia Cesarini^1;2, Elena Bonora², Cesare Rossi¹, Marco seri^1;2

¹U.O. Genetica Medica - IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; ²Dipartimento di Scienze Mediche e Chirurgiche, Alma Mater Studiorum - Università di Bologna, Italy, Bologna, Italy; ³U.O. Genetica Medica - AUSL della Romagna - Cesena (FC), Cesena, Italy

Background/Objectives: X-linked hypophosphatemia (XLH) is an X-linked dominant disorder caused by inactivating mutations in the PHEX gene. The clinical phenotype ranges from isolated hypophosphatemia to severe manifestations of rickets, such as bowing of the lower extremities, bone pain, low stature, and dental anomalies.

Methods: Medical records from 2008 to 2021 were retrospectively reviewed to collect clinical, biochemical and molecular findings for patients carrying pathogenic PHEX variants, detected through Sanger or Next Generation Sequencing on DNA extracted from peripheral blood lymphocytes.

Results: We report a series of seven unrelated patients from our clinical center carrying pathogenic PHEX variants. In particular, we identified one intragenic deletion and five distinct nonsense or frameshift variants (one of which was present in two unrelated patients); three of the variants we identified were novel and one was present as a somatic mosaicism in a male patient. To our knowledge, only 12 cases of somatic mosaicism for a PHEX mutation in hemizygous males have been reported in the literature; we reviewed their clinical and biochemical features, confirming the wide phenotypic variability among mosaic male patients with XLH.

Conclusion: While some reports suggested a dosage effect, with a milder clinical phenotype in mosaic males, our case and a review of the literature show a broad range of severity in these patients, overlapping that of male and female patients with germline mutations.

References:.

Grants:.

Conflict of Interest: None declared.

P04.031.B Exome sequencing in adult nephropathy of unknown origin: a population study

Alice DOREILLE^1;2, Yannis Lombardi¹, Hugo Garcia¹, Radoslava Saraeva-Lamri³, Marine Dancer³, Laure Raymond³, Laurent Mesnard ^1;2

¹Tenon Hospital, Paris, France; ²Sorbonne Université, Paris, France; ³Eurofins Biomnis, Génétique, Lyon, France

Background/Objectives: Despite the importance of an etiological diagnosis, up to 20% of nephropathies remain of undetermined origin. Increased availability of genome-wide testing, such as exome sequencing (ES), may allow many cases of undetermined kidney disease to be reclassified. Here, we report the largest French cohort of ES in adult nephrology.

Methods: Between September 2018 and February 2021, 538 unrelated patients underwent an ES (Twist Human Core Exome kit, 100 base pair-end sequenced on NextSeq500- Illumina). CNV research was done from the same assay, calling was based on model built by GATK4. Variant co-segregation was investigated by Sanger sequencing in relatives when needed.

Results: Of 538 unrelated patients, ES resulted in a molecular diagnosis in 135 patients (diagnostic yield of 25%). Seven patients had a conclusive result with identification of a pathogenic CNV (5% of positive diagnoses). The mean age of the patients was 43 years [± 13]. 80% of the patients were analysed in solo exome. The genetic diagnosis had various consequences: allowing for a pre-symptomatic diagnosis (63%), assisting in the selection of a potential related living donor (7.5%), not considering immunosuppressive therapy (11%), ruling-out potential recurrence on the graft (20%), prescribing further investigations in the context of retrophenotyping (36%).

Conclusion: With a high diagnostic yield, major clinical and therapeutic consequences and a measured cost, our study shows the interest of ES with analysis integrating CNV detection in indeterminate adult nephropathies. The diagnoses made were often unexpected, validating the interest of a genome-wide approach.

References:.

Grants: None.

Conflict of Interest: None declared.

P04.032.C Exome sequencing in isolated population: new insights into nephropathies of unknown origin in New Caledonia

David De Saint Gilles¹, nicolas quirin², Marine Dancer³, Nadège BATAIL ³, yves doussy⁴, thomas lamy², Laure Raymond³, Laurent Mesnard¹, Alice DOREILLE¹

¹Tenon Hospital, Paris, France; ²Medipole, Dumbéa Sur Mer, New Caledonia; ³Eurofins Biomnis, Lyon, France; ⁴ATIR, Dumbéa, New Caledonia

Background/Objectives: Recently, exome sequencing (ES) has shown a good diagnostic yield in adult nephrology. New Caledonia has a very high incidence of Chronic Kidney Disease (CKD) with a clear predominance of indeterminate nephropathies (40%) and a young population (60% of dialyzed patients < 65 y/o). Given its insularity, genetics offer very interesting perspectives for New Caledonian patients with nephropathy of unknown origin.

Methods: Since November 2019, ES is offered to adult patients with CKD of unclear origin with early-onset (< 45 y/o) or with a family history of kidney disease, regardless of age.

Results: From November 2019 to January 2022, 84 unrelated patients received a solo ES as a first-line exploration. The sequenced population was predominantly of Melanesian (60%) and Polynesian (30%) origin. ES identified pathogenic diagnostic variants in 15 patients (19%). Twenty-two patients (26%) presented a variant of unknown significance (VUS). None of the 84 patients carried APOL1 risk alleles. This exome-wide approach had major consequences (allow a new look on the whole clinical picture, description of new pathogenic variants, selection of a related living donor for 15 patients, a renal biopsy could be avoided in one relative).

Conclusion: With a diagnostic yield of 19% and major clinical impact, nephropathies of unknown origin in New Caledonia appear as an interesting indication of ES. As the Oceanian populations are absent from Gnomad most variants required co-segregation and retrophenotyping studies to reach ACMG 4/5 rank. Thus an important number of variants remained VUS.

References:.

Grants:.

Conflict of Interest: None declared.

P04.033.D Stringent variant assessment in pulmonary fibrosis reveals a stronger genetic predisposition in female than in male patients

Samuel Wilkinson ¹, Amy Slater², Matthew Edwards³, Shibu John⁴, Deborah Morris-Rosendahl⁵

¹Royal Brompton & Harefield NHS Foundation Trust, Clinical Genetics and Genomics, London, United Kingdom; ¹Royal Brompton & Harefield NHS Foundation Trust, Clinical Genetics and Genomics, London, United Kingdom; ³Royal Brompton & Harefield NHS Foundation Trust, London, United Kingdom; ¹Royal Brompton & Harefield NHS Foundation Trust, Clinical Genetics and Genomics, London, United Kingdom; ¹Royal Brompton & Harefield NHS Foundation Trust, Clinical Genetics and Genomics, London, United Kingdom

Background/Objectives: Pulmonary fibrosis (PF) is a severely progressive and fatal fibrotic interstitial lung disease, with a late age of onset (55-75 years) and an average survival rate of 3 years post diagnosis. The phenotype is strongly associated with a reduction in telomere length and pathogenic variants in telomere- associated genes, have been found in approximately 8-15% of familial PF cases. Genetic testing provides diagnostic accuracy in symptomatic patients, the possibility of predictive testing in family members and can aid in disease course prognostication and risk stratification when considering lung transplantation.

Methods: 180 adult patients with PF (mean age 59 years, 102 female and 78 male) were referred for genetic testing and sequenced on our custom “Respigene” panel for inherited respiratory conditions. Analysis was bioinformatically targeted to 25 genes associated with familial pulmonary fibrosis (FPF).

Results: Sixteen patients (9%, 11 female and 5 male) were found to have a pathogenic or likely pathogenic variant in the RTEL1, TERT, PARN or SFTPC genes; 48 patients (27%, 27 female, 21 male) harboured at least one variant of uncertain significance (VUS). The MUC5B rs3570590 risk-associated variant was detected in 23.69% of PF patients, compared to 18.94% in patients with a non-ILD respiratory condition.

Conclusion: Female patients appear more likely to have a genetic cause to their PF than male patients. Functional studies are required to refine the pathogenicity of the many VUSs detected. The presence of the MUC5B risk allele at a significantly higher frequency in PF patients than in patients with other respiratory conditions, was confirmed.

References:.

Grants:.

Conflict of Interest: None declared.

P02 Skeletal, Connective Tissue, Ectodermal and Skin Disorders

P05.001.B Disruption of zfhx4 leads to defects in zebrafish craniofacial development matching human characteristics of nonsyndromic cleft lip with cleft palate

Selina Hölzel ^1;2, Nina Ishorst^1;2, Carola Greve^1;3, Franziska Degenhardt^1;4, Öznur Yilmaz², Tobias Lindenberg², Dmitriy Drichel⁵, Carlo Maj⁶, Michael Nothnagel^5;7, Jayne Y. Herir-Kwa⁸, Joris A. Veltman^8;9, Berina Zametica¹, Teresa Kruse¹⁰, Stefanie Nowak¹, Carine Carels^11;12, Iris van Rooij¹³, Kerstin Ludwig¹, Benjamin Odermatt², Elisabeth Mangold¹

¹Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; ²Institute of Anatomy, Division of Neuroanatomy, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; ³LOEWE Centre for Translational Biodiversity Genomics, Frankfurt am Main (current address), Frankfurt am Main, Germany; ⁴Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital Essen, University of Duisburg-Essen (current address), Duisburg, Germany; ⁵Cologne Center for Genomics, University of Cologne, Cologne, Germany; ⁶Institute for Genomic Statistics and Bioinformatics, University of Bonn, School of Medicine and University Hospital Bonn, Bonn, Germany; ⁷University Hospital Cologne, Cologne, Germany; ⁸Department of Human Genetics, Donders Institute, Radboud University Medical Center, Nijmegen, Netherlands; ⁹Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne, United Kingdom; ¹⁰Department of Orthodontics, University of Cologne, Cologne, Germany; ¹¹Department of Human Genetics, KU Leuven, Leuven, Belgium; ¹²Orthodontics, University Hospitals KU Leuven, Leuven, Belgium; ¹³Department for Health Evidence, Radboud Institute for Health Sciences, Radboud university medical center, Nijmegen, Netherlands

Background/Objectives: Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is a frequent congenital anomaly with high heritability estimates.^1,2 Despite the discovery of several common nsCL/P risk loci, major fractions of its complex genetic background yet remain unrevealed.³.

Methods: In order to identify copy number variants that might be causal for nsCL/P we reanalyzed an exome sequencing dataset comprising 50 Central European nsCL/P trios. Here, we identified a heterozygous 86 kb de novo deletion that affects exons 3-11 of 11 of the ZFHX4 gene. ZFHX4 encodes the transcription factor Zinc Finger Homeobox 4 which recently has been shown to be enriched for de novo mutations in patients with nonsyndromic orofacial clefting.⁴ To functionally characterize the role of ZFHX4 in craniofacial development, we knocked down the zebrafish orthologue zfhx4 in wild type zebrafish larvae (zfl) by a translational blocking morpholino (TB MO).

Results: Cartilage staining of TB MO injected zfl at 4 days post-fertilization showed underdeveloped and abnormally shaped cartilaginous jaw and ethmoid plate structures, and therefore matched human characteristics of nsCL/P. Moreover, preliminary data of transient CRISPR/Cas9 zfhx4 F_o-knockout in zfl replicated these observations.

Conclusion: In conclusion, our human genetic findings and subsequent functional studies in zfl indicate the importance of ZFHX4 in the process of craniofacial development and confirm its role as susceptibility gene for nsCL/P.

References: ¹Mangold, et al., 2011. Trends Mol Med0., ²Grosen, et al., 2011. Epidemiology., ³Welzenbach, et al., 2021. HGG Advances., ⁴Bishop, et al., 2020. Am J Hum Genet.

Grants: S.H.: BonnNI Q-614.1254.; E.M.: DFG, MA 2546/6-1.; K.U.L.: DFG, LU 1944/3-1.

Conflict of Interest: None declared.

P05.002.C LRP4 intracellular variant may lead to high bone mass with bone fragility: a functional analysis

Caroline Caetano da Silva ¹, Genevieve Baujat², Adrien Bloch¹, Manon Ricquebourg¹, Kelvin Valmorin¹, Martine Cohen-Solal¹, Corinne Collet^1;3

¹INSERM U1132, Paris, France; ²INSERM UMR 1163, IMAGINE Institute, Necker Enfants Malades Hospital, Paris, France; ³UF de Génétique Moléculaire, Hôpital Robert Debré, Département de Génétique, Paris, France

Background/Objectives: The LRP4 receptor is associated to bone and musculoskeletal diseases¹. We identified a novel missense variant located in the intracellular domain in LRP4 in a 6-years-old patient, with an atypical bone phenotype characterized by osteocondensation and fractures. Another patient presented a rare LRP4 variant in extracellular domain with high bone mass, without fractures. Our goal was to confirm the pathogenicity of the variants and to unravel the dysregulates pathways.

Methods: A CRISPR-Cas9 approach was used to generate mutated osteosarcoma cells (U2OS) for functional analysis.

Results: We generated two heterozygous mutant cell lines, one with the mutation in intracellular domain c.5317_5320del p.(Thr1773Argfs*3) and other in the extracellular domain c.4699_4727del p.(Arg1567Glnfs*7). Both mutant cells presented a slightly decrease in apoptosis compared to control. An increased mineralization was observed in the intracellular mutant, as well a higher RUNX2 and ALPL expression by RT-qPCR. The extracellular mutant had a lower mineralization/differentiation and an increase in BMP2 expression. RNAseq analysis in both mutant cells revealed 1700 and 1400 under and over expressed genes for intracellular mutant and 100 and 40 under and over expressed genes for extracellular mutant.

Conclusion: Mineralization and expression of target genes differ according to the variant localization, showing that the pathways and the pathogenicity are not the same. This approach will allow to study new functions of LRP4 in bone.

References: 1. Shen, et al. “LRP4 in neuromuscular junction and bone development and diseases.” Bone (2015). https://doi.org/10.1016/j.bone.2015.05.012.

Grants: European Union’s Horizon 2020, grant agreement 766347.

Conflict of Interest: None declared.

P05.003.D Developmental genomics on split-hand/foot malformation, further indication of allelic series and gene dosage effects

Ruizhi Duan ¹, Hadia Hijazi^1;2, Haowei Du¹, Jawid Fatih¹, Christopher Grochowski¹, Shalnini Jhangiani³, Jennifer Posey¹, Zeynep Coban Akdemir^1;4, V. Reid Sutton¹, Claudia Carvalho^1;5, Davut Pehlivan^1;6, James Lupski^1;3;6;7

¹Baylor College of Medicine, Department of molecular and human genetics, Houston, United States; ²Johns Hopkins University, McKusick-Nathans Institute of Genetic Medicine, Baltimore, United States; ³Baylor College of Medicine, Human Genome Sequencing Center, Houston, United States; ⁴UTHealth School of Public Health, Houston, United States; ⁵Pacific Northwest Research Institute, Seattle, United States; ⁶Texas Children’s Hospital, Houston, United States; ⁷Baylor College of Medicine, Department of Pediatrics, Houston, United States

Background/Objectives: Split-hand/foot malformation (SHFM) is a rare form of congenital limb anomaly with unclear molecular etiology, presumably because of a failure to maintain the central portion of apical ectodermal ridge (AER) during limb bud proximal-distal (P/D) axial development.

Methods: Family-based genomics and rare variant analyses were implemented using exome sequencing (ES) combined with whole-genome array-based comparative genomic hybridization (aCGH) to investigate 5 SHFM cases that originated from diverse world geographical populations.

Results: We identified one novel pathogenic variant of WNT10B in two unrelated Turkish families; one family with a de novo 7Mb large chromosomal deletion encompassing the entire HOXD territory; two families involving copy number gain of Chr17p13.3 including BHLHA9. Notably, breakpoint junction analyses for all three CNV alleles provided evidence for microhomology-mediated break-induced replication (MMBIR) as the putative molecular mutational mechanism facilitated by Alu/Alu-mediated rearrangement (AAMR). In contrast to heterozygous duplication of BHLHA9 associated with SHFLD3, homozygous duplication CNV was observed in association with the Gollop-Wolfgang Complex, implicating semi-dominant inheritance.

Conclusion: Genes acting on limb patterning are sensitive to a gene dosage effect and often associated with an allelic series. Novel variant identifications involving these genes/loci provide potential insights that further elucidate the developmental genomics of SHFM. We extend a gene dosage model which could potentially, in an adjuvant way, assist interpreting the interconnections among allelic series, clinical phenotypical severity, and reduced penetrance of the BHLHA9-related CLM spectrum.

References: da Rocha et al. Clinical Genetics. 2021; 100(5):615-623.

Grants: BHCMG, UM1 HG006542; BCM-GREGoR; U01 HG011758; NIH R35NS1050.

Conflict of Interest: Ruizhi Duan: None declared, Hadia Hijazi: None declared, Haowei Du: None declared, Jawid Fatih: None declared, Christopher Grochowski: None declared, Shalnini Jhangiani: None declared, Jennifer Posey J.E.P. was supported by NHGRI K08 HG008986, and is the principal investigator of Baylor College of Medicine Genomics Research Elucidates Genetics of Rare (BCM-GREGoR; U01 HG011758), Zeynep Coban Akdemir: None declared, V. Reid Sutton: None declared, Claudia Carvalho: None declared, Davut Pehlivan D.P. is supported by the International Rett Syndrome Foundation (IRSF grant #3701-1), James Lupski United States (U.S.) National Human Genome Research Institute (NHGRI) and National Heart Lung and Blood Institute (NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542) and Baylor College of Medicine Genomics Research Elucidates Genetics of Rare (BCM-GREGoR; U01 HG011758), U.S. National Institute of Neurological Disorders and Stroke (NINDS) (R35NS105078 to J.R.L.); and Muscular Dystrophy Association (MDA), (512848 to J.R.L.) and Spastic Paraplegia Foundation (SPF) (to J.R.L.), J.R.L. has stock ownership in 23andMe, J.R.L. is a paid consultant for Regeneron Genetics Center; The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics (BG); J.R.L. serves on the Scientific Advisory Board (SAB) of BG., J.R.L. is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders and bacterial genomic fingerprinting.

P05.004.A Weill-Marchesani syndrome: natural history and genotype-phenotype correlation from 18 cases

Pauline Marzin ^1;2, jean-luc alessandri³, Klaus Dieterich⁴, Christine Francannet⁵, Sandra Mercier⁶, Caroline Michot^1;2, Oana Moldovan⁷, Gianmaria Miolo⁸, Massimiliano Rossi⁹, Sophie Rondeau^10;11, Valérie Cormier-Daire^1;2

¹Institut Imagine, AP-HP, Hôpital Necker-Enfants Malades, Fédération de Génétique e, Paris, France; ²Université de Paris, UMR1163, INSERM, Paris, France; ³CHU de la Réunion - Hôpital Félix Guyon, Bellepierre, Pôle Femme-Mère-Enfants, Saint-Denis, France; ⁴Univ. Grenoble Alpes, Inserm, U1209, CHU Grenoble Alpes, Institut of Advanced Biosciences, Grenoble, France; ⁵CHU de Clermont-Ferrand - Hôpital d’Estaing, Service de génétique médicale, Pôle Femme et Enfant, Clermont-Ferrand, France; ⁶CHU de Nantes - Hôtel Dieu, Service de génétique médicale - Unité de Génétique clinique, Nantes, France; ⁷Hospital de Sta. Maria - Centro Hospitalar de Lisboa Norte, EPE, Serviço de Genética Médica (Centro de desenvolvimento infantil), Lisbones, Portugal; ⁸Azienda Ospedaliera Santa Maria degli Angeli, Dipartimento di Medicina di Laboratorio di Citogenetica e Genetica Molecolare, Porderone, Italy; ⁹CHU de Lyon HCL - GH Est-Hôpital Femme Mère Enfant, Service de génétique, Bron Cedex, France; ¹⁰Institut Imagine, AP-HP, Hôpital Necker-Enfants Malades, Fédération de Génétique, Paris, France; ¹¹Université de Paris, UMR1163, INSERM, Paris, France

Background/Objectives: Weill-Marchesani syndrome (WMS) belongs to the acromelic dysplasia group, defined by short stature, brachydactyly and progressive joint stiffness. WMS is characterized by ophthalmological abnormalities such as microspherophakia, severe myopia secondary to lens shape anomaly, and lens ectopia. Cardiovascular defects have been reported. Monoallelic variations in FBN1 are associated with a dominant WMS, while biallelic variations in ADAMTS10, ADAMTS17 and LTBP2 are responsible for recessive WMS. These four genes code for components of the extracellular matrix.

Objectives: Natural history description of WMS and genotype-phenotype correlation establishment.

Methods: Retrospective multicenter study. Inclusion criteria: clinical diagnosis of WMS with identified mutations.

Results: 18 individuals, 11 females and 7 males, with a mean age of 20 years (from 1.5 to 59 years) were included. Eight have a mutation in FBN1 without specific localization, 6 in ADAMTS10, 4 in ADAMTS17, none in LTBP2. All individuals presented with eye anomalies including lens ectopia (10/18), high myopia (9/18), microspherophakia (8/18), glaucoma (5/18), and cataract (4/18). 10/18 have a short stature (-2 to -4 DS), 12/18 joints limitation, 12/18 brachydactyly. 7/18 individuals have cardiac defect such as valvulopathy (pulmonary stenosis 2/7, aortic stenosis 1/7, mitral insufficiency 3/7, mitral thickening 1/7). Other manifestations included: recurrent laryngitis (1), hepatomegaly (1), carpal tunnel syndrome detected at 22 years of age (1).

Conclusion: Apart from ophthalmological findings which are mandatory for diagnosis, phenotype of WMS seems to be more variable than initially described, notably, only half of patients presented with short stature. No genotype-phenotype correlation emerges from this cohort.

References:.

Grants:.

Conflict of Interest: None declared.

P05.005.B Potential therapeutic targets for hypermobile Ehlers-Danlos syndrome from proteome and secretome analyses of patients’ dermal fibroblasts

Valeria Cinquina ¹, Nicola chiarelli¹, Nicoletta Zoppi¹, Daniele Capitanio², Virginia Bassi¹, Marina Venturini³, Cecilia Gelfi², Marco Ritelli¹, Marina Colombi¹

¹University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy; ²University of Milan, Department of Biomedical Sciences for Health, Milano, Italy; ³Spedali Civili University Hospital, Department of Clinical and Experimental Sciences, Brescia, Italy

Background/Objectives: Hypermobile Ehlers-Danlos syndrome (hEDS) is a connective tissue disorder without a known genetic etiology and specific therapies, which is mainly characterized by generalized joint hypermobility and musculoskeletal complaints. Patients’ dermal fibroblasts exhibit a widespread extracellular matrix (ECM) disarray and myofibroblast-like characteristics including α-SMA cytoskeleton organization. Control fibroblasts treated with hEDS cells-derived conditioned media (hEDS-CM) acquire this pathological phenotype.

Methods: A comprehensive proteomic study based on top-down and bottom-up approaches was performed by comparing the intra- and extracellular proteome of patients’ and controls’ fibroblasts. We also performed in vitro studies to define the effect of the matrix metalloproteinases (MMPs) inhibitor doxycycline on ECM organization and fibroblast-to-myofibroblast transition (FMT).

Results: Cellular proteome analyses unveiled protein changes essential to actin cytoskeleton dynamics, energy metabolism and redox balance, proteostasis, intracellular trafficking and secretion. Secretome profiling of hEDS-CM mainly revealed an MMPs dysfunction as a possible disease driver by causing a detrimental feedback loop of excessive ECM degradation coupled with myofibroblast differentiation. Doxycycline-mediated MMPs inhibition rescued in patients’ cells a control-like ECM organization and induced a partial reversal of their myofibroblast-like phenotype. Furthermore, the addition of doxycycline to hEDS-CM abolishes its capability to induce in control cells ECM disarray and FMT.

Conclusion: Altogether, these data provide evidence on several putative disease targets for the development of therapeutic strategies with a potential benefit for patients’ management.

References:.

Grants: This research was funded by The Ehlers-Danlos Society (grant numbers: 2018.02c. LOI.26; Molecular Studies in hEDS and HSD $1 Million Grant).

Conflict of Interest: Valeria Cinquina: None declared, Nicola chiarelli: None declared, Nicoletta Zoppi: None declared, Daniele Capitanio: None declared, Virginia Bassi: None declared, Marina Venturini: None declared, Cecilia Gelfi: None declared, Marco Ritelli: None declared, Marina Colombi Principal investigator funded by The Ehlers-Danlos Society (grant numbers: 2018.02c. LOI.26; Molecular Studies in hEDS and HSD $1 Million Grant).

P05.006.C Mutation spectrum in Bulgarian patients with skeletal tissue disorders, revealed by clinical exome sequencing

Ivanka Dimova ¹, Kalina Mihova², Kunka Kamenarova², Nevyana Ivanova², Darina Kachakova-Yordanova², Radka Kaneva²

¹Medical University Sofia, Sofia, Bulgaria; ²Medical University Sofia, Molecular Medicine Center, Sofia, Bulgaria

Background/Objectives: Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. We aimed in revealing the type and frequency of genetic mutations in Bulgarian patients with skeletal tissue disorders.

Methods: We performed next generation sequencing (NGS) by using TruSight One kit (clinical exome) and MiSeq platform of Illumina in 26 patients with clinical diagnosis, suspicious for skeletal tissue disorder.

Results: We found pathogenic and likely pathogenic variants in 10 and 2 patients, respectively. Six were missense mutation, 3 – nonsense, 2 - frameshift and 1 splicing variant. The highest frequency of mutations was detected for Osteogenesis imperfecta. The Table below represents our findings.

Gene	Gene variant	Genetic diagnosis
Pathogenic variants
COL1A1	c.252dupC (p.Glu85Argfs*84) c.581G>A (p.Gly194Asp) c.1251delC (p.Ser418Leufs*123) c.2644C>T (p. p.Arg882Ter)	Osteogenesis imperfecta
FBN1	c.8051+2T>A c.1546C>T (p.Arg516Ter)	Marfan and associated syndromes
COL2A1	c.1510G>A (p.Gly504Ser)
FGFR2	c.314A>G (p. Tyr105Cys)	Crouzon syndrome
FGFR3	c.1626C>G (p.Asn542Lys)	Hypochondroplasia-like phenotype
NSD1	c.1831C>T (p.Arg611Ter)	Sotos syndrome
Probably pathogenic variants
COL5A1	c.4819G>A (p.Gly1607Ser)	Ehlers-Danlos syndrome, classical type
SKI	c.1877A>T (p.Lys626Met)	Shprintzen-Goldberg syndrome

Conclusion: We put genetic diagnosis in 46% of the patients with clinical diagnosis of skeletal tissue disorder by using clinical exome sequencing. These findings demonstrate the utility of NGS testing for individuals with a suspected skeletal dysplasia or growth disorder.

References: Scocchia, A., et al. Diagnostic utility of next-generation sequencing-based panel testing in 543 patients with suspected skeletal dysplasia. Orphanet J Rare Dis 16, 412 (2021).

Grants: Д01-285-17.12.2019.

Д01-395-18.12.2020.

Д01-302-17.12.2021.

Conflict of Interest: None declared.

P05.007.D The familiar and the sporadic form of Trichorhinophalangeal syndrome- case reports

Lenka Horáková ¹, Gabriela Křečková¹, Monika Koudová¹, Marie Trková¹, Ivo Mařík², Jitka Štekrová³, Katerina Hirschfeldova³

¹GENNET - Center of Medical genetics and reproductive medicine, Prague, Czech Republic; ²Ambulatory Centre for Defects of Locomotor Apparatus, Prague, Czech Republic; ³First Faculty of Medicine Charles University, The Institute of Biology and Medical Genetics, Prague, Czech Republic

Background/Objectives: Trichorhinophalangeal syndrome type I (TRPS I) is a rare autosomal dominant disorder caused by mutation in the TRPS1 gene coding a transcriptional repressor of GATA-regulated genes. TRPS1 haploinsufficiency reduces activity of genes regulating the growth of bone and cartilage with clinical picture of TRPS I: abnormal bones in the fingers and toes, joint abnormalities, distinctive facial features and other symptoms (fine, sparse hair, pear-shaped nose, prominent ears, brachydactyly, cone shaped epiphyses of the phalanges, dystrophic nails, short stature, small breasts, hip dysplasia). We report 2 cases. The first case is the 16 years old girl with growth retardation and typical facial features. The proband´s mother manifested the same facial signs and. The 2nd sporadic case is 12 years old girl with similar facial signs, disproportion between leg length and scoliosis.

Methods: In the first case we carried out a NGS amplicon analysis of TRPS1 gene. In the second case SNP array (OmniExpress-24 v1.3 chip, Illumina) was performed and result was confirmed by MLPA (Salsa MLPA KIT Illumina P-228 TRPS1-LGS).

Results: In the first case a missense mutation c.2761C>T (p. Arg921Ter) in exon 6 of TRPS1 gene of maternal origin and in the second case de novo 155kb microdeletion involving exons 3-5 of TRPS1 gene were found.

Conclusion: Correct diagnosis based on characteristic facial features in combination with targeted genetic testing is essential for confirmation of diagnosis with adequate follow up and therapeutic care and is very important for possibility to provide appropriate genetic counselling.

References:.

Grants:.

Conflict of Interest: None declared.

P05.008.A Molecular characterization and investigation of the role of genetic variation in phenotypic variability and response to treatment in a large pediatric Marfan syndrome cohort

Josephina (Jeannette) Meester ¹, Silke Peeters¹, Lotte Van Den Heuvel¹, Aline Verstraeten¹, Lut Van Laer¹, Bart Loeys^1;2

¹University of Antwerp and Antwerp University Hospital, Center of Medical Genetics, Antwerp, Belgium; ²Radboud University Medical Center, Department of Human Genetics, Nijmegen, Netherlands

Background/Objectives: In a large cohort of 373 paediatric patients with Marfan syndrome (MFS) with a severe cardiovascular phenotype, we explored the proportion of patients with MFS with a pathogenic FBN1 variant and analysed whether the type/location of FBN1 variants was associated with specific clinical characteristics and response to treatment. Patients were recruited on the basis of the following criteria: aortic root z-score > 3, age 6 months to 25 years, no prior or planned surgery, and aortic root diameter < 5 cm.

Methods: Targeted resequencing and deletion/duplication testing of FBN1 and related genes were performed.

Results: We identified (likely) pathogenic FBN1 variants in 91% of patients. Ectopia lentis was more frequent in patients with dominant-negative (DN) variants (61%) than in those with haploinsufficient variants (27%). For DN FBN1 variants, the prevalence of ectopia lentis was highest in the N-terminal region (84%) and lowest in the C-terminal region (17%). The association with a more severe cardiovascular phenotype was not restricted to DN variants in the neonatal FBN1 region (exon 25-33) but was also seen in the variants in exons 26 to 49. No difference in the therapeutic response was detected between genotypes.

Conclusion: Important novel genotype-phenotype associations involving both cardiovascular and extra-cardiovascular manifestations were identified, and existing ones were confirmed. These findings have implications for prognostic counselling of families with MFS.

References: PMID: 35058154.

Grants: FWO Flanders: 12X8520N and G044720N.

Conflict of Interest: None declared.

P05.009.B Serum calcification propensity T50 associates with disease severity in patients with pseudoxanthoma elasticum

Lukas Nollet ^1;2;3, Matthias Van Gils^1;2;3, Suzanne Fischer⁴, Laurence Campens⁵, Swapna Karthik⁶, Andreas Pasch^6;7;8, Julie De Zaeytijd⁹, Bart Leroy^1;9;10, Daniel Devos¹¹, Tine De Backer⁵, Paul Coucke^1;2, Olivier Vanakker^1;2;3

¹Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; ²Department of Biomolecular Medicine, Ghent University, Ghent, Belgium; ³Ectopic Mineralization Research Group, Ghent, Belgium; ⁴Laboratory of Experimental Cancer Research, Ghent University, Ghent, Belgium; ⁵Department of Cardiology, Ghent University Hospital, Ghent, Belgium; ⁶Calciscon AG, Nidau, Switzerland; ⁷Institute of Physiology and Pathophysiology, Johannes Keppler University, Linz, Austria; ⁸Department of Nephrology, Lindenhofspital, Bern, Switzerland; ⁹Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium; ¹⁰Division of Ophthalmology, The Children’s Hospital of Philadelphia, Philadelphia, United States; ¹¹Department of Radiology, Ghent University Hospital, Ghent, Belgium

Background/Objectives: Pseudoxanthoma elasticum (PXE) is a currently intractable Mendelian disorder characterized by progressive ectopic calcification in the skin, eyes and arteries. Therapeutic trials in PXE are severely hampered by the lack of reliable biomarkers. Serum calcification propensity T50 is a novel blood test measuring the functional anti-calcifying buffer capacity of serum. Here, we evaluated T50 in PXE patients aiming to investigate its determinants and suitability as a potential biomarker for disease severity.

Methods: Fifty-seven PXE patients were included and demographic, clinical, imaging and biochemical data were collected from medical health records. PXE severity was assessed using Phenodex scores. T50 was measured using a validated, nephelometry-based assay. Multivariate models were then created to investigate T50 determinants and associations with disease severity.

Results: Mean age of patients was 45.2 years, 68.4% was female and mean serum T50 was 347 minutes. Multivariate regression analysis identified serum fetuin-A (p < 0.001), phosphorus (p = 0.007) and magnesium levels (p = 0.034) as significant determinants of T50, while no correlations were identified with serum calcium, eGFR, plasma PPi levels or ABCC6 genotype. After correction for covariates, T50 was found to be an independent predictor of ocular (p = 0.013), vascular (p = 0.013) and overall disease severity (p = 0.016) in PXE.

Conclusion: In this cross-sectional cohort study, shorter serum T50 – indicative of higher calcification propensity – associated with a more severe phenotype in PXE patients. This study indicates for the first time that serum T50 might be a reliable and clinically relevant biomarker in PXE and may act as a surrogate endpoint in future therapeutic trials.

References:.

Grants:.

Conflict of Interest: Lukas Nollet: None declared, Matthias Van Gils: None declared, Suzanne Fischer: None declared, Laurence Campens: None declared, Swapna Karthik Swapna Karthik is an employee of Calciscon AG., Swapna Karthik is a stockholder of Calciscon AG., Andreas Pasch Andreas Pasch is an employee of Calciscon AG., Andreas Pasch is a stockholder of Calciscon AG., Julie De Zaeytijd: None declared, Bart Leroy: None declared, Daniel Devos: None declared, Tine De Backer: None declared, Paul Coucke: None declared, Olivier Vanakker: None declared.

P05.010.C Genetic and functional studies of novel candidate genes for Psoriatic Arthritis Mutilans

Sailan Wang ¹, Pernilla Nikamo¹, Raquel Vaz¹, Fulya Taylan¹, Jesper Eisfeldt¹, Xiaowei Zheng¹, Leena Laasonen¹, Isabel Tapia-Paez¹, Mona Stahle¹

¹karolinska institutet, Stockholm, Sweden

Background/Objectives: Psoriatic arthritis mutilans (PAM) is the most severe and rare clinical subtype of psoriatic arthritis (PsA) characterized by severe destruction of the distal joints. The prevalence of PAM in the Nordic countries is estimated to 3.69 cases per million inhabitants.

Methods: In this project, we aim to study the genetic basis of PAM in a unique Nordic PAM cohort consisting of 61 well-characterized patients¹ by the whole genome sequence (WGS) and whole exome sequence (WES).

Results: Using next generation sequencing we found three very rare variants in the same gene in four unrelated patients. The gene found suggest involvement of the Reactive oxygen species (ROS) in development of PAM. To measure ROS generation in patients’ blood we are using the very sensitive Electron Paramagnetic Resonance (EPR) method. In addition, we are creating cell models to study the effects of the mutations in global expression, and we plan to use zebrafish models to study the consequences of these variants in vivo.

Conclusion: In summary, identifying the genetic cause of PAM is very valuable as it gives affected individuals the opportunity for diagnosis, important information about prognosis and treatment.

References: 1.Gudbjornsson, B et al. “Psoriatic arthritis mutilans (PAM) in the Nordic countries: demographics and disease status. The Nordic PAM study.” Scandinavian journal of rheumatology vol. 42,5 (2013): 373-8. https://doi.org/10.3109/03009742.2013.771211.

Grants: 1) KI- China Scholarship Council (CSC202008320311 to Sailan Wang). 2) 2021: Sällsyntafonden. Funding: 35 000 SEK. Role: solo applicant.

Conflict of Interest: None declared.

P05.011.D Expanding the allelic and locus heterogeneity of Multiple osteochondromas

Artem Borovikov ¹, Nailya Galeeva¹, Tatiana Markova¹, Elena Zinina¹, Tatiana Nagornova¹, Ekaterina Zakharova¹, Aysylu Murtazina¹, Ilya Kanivets^2;3, Sergey Korostelev², Maria Orlova¹, Aleksander Polyakov¹, Elena Dadali¹, Olga Schagina¹

¹Research Centre for Medical Genetics, Moscow, Russian Federation; ²Genomed LTD, Moscow, Russian Federation; ³FSBEI FPE RMACPE MOH Russia, Moscow, Russian Federation

Background/Objectives: Multiple osteochondromas (MO) is a skeletal dysplasia characterized by the growth of two or more osteochondromas, caused by pathogenic nucleotide variants in EXT1 or EXT2 genes in 70%-90% of patients. Another rarer disease characterized by multiple osteochondromas but with additional enchondromas is metachondromatosis, associated with the loss-of-function variants in the PTPN11 gene. The clinical similarity of the two diseases suggests that atypical forms of metachondromatosis can be misdiagnosed as MO. This study aims to characterize the genotype profile of the Russian population and find additional molecular causes in patients with MO.

Methods: DNA sequencing (Sanger Method and NGS panel) and MLPA analysis were performed to identify SNV and CNV in the sample of 348 patients from 153 families. Patients were divided into groups according to the severity of the disease, using a three-degree clinical scale with two subgroups.

Results: Germline pathogenic or likely pathogenic variants were identified in 137 (89%) families. 122 unique disease-causative variants (53 novel and 69 known) and 15 recurrent (4 novel) were identified including frameshift (34,4%), nonsense (26,2%), missense (12,3%), splicing (15,5%) variants, genomic rearrangements (5,7%) and in-frame deletion (0,8%). In 16 families with the clinical picture of MO with no significant findings in EXT1 and EXT2 genes, five loss-of-function variants in the PTPN11 gene were found.

Conclusion: Our work expanded the allelic and locus heterogeneity of MO and showed that some of the undiagnosed patients with MO could have pathogenic variants in the PTPN11 gene.

References:.

Grants:.

Conflict of Interest: None declared.

P05.013.B A biallelic gain-of-function variant in MSGN1 causes a new skeletal dysplasia syndrome

Asuman Koparir ¹, Caroline Lekszas¹, Kemal Keseroglu², Aboulfazl Rad³, Atefeh Hasanzadeh⁴, Ehsan Ghayoor Karimiani⁵, Nakul Narendran⁶, Ertugrul Ozbudak^7;8, Barbara Vona^1;3;9;10, Thomas Haaf¹

¹Institute of Human Genetics, Julius Maximilians University Würzburg, Würzburg, Germany; ²Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; ³Department of Otolaryngology-Head & Neck Surgery Tübingen Hearing Research Centre, Eberhard Karls University Tübingen, Tübingen, Germany; ⁴Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran; ⁵Department of Medical Genetics, Next Generation Genetic Polyclinic, Mashhad, Mashhad, Iran; ⁶University of Cincinnati College of Medicine, Cincinnati, United States; ⁷Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; ⁸Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, United States; ⁹Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany; ¹⁰Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany

Background/Objectives: Mesogenin1 (MSGN1, OMIM: *612209), a basic-Helix–Loop–Helix transcription factor, is expressed in the presomitic mesoderm (PSM) and plays a crucial role in formation of PSM progenitor cells during somitogenesis. We aimed at identifying the molecular basis of a patient with severe skeletal dysplasia in a single family through utilization of whole-exome sequencing and subsequent Sanger sequencing.

Methods: The proband displays short stature and multiple skeletal problems, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. To test the pathogenicity of the detected mutation, we overexpressed either wild-type (WT) or the mutant msgn1 in zebrafish via mRNA injection and analyzed tbxta (i.e., T/brachyury/ntl) expression in the tailbud.

Results: A novel homozygous c.374G>T (p. Arg125Leu) missense variant was detected in MSGN1 (NM_001105569.2) in a patient. MSGN1 resides in a ~13.2 Mb homozygous interval on chromosome 2. We found overexpression of WT or the mutant msgn1 significantly reduces tbxta expression in the tailbud compared to uninjected WT embryos (p < 0.0001). In addition, we also found the mutant msgn1 has a more severe effect than WT msgn1 (p = 0.0186, Kruskal-Wallis), causing a gain-of-function.

Conclusion: In contrast to loss-of-function effect, gain-of-function of MSGN1 explains mild affected vertebrae of the proband. Furthermore, as somite segments from 9 to 13 are known to contribute to the forelimb muscle, these might potentially be related with mesomelic shortening in our case. Our findings highlight a new skeletal dysplasia syndrome caused by a gain-of-function mutation in MSGN1.

References:.

Grants:.

Conflict of Interest: None declared.

P05.014.C MN1-related disorders in two Bulgarian patients

Mila Sleptsova ¹, Tihomir Todorov¹, Slavena Atemin^1;2, Mihaela Mladenova^1;2, Daniela Avdjieva-Tzavella³, Albena Todorova^1;2

¹Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria; ²Department of Medical Chemistry and Biochemistry, Medical University Sofia, Sofia, Bulgaria; ³Department of Clinical Genetics, University Pediatric Hospital Sofia, Sofia, Bulgaria

Background/Objectives: Pathogenic variants in the MN1 gene are a known cause of CEBALID syndrome, which is characterised mainly by craniofacial and structural brain abnormalities, and global developmental and variable intellectual delay. Predominantly nonsense and frameshift variants have been reported as pathogenic so far. The syndrome is with autosomal dominant inheritance (Mak et al., 2019). We report two patients which presented with similar clinical characteristics and were found to carry heterozygous variants in the MN1 gene.

Methods: Whole Exome Sequencing was performed for both patients and a panel of genes was analysed. Variants flagged through the analysis were confirmed and segregated in the patients’ families via Sanger sequencing.

Results: The first case is a patient with craniofacial dysostosis, low-set ears, polycystic kidney dysplasia and congenital intellectual delay. The following variant in MN1 was found in a heterozygous state: c.3743G>A, p.Trp1248Ter. The segregation analysis confirmed the variant occurs de novo.

The second case is a patient which presents with craniofacial malformations. The c.2486C>A, p.Ser829Tyr variant was found in a heterozygous state. The segregation analysis confirmed the variant occurs de novo.

Conclusion: Those cases provide insight into the diversity of MN1 variants that could be disease-causing. Although no missense variants have been reported as pathogenic so far, functional studies might be worth looking into.

References: Mak, C., Doherty, D., Lin, A., et al., 2019. MN1 C-terminal truncation syndrome is a novel neurodevelopmental and craniofacial disorder with partial rhombencephalosynapsis. Brain, 143(1), pp.55-68.

Grants:.

Conflict of Interest: None declared.

P05.015.D Functional analyses of pathogenic variants in SNRPE associated with the rare hair loss disorder hypotrichosis simplex

Nicole Cesarato ¹, Yasmina Gossmann¹, F. Buket Basmanav¹, Regina C. Betz¹

¹Institute of Human Genetics, Bonn, Germany

Background/Objectives: Pathogenic variants in SNRPE cause autosomal-dominant hypotrichosis simplex. The extent of scalp and body hair involvement shows great interindividual variability, even within the same family. In particular, all of the affected individuals present with scanty or no eyebrows, while some of them show also hypotrichosis of scalp and body hair.1 SNRPE encodes a core protein of the U snRNPs, key factors of the minor and major spliceosomes. It is yet not clear how pathogenic variants in this gene cause the hair loss phenotype and which specific signaling pathways are involved. We aim at understanding the function of SNRPE in relevance to hair biology.

Methods: We transfected HEK293T and/oror HaCaT cells with wild type and mutant SNRPE constructs and performed immunofluorescence, co-immunoprecipitation and WB. We silenced the endogenous SNRPE mRNA, performed RNAseq.

Results: SNRPE mutants are less expressed than the wild type and are partially degraded via the proteasome; they are also incorporated in the minor spliceosome. They show mislocalization in the cytosol, seeming to fail to enter the nucleus. RNAseq data on HaCaT cells downregulated for the endogenous SNRPE show an alteration of many physiological processes and a reduction in KRT81 expression levels, previously linked to monilethrix.

Conclusion: The mechanism by which mutations in SNRPE cause hypotrichosis is still unclear. However, mutant forms of SNRPE are probably unable to exert their function. Downregulation of SNRPE in HaCaT affects the expression of KRT81, known to cause monilethrix.

References: 1 Pasternack et al., 2013.

Grants: I-1443-422.13/2017 (German-Israeli Foundation).

Conflict of Interest: None declared.

P05.016.A Age-specific effects of body size on fracture risk in later life: A lifecourse Mendelian randomization study

Grace Marion Power ¹, Jonathan Tobias^1;2, Timothy M. Frayling³, Jessica Tyrrell³, April Hartley², Jon Heron¹, George Davey Smith¹, Tom G. Richardson¹

¹University of Bristol, MRC Integrative Epidemiology Unit, Population Health Sciences, Bristol, United Kingdom; ²University of Bristol, Musculoskeletal Research Unit, Bristol, United Kingdom; ³University of Exeter, Genetics of Complex Traits, College of Medicine and Health, Exeter, United Kingdom

Background/Objectives: Musculoskeletal conditions, including fractures, can have severe and long-lasting consequences. Higher body mass index in adulthood is widely acknowledged to be protective for most fracture sites. However, the association between weight and bone health is complex and confounding bias may have distorted earlier findings. Employing a lifecourse Mendelian randomization (MR) approach, this investigation explores how prepubertal and adult body size independently influence fracture risk in later life.

Methods: Using data from a large UK-based prospective cohort, univariable and multivariable MR were conducted to simultaneously estimate the effects of age-specific genetic proxies for body size (n = 453,169) on the odds of fracture in later life (n = 416,795). A two-step MR framework was additionally applied to elucidate potential mediators.

Results: Univariable and multivariable MR indicated strong evidence that higher body size in childhood reduced fractures in later life (OR, 95% CI: 0.89, 0.82 to 0.96, P = 0.005 and OR, 95% CI: 0.76, 0.69 to 0.85, P = 1x10-6, respectively). Conversely, higher body size in adulthood increased fracture risk (OR, 95% CI: 1.08, 1.01 to 1.16, P = 0.023 and OR, 95% CI: 1.26, 1.14 to 1.38, P = 2x10-6, respectively). Two-step MR analyses suggested that the effect of higher body size in childhood on reduced fracture risk was mediated by its influence on higher estimated bone mineral density in adulthood.

Conclusion: This investigation provides novel evidence that higher body size in childhood reduces fractures in later life and higher body size in adulthood is a risk factor, opposing findings from earlier research. Protective effect estimates previously observed are likely attributed to childhood effects.

References:.

Grants: MR/N0137941/1;MC_UU_00011/1;MC_UU_00011/1;MR/T002239/1;SBF004\1079.

Conflict of Interest: Grace Marion Power GMP is employed part-time by Clifton Insight outside of this work., Jonathan Tobias: None declared, Timothy M. Frayling: None declared, Jessica Tyrrell: None declared, April Hartley: None declared, Jon Heron: None declared, George Davey Smith: None declared, Tom G. Richardson TGR is employed part-time by Novo Nordisk outside of this work.

P05.017.B Genetic risk factors for developmental dysplasia of the hip in the Trøndelag Health Study

Kaya Kvarme Jacobsen ¹, Sigrid Børte^2;3, Lene Bjerke Laborie^4;5, Hege Kristiansen^6;7, Annette Schäfer⁶, Trude Gundersen^8;9, Tetyana Zayats^10;11;12, Bendik Kristoffer Slagsvold Winsvold^2;3;13, Karen Rosendahl^14;15

¹Helse Førde, Department of Orthopedic Surgery, Førde, Norway; ²Oslo University Hospital, Department of Research and Innovation, Division of Clinical Neuroscience, Oslo, Norway; ³Norwegian University of Science and Technology (NTNU), K. G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, Faculty of Medicine and Health Sciences, Trondheim, Norway; ⁴Helse Bergen, Department of Radiology, Bergen, Norway; ⁵Helse Vest, Bergen, Norway; ⁶Helse Førde, Department of Pediatrics, Førde, Norway; ⁷University of Bergen, Department of Clinical Science, Bergen, Norway; ⁸Helse Bergen, Department of Orthopedic Surgery, Bergen, Norway; ⁹University of Bergen, Department of Clinical Medicine, Bergen, Norway; ¹⁰Massachusetts General Hospital, Boston, United States; ¹¹Broad Institute, Stanley Center for Psychiatric Research, Boston, United States; ¹²University of Oslo, PROMENTA, Oslo, Norway; ¹³Oslo University Hospital, Department of Neurology, Oslo, Norway; ¹⁴University Hospital of Northern Norway, Department of Radiology, Tromsø, Norway; ¹⁵University of Tromsø, Department of Medicine, Tromsø, Norway

Background/Objectives: Developmental dysplasia of the hip (DDH) is a congenital condition affecting 2-3% of all infants¹. DDH increases the risk of osteoarthritis, is the cause of 30% of all total hip arthroplasties (THAs) in adults <40 years of age and can result in loss of life quality². We aim to explore the genetic background of DDH in order to improve diagnosis, management and longterm outcome.

Methods: We used the large, ongoing, longitudinal Trøndelag Health Study (HUNT) database³, Case definition was based on ICD-9/-10 diagnoses of DDH, or osteoarthritis secondary to DDH. Analyses were performed using SAIGE software, with covariates including sex, batch, birth year and principal components. We included only SNPs with MAF ≥ 0.01, R² ≥ 0.8 and HWE ≥ 0.0001. Significance level was set at p < 5x10^-8. The regional ethical committee approved the study.

Results: Analysis included 69,500 individuals, of which 408 cases, and 8,531,386 SNPs. Two SNPs near COL11A1 were significantly associated with DDH; rs713162 (β = -0.43, SE = 0.07, p = 8.4x10^-9) and rs6577334 (β = -0.43, SE = 0.08, p = 8.9x10^-9). COL11A1 has previously been associated with acetabular dysplasia and osteoarthritis^4,5.

Conclusion: This large, genome-wide case-control study indicates an association between COL11A1 and DDH and is an important contribution to investigating the etiology of DDH, with further research needed.

References: ¹Rosendahl et al, 1996, Pediatric Radiology.

²Engesæter et al, 2011, Acta Orthopaedica.

³Ferreira et al, 2017, Nat Genet.

⁴Raine et al, 2013, BMC Musculoskeletal Disorders.

⁵Versteeg et al, 2016, Osteoarthrits and Cartilage.

Grants: Helse Vest (F-12550-D11544).

Conflict of Interest: None declared.

P05.018.C Satisfaction with resilient denture liner versus acrylic resin telescopic prostheses for patients with ectodermal dysplasia: A non randomized crossover clinical trial

Yasmine H. Mohsen ¹, Mohamed Abdel Kader¹, Nouran Abdel Nabi², Iman A. W. Radi²

¹Human Genetics and Genome Research Institute, National Research Centre, Orodental Genetics, Giza, Egypt; ²Faculty of Dentistry, Cairo University, Prosthodontic Department, Cairo, Egypt

Background/Objectives: Ectodermal dysplasia is a rare genetic disorder that manifests dry skin, sparse hair, xerostomia, complete or partial anodontia of deciduous and/or permanent teeth and malformed teeth. The crowns are usually conical anteriorly. The roots are stunted having a huge pulp chamber. Enamel usually forms a delicate layer, decreasing in thickness towards cervical line. Xerostomia is mostly present due to absence of salivary glands or reduction in their number. Alveolar ridges are commonly flat due lack of tooth formation, it is the main reason for the low vertical dimension of the face and the senile appearance of the patient. These oral features can hinder proper restoration of teeth.

Methods: In this non-randomized clinical trial, ectodermal dysplasia patients with partial anodontia were recruited. Overdentures were constructed for the patients, that were later relined opposing to the teeth with a soft liner. Follow up was done 1 week and 3 months following denture delivery and denture relining. After each stage, patient satisfaction, retention, and periodontal health parameters were assessed. Patient satisfaction was assessed with a validated, reliable questionnaire.

Results: A statistical significant difference was found between the groups regarding retention (P = 0.025), probing depth (P < 0.001), and gingival index (P = 0.011) favouring the acrylic resin coping-retained overdentures 3 months after overdenture delivery.

Conclusion: The resilient denture liner-retained maxillary complete overdenture improved patient satisfaction and tooth mobility of anterior teeth, while minimally jeopardizing the periodontal condition of the abutment teeth in children with ectodermal dysplasia.

References:.

Grants: Supported by a research grant from the National Research Centre, Cairo, Egypt.

Conflict of Interest: Yasmine H. Mohsen National research centre, Mohamed Abdel Kader: None declared, Nouran Abdel Nabi: None declared, Iman A. W. Radi: None declared.

P05.019.D Identification of a novel deep intronic variant in CRTAP by genome sequencing as the cause of osteogenesis imperfecta

Gandham SriLakshmi Bhavani ¹, Prajna Udupa¹, Shalini Nayak¹, Neethukrishna Kausthubham¹, Ashwin Dalal², Katta Girisha¹

¹Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Department of Medical Genetics, Manipal, India; ²Centre for DNA Fingerprinting and Diagnostics, Diagnostics Division, HYDERABAD, India

Background/Objectives: CRTAP is one of the 21 genes known to be associated with Osteogenesis Imperfecta (OI). Biallelic variants in CRTAP (cartilage-associated protein) cause autosomal recessive OI type VII. CRTAP forms a complex with P3H1 in the endoplasmic reticulum and binds to pro-alpha collagen chains to mediate post-translational modification of collagen triple helix.

Methods: Detailed clinical evaluation and a complete skeletal survey were performed. We did exome sequencing followed by genome sequencing to identify the genetic aetiology. RNA was extracted from the fetal fibroblasts for transcript analysis.

Results: Antenatal sonography at 19 weeks of gestation showed shortening of long bones and rocker bottom feet. Perinatal pathological evaluation showed protruding thoracic cage, depressed nasal bridge with anteverted nares, and long philtrum. Radiographic findings were short and bowed legs with multiple fractures at different stages of healing, and generalized osteopenia. Exome sequencing did not reveal any significant pathogenic variants. Genome sequencing identified a deep intronic homozygous variant in intron 3, c.794-1403A>G. RNA studies revealed that it affects splicing and results in two different abnormal transcripts. The detected variant generates the novel splice donor site and utilizes upstream acceptor sites resulting in the formation of two different transcripts of 68 bp and 75 bp cryptic exons in CRTAP mRNA.

Conclusion: The deep intronic variant is associated with alteration of splicing, leading to the generation of two abnormal transcripts. This novel splicing effect in CRTAP is the likely cause of lethal form of OI in the fetus.

References:.

Grants:.

Conflict of Interest: None declared.

P05.020.A Pathogenic LEF1 variants disrupt WNT signaling to cause ectodermal dysplasia associated with limb malformations

Salem Alawbethani^1;2, Maria Asif ^1;2;3, William Dufour^4;5, Anne-Sophie Jourdain^4;6, Genevieve Baujat⁷, Christian Becker¹, Birgit Budde¹, Lyndon Gallacher^8;9, Theodoros Georgomanolis¹, Jamal Ghoumid^4;5, Wolfgang Höhne¹, Stanislas Lyonnet⁷, Iman Ali Ba-Saddik¹⁰, Sylvie Manouvrier-Hanu^4;5, Susanne Motameny¹, Angelika A Noegel^2;3, Lynn Pais¹¹, Clemence Vanlerberghe^4;5, Prerana Wagle¹², Sue White^8;9, Marjolaine Willems¹³, Peter Nürnberg^1;3, Fabienne Escande^4;6, Florence Petit^4;5, Muhammad Sajid Hussain^1;2;3

¹CCG - Cologne Center for Genomics, Köln, Germany; ²Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany; ³Center for Molecular Medicine Cologne, Köln, Germany; ⁴University of Lille, Lille, France; ⁵Chu De Lille, Lille, France; ⁶Chu, Lille, France; ⁷Necker Hospital, Paris, France; ⁸Victorian Clinical Genetics Services, Parkville, Australia; ⁹University of Melbourne, Parkville, Australia; ¹⁰Department of Pediatrics, Faculty of Medicine and Health Sciences, University of Aden, Aden, Yemen; ¹¹Broad Institute of MIT and Harvard, Cambridge, United States; ¹²Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany; ¹³Hospital Center University De Montpellier, Montpellier, France

Background/Objectives: Ectrodactyly ectodermal dysplasia without cleft lip/palate (MIM 129810) is reported with clinical manifestation of split-hand/foot malformation accompanied by ectodermal anomalies like hypotrichosis and abnormal dentition. This disorder has received insufficient attention in the past. We aim to delineate the underlying genetic determinants of this disorder.

Methods: Whole exome and transcriptome sequencing, Pull down assay, Immunoblotting, Immunofluorescence.

Results: We recruited 12 individuals from 5 unrelated families manifesting a syndrome with variable expression of limb malformations and/or ectodermal dysplasia. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia in some individuals. We identified four novel LEF1 variants — monoallelic in 11 affected individuals and biallelic in one. We have shown that out of four, only the p.Met23dup impaired interaction with β-catenin due to its location in a highly conserved β-catenin binding domain of LEF-1. Whole transcriptomic profiling further confirmed that Wnt/ β-catenin signaling pathway is impaired. We have seen significant differential expression of transcripts already known as downstream targets of the Wnt/ β-catenin signaling pathway and HOX family — both Wnt and HOX are crucial for embryonic developmental events.

Conclusion: Our functional data show that two molecular mechanisms are at play: haploinsufficiency or loss of DNA-binding are responsible for a mild to moderate phenotype, while loss of β-Catenin binding due to biallelic variants is associated with a severe phenotype. Our findings establish mono- and biallelic variants in LEF1 as a cause for a syndrome comprising limb malformations and ectodermal dysplasia.

References:.

Grants: CHU Lille, CMMC, DAAD, NHGRI, NHLBI.

Conflict of Interest: None declared.

P05.021.B Contribution of SHOX gene sequencing in the etiological diagnosis of short stature

Camille Porteret ¹, Sylvie Patri¹, Florence Compain¹, Pascaline Letard¹, Sylvie Roullaud², Gwenaël Le Guyader¹, Fabienne Dufernez¹, Matthieu Egloff¹

¹Poitiers University Hospital, Poitiers, France; ²Angoulême Hospital, Angoulême, France

Background/Objectives: Analysis of the SHOX gene is common in clinical practice, as an abnormality justifies a treatment by growth hormone. Our study aims to determine the interest of SHOX sequencing when large rearrangements analyses are inconclusive, and in relation with the precise clinical context¹.

Methods: We analyzed the clinical and genetic data of 163 probands referred to our laboratory for analysis of the SHOX locus for Idiopathic Short Stature (ISS) or Léri-Weill dyschondrosteosis (LWD), after Turner syndrome was excluded by karyotyping. All patients underwent CNV analysis by MLPA followed by gene sequencing.

Results: Out of the 163 patients analyzed, 15 had a CNV affecting SHOX or its regulatory regions: 11 pathogenic deletions and 4 duplications (2 likely benign and 2 VUS). In the 152 patients without pathogenic CNV, 5 had SNVs or indels. The only patient carrying a variant classified as pathogenic also exhibited major clinical signs of LWD. The remaining variants were classified as benign for one rare polymorphism, likely benign because of inheritance.

Conclusion: In our cohort, the diagnostic yield of SHOX sequencing was 33% (1/3) for patients with signs of LWD, whereas it was zero in patients with ISS. Thus this analysis seems questionable in the context of ISS and could be limited to patients with significant signs of LWD (i.e. a positive “Rappold revisited” score¹).

References: ¹Hirschfeldova et al. Journal of Human Genetics, 2016.

Grants:.

Conflict of Interest: None declared.

P05.022.C Body mass index and the risk of rheumatic disease: linear and nonlinear Mendelian randomization analyses

Torgny Karlsson ¹, Fatemeh Hadizadeh¹, Mathias Rask-Andersen¹, Åsa Johansson¹, Weronica Ek¹

¹Uppsala University, Department of Immunology, Genetics and Pathology and Science for Life Laboratory, Uppsala, Sweden

Background/Objectives: While the association between obesity and risk of rheumatic disease is well established, the causal relation is not fully established. Here we estimate the causal effect of body mass index (BMI) on the risk of developing five types of rheumatic disease.

Methods: Linear and nonlinear mendelian randomization (MR) was used to estimate the effect of BMI on the risk of developing of rheumatic disease and to identify sex-specific effects. Primary MR analyses were performed in 361,952 participants from the UK Biobank cohort.

Results: We found that a one standard deviation increase in BMI increases the risk for rheumatoid arthritis (IRR = 1.52; 95% CI = 1.36-1.69), osteoarthritis (IRR = 1.49; 1.43-1.55), psoriatic arthropathy (IRR = 1.80; 1.31-2.48), gout (IRR = 1.73; 1.56-1.92), and inflammatory spondylitis (IRR = 1.34; 1.14-1.57). BMI was also found to be a stronger risk factor for psoriatic arthropathy (sex-interaction P = 3.3 × 10-4) and gout (P = 4.3 × 10-3) in females compared to males. Nonlinear effects of BMI were identified for osteoarthritis and gout in males, and for gout in females. The nonlinearity for gout was more extreme in males compared to females, and was similar to what was observed for urate levels.

Conclusion: Higher BMI causes an increased risk for rheumatic disease, an effect that is more pronounced in females compared to males for both gout and psoriatic arthropathy. These results give further insight into etiology and pathology of rheumatic disease.

References:.

Grants: This work was supported by the Swedish Research Council, the Marcus Borgströms, the Åke Wibergs, A and M Rudbergs foundations, Vleugels foundation and Hedströms foundation.

Conflict of Interest: None declared.

P05.023.D MED12 somatic mutation in Paget’s disease of bone

Nerea Gestoso-Uzal ^1;2;3, Luis Antonio Corchete^2;3, Ana-Belén Herrero^1;2;3, Javier del Pino-Montes^2;4, Juan Francisco Blanco-Blanco^2;5, Rogelio González-Sarmiento^1;2;3

¹Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ²Biomedical Research Institute of Salamanca (IBSAL), University Hospital of Salamanca-USAL-CSIC, Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain; ⁴Rheumatology Service, University Hospital of Salamanca-IBSAL, Salamanca, Spain; ⁵Traumatology and orthopedic surgery service, University Hospital of Salamanca-IBSAL, Salamanca, Spain

Background/Objectives: Paget’s disease of bone (PDB) is a metabolic bone disease characterized by an increase in bone turnover in a disorganized way. Germinal mutation in SQSTM1 gene constitutes the most important known genetic factor predisposing to PDB, but it can not explain the etiopathogenesis of all the patients. Moreover, PDB is a focal disorder that does not affect to all the bone tissue. The aim of this study was to identify somatic mutations that can determine the development of PDB.

Methods: DNA was extracted from both pagetic and normal bone tissue from one PDB patient following a standard phenol/chloroform procedure. Whole exome sequencing was performed to identify pagetic bone-exclusive mutations.

Results: We identified 40 somatic variants in the pagetic bone that did not appear in the normal tissue, including 11 exonic variants. We found just one nonsense mutation, c.103C>T; p.E35*, in MED12 gene. Somatic mutation of MED12 has been associated with uterine myomas, characterized by uncontrolled but benign cell proliferation. Moreover, it has been reported that MED12 mutation causes a blockade of autophagy, as it occurs with reported mutations in SQSTM1(1).

Conclusion: Our results suggest that somatic alteration of MED12 gene could be responsible of PDB development.

References: 1. A. El Andaloussi, A. Al-Hendy, N. Ismail, T. G. Boyer, S. K. Halder, Reprod. Sci. 27, 823 (2020).

Grants: This study was funded by FIS-FEDER: PI18/01476.

Conflict of Interest: None declared.

P05.024.A Syntaxin-18 defects in human and zebrafish cause traffic jams and unravel key roles in early bone development

Brecht Guillemyn ¹, Hanna De Saffel¹, Jan Willem Bek¹, Piyanoot Tapaneeyaphan¹, Adelbert De Clercq¹, Tamara Jarayseh¹, Sophie Debaenst¹, Andy Willaert¹, Peter Byers², Paul Coucke¹, Bettina Blaumeiser³, Delfien Syx¹, Fransiska Malfait¹, Sofie Symoens¹

¹Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent, Belgium; ²University of Washington, Department of Laboratory Medicine and Pathology - Department of Medicine, Seattle, United States; ³University of Antwerp, Department of Medical Genetics, Antwerp, Belgium

Background/Objectives: Membrane fusion is a key process in all living organisms. SNAREs are required for fusogenic action, where they zipper up at the juncture of membranes, enabling fusion and cargo transfer. Syntaxin-18 (STX18), an endoplasmic reticulum-resident SNARE, is crucial for membrane fusion events, vesicular transport and secretion of collagens. Currently, STX18 is not linked to any human disease.

Methods: Whole exome sequencing (WES) was applied for a fetus with lethal osteogenesis imperfecta, presenting with multiple fractures, abnormal cartilage formation, tibial bowing, irregularly formed mandible, and severe cranial undermineralization. Protein modelling and overexpression studies were performed and stx18 zebrafish crispants (uppercut18) were generated using CRISPR/Cas9.

Results: WES revealed a homozygous missense variant p.(Arg10Pro) in STX18, affecting a critical residue in the cytoplasmic N-terminal alfa-helical domain of syntaxin-18, leading to stable mutant STX18 protein. Uppercut18 had severe craniofacial malformations, curved backbones and underdeveloped fins, and all died at 11-12dpf. Strikingly, uppercut18 revealed impaired cartilage and skeletal development; and interestingly, displayed (1) altered bone remodelling, (2) general upregulated mRNA and protein levels of components acting within the forming stx18-complex and secretory pathway, and (3) altered behaviour including decreased movement and an increased light-to-dark-transition response.

Conclusion: Our study provides evidence for a link between genetic defects in STX18 and human bone disease, expanding the phenotype of the SNAREopathies, and our in vivo data describes for the first time an essential role for syntaxin-18 during skeletal development and its involvement in neurological pathways.

References: https://doi.org/10.1126/science.1161748, https://doi.org/10.7554/eLife.02784, https://doi.org/10.1016/j.ydbio.2016.11.010.

Grants: Research Foundation Flanders (12Q5920N,1842318N), Ghent University.

Conflict of Interest: None declared.

P05.025.B Comprehensive molecular screening using NGS custom gene panel identifies ultra-rare types of Osteogenesis imperfecta and OI-like disorders

Kinga Sałacińska ¹, Lena Rutkowska¹, Iwona Pinkier¹, Dominik Salachna¹, Izabela Michałus², Elżbieta Jakubowska-Pietkiewicz³, Łukasz Kępczyński¹, Agnieszka Gach¹

¹Polish Mother’s Memorial Hospital - Research Institute, Department of Genetics, Lodz, Poland; ²Polish Mother’s Memorial Hospital - Research Institute, Department of Endocrinology and Metabolic Diseases, Lodz, Poland; ³Medical University of Lodz, Department of Paediatric Propedeutics and Bone Metabolic Diseases, Lodz, Poland

Background/Objectives: Osteogenesis imperfecta (OI) is a rare connective tissue disorder, presenting genetic and phenotypic heterogeneity. The molecular etiology reaches XXI types, of which types I-IV (90% of patients) are associated with mutations in genes encoding collagen type I. Other cases are caused by aberrations in one out of 17 non-collagen genes influencing collagen biosynthesis or related molecular pathways, which prevalence is below 1 per 1 000 000 births or remains unknown.

Methods: The study included 158 patients suspected with Osteogenesis imperfecta, aged 6 months to 44 years, presenting a broad spectrum of clinical manifestation. NGS custom gene panel, encompassing all known genes for OI types I-XIX and OI-like disorders, was performed.

Results: We have identified 9 families with pathogenic mutations located in six non-collagen genes (IFITM5, SERPINF1, FKBP10, WNT1, P4HB, LRP5) associated with an ultra-rare OI type V, VI, XI, XV and OI-like disorders as Cole-Carpenter syndrome, Primary Osteoporosis, Idiopathic Juvenile Osteoporosis. This unique group of patients constituted 7% of the study population with a proven molecular diagnosis of congenital bone fragility. Of 10 non-collagen variants, four have not been reported previously.

Conclusion: Obtained results reflect phenotypic and genetic variety of skeletal disorders, as primary patients with mutations in non-collagen genes were classified as having OI type I (LRP5, WNT1, P4HB), type III (FKBP10, IFITM5) and type IV (SERPINF1, IFITM5). Thus, comprehensive molecular screening is crucial for correct diagnosis, genetic counselling and appropriate treatment.

References: PMID: 24715559; PMID: 20301472; OMIM Database.

Grants: Young Scientist Grant 2016/IV/57-MN.

Conflict of Interest: None declared.

P05.026.C Biallelic loss of function variants in EXOC6B are associated with impaired primary ciliogenesis and cause spondylo-epi-metaphyseal dysplasia with joint laxity type 3

Pelin Simsek-Kiper¹, Prince Jacob ², Priyanka Upadhyai², ekim taskiran³, Vishal Singh Guleria², beren karaosmanoglu³, gozde imren³, rahşan göçmen⁴, Gandham SriLakshmi Bhavani², Neethukrishna Kausthubham², hitesh shah⁵, Gulen Eda Utine¹, koray boduroglu¹, Katta Girisha²

¹Department of Pediatric Genetics, Faculty of Medicine Hacettepe University, Ankara, Turkey, Ankara, Turkey; ²Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India, Manipal, India; ³Department of Medical Genetics, Faculty of Medicine Hacettepe University, Ankara, Turkey, Ankara, Turkey; ⁴Department of Radiology, Faculty of Medicine Hacettepe University, Ankara, Turkey, Ankara, Turkey; ⁵Department of Pediatric Orthopaedics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India, Manipal, India

Background/Objectives: Spondylo-epi-metaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is characterized by multiple joint dislocations and is caused by biallelic pathogenic variants in EXOC6B. Only four molecularly-confirmed individuals have been reported hitherto and the underlying mechanism is yet to be elucidated.

Methods: Using exome sequencing, we identified c.2122+15447_2197-59588del and c.401T>G biallelic variants in EXOC6B in two individuals from unrelated families with different ethnicities. Immunofluorescence and immunoblotting assays were performed in proband 1 (P1), and osteogenic differentiation assay and total mRNA sequencing in proband 2 (P2). RT-PCR and RT-qPCR were performed for both in patient derived fibroblasts.

Results: P1 at age 3 years demonstrated leptodactyly (slender metacarpals and metatarsals), delayed carpal ossification, lumbar lordosis with bilateral hip, and knee dislocations. P2 at age 13 years had intellectual disability with central nervous system anomalies including hydrocephalus, hypoplastic mesencephalon, and thin corpus callosum in addition to the known features of SEMDJL3. Increased levels of EXOC6B mRNA, absent protein in fibroblast-derived cell lysates, shortening of primary cilia length with minor variability of primary cilia frequency in P1 as compared to control derived fibroblasts were observed. The expression of EXOC6B mRNA and osteogenic differentiation potential was reduced in dermal fibroblasts of P2 as compared to that of control fibroblasts. Pathways related to the extracellular matrix were also distrubed in P2.

Conclusion: Our study provides the first evidence of the impairment of exocytosis potentially deregulating primary ciliogenesis in two subjects with SEMDJL3 that might represent yet another ciliopathy with central nervous system involvement and joint dislocations.

References:.

Grants: India Alliance Fellowship (IA/CRC/20/1/600002).

Conflict of Interest: Pelin Simsek-Kiper: None declared, Prince Jacob: None declared, Priyanka Upadhyai: None declared, ekim taskiran: None declared, Vishal Singh Guleria: None declared, beren karaosmanoglu: None declared, gozde imren: None declared, rahşan göçmen: None declared, Gandham SriLakshmi Bhavani: None declared, Neethukrishna Kausthubham: None declared, hitesh shah: None declared, Gulen Eda Utine: None declared, koray boduroglu: None declared, Katta Girisha Founding Director of Suma Genomics Pvt. Ltd. (Incubatee at Manipal Universal Technology Business Incubator).

P05.027.D Genetic anomalies and diagnostic yield in an 11-year birth cohort of craniosynostosis patients

Linda Gaillard ¹, Anne Goverde², Annelies de Klein², Irene Mathijssen¹, Marieke van Dooren²

¹Erasmus Medical Center-Sophia Children’s Hospital, University Medical Center, Department of Plastic, Reconstructive and Hand Surgery, Rotterdam, Netherlands; ²Erasmus Medical Center-Sophia Children’s Hospital, University Medical Center, Department of Clinical Genetics, Rotterdam, Netherlands

Background/Objectives: Craniosynostosis is a rare congenital anomaly, defined by premature fusion of one or more cranial sutures. Craniosynostosis can occur in isolation or as part of a clinical syndrome. The genetic cause of craniosynostosis may impact the clinical course and treatment of patients with craniosynostosis. Knowledge on the genetic etiology therefore is key to ensure adequate counseling and to improve clinical management of craniosynostosis patients. In line with this, the Dutch craniosynostosis guideline recommends genetic diagnostic testing in patients with craniosynostosis. This study aims to assess both the prevalence of the different subtypes of craniosynostosis in an 11-year birth cohort of craniosynostosis patients as well as the diagnostic yield of genetic testing.

Methods: We conducted a retrospective cohort study among patients who presented at the outpatient clinic of the Erasmus University Medical Center, The Netherlands. We included all patients, born between 2010-2021, with radiologically confirmed craniosynostosis and assessed diagnostic yield of genetic testing.

Results: We included 993 patients (n = 334 female/659 male), of whom 856 presented with single-suture craniosynostosis (449 sagittal, 268 metopic, 116 unicoronal, 14 unilambdoid, 9 frontosphenoidal) and 136 patients presented with multisutural craniosynostosis, and one unknown (preliminary results).

Conclusion: This study will discuss the prevalence of chromosomal and monogenic (likely) pathogenic variants and provide an update on the diagnostic yield of genetic testing in our 11-year birth cohort craniosynostosis patients. Finally, we will compare the diagnostic yield for different types of craniosynostosis with the long term aim of improving genetic testing strategies and counseling.

References:.

Grants:.

Conflict of Interest: None declared.

P05.028.A The stop-loss variant c.690A>C in RAB33B results in milder phenotype of Smith-McCort dysplasia-2

Ashwin Dalal ^1;2, Prince Jacob², Katta Girisha², Prajna Udupa², debashish ghosh², Gandham SriLakshmi Bhavani², hitesh shah²

¹Centre for DNA Fingerprinting and Diagnostics - CDFD, Hyderabad, India; ²Kasturba Medical College, Manipal, Manipal, India

Background/Objectives: Smith-McCort dysplasia-2 (SMC-2) is a rare spondylo-epiphyseal-metaphyseal dysplasia, caused by biallelic loss of function variants in RAB33B. RAB33B has dual functions in membrane trafficking and in the autophagy process. Studies investigating the dynamics of the autophagy with relation to RAB33B in patients with SMC2 have not been reported.

Methods: We identified a biallelic stop-loss variant, c.690A>C in RAB33B in two siblings (P1 and P2) by exome sequencing. Functional studies were done using immunofluorescence, immunoblotting, and qPCR in fibroblast cells of P1 and P2.

Results: P1 and P2 at age 13 years and 15 years respectively presented with short trunk, barrel-shaped chest, brachydactyly, plump interphalangeal joints, limited extension of elbow joints, and progressive joint pain. Convex vertebral endplates, reduced hip joint spaces, short metacarpals (4^th and 5^th), abnormal carpal bone morphology were observed in their radiographs. The mutant RAB33B showed loss of its Golgi body localization which led to aggregation of collagens in cytosol. Abundance of cytoplasmic RAB33B led to increased flux of autophagy, causing accelerated clearance of the aggregated collagen by aggrephagy. Thus, the equilibrium of RAB33B localization to Golgi body and phagophore membrane is modulated by the mutant RAB33B, leading to differential crosstalk of Golgi body-RAB33B-autophaosome in the patient fibroblasts.

Conclusion: Mislocalization of mutant RAB33B at Golgi body with simultaneous increased autophagy probably underlie the milder phenotype of SMC-2. Our work highlights the emerging role of autophagy in collagen degradation and in skeletal dysplasia.

References:.

Grants:.

Conflict of Interest: None declared.

P05.029.B Inversion of LMX1B - a novel cause of nail-patella syndrome in a Swedish family

Hillevi Lindelöf ^1;2, Eva Horemuzova¹, Ann Nordgren^1;2, Ulrika Voss³, Anna Hammarsjö^1;2, Giedre Grigelioniene^1;2

¹Karolinska Institutet, Department of molecular medicine and surgery, Stockholm, Sweden; ²Karolinska University Hospital, Department of Clinical Genetics, Stockholm, Sweden; ³Karolinska University Hospital, Department of Pediatric Radiology, Stockholm, Sweden

Background/Objectives: Nail-patella syndrome (OMIM #161200) is a rare autosomal dominant disorder with an estimated prevalence of 1 in 50 000. The characteristic clinical findings are different nail and skeletal abnormalities including underdeveloped nails, hypoplastic or absent patellae and iliac horns. It is caused by missense or truncating variants in LMX1B, resulting in haploinsufficiency. In this case report, we present an unusual cause of NPS in a family of five affected individuals where Sanger sequencing failed to detect any pathogenic variants in the LMX1B gene.

Methods: We describe a large inversion disrupting the LMX1B gene in five affected family members with mild but variable clinical features of nail-patella syndrome.

Results: Whole genome sequencing revealed an inversion stretching approximately 4,2 Mb and disrupting LMX1B and ABL1 which was confirmed by Sanger sequencing of the breakpoints.

Conclusion: This expands the molecular spectrum of nail-patella syndrome, indicating that genomic rearrangements should be considered a possible cause in patients where standard genetic investigations fail to detect any pathogenic variants in LMX1B.

References: Sweeney, E., Fryer, A., Mountford, R., Green, A., & McIntosh, I. (2003). Nail patella syndrome: a review of the phenotype aided by developmental biology. J Med Genet, 40(3), 153-162.

Grants: The Swedish Rare Diseases Research foundation (Sällsyntafonden), Sällskapet Barnavård and Karolinska Institutet, Swedish Research Council, through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet, Promobilia Foundation and Frimurare Barnhuset foundation in Stockholm.

Conflict of Interest: None declared.

P05.031.D de novo c.784G>A variant in LMNA disturbs nuclear proteostasis and results in progeroid manifestations

Shruti Pande ¹, Debasish Kumar Ghosh¹, Jeevan Kumar¹, Dhanya Yesodharan², Sheela Nampoothiri², Periyasamy Radhakrishnan³, Chilakala Gangi Reddy⁴, Akash Ranjan⁴, Katta Girisha^3;5

¹Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Department of Medical Genetics, Udupi, India; ²Amrita Institute of Medical Sciences and Research Centre Cochin, Kerala, India, Department of Pediatric Genetics, Cochin, India; ³Suma Genomics Private Limited Manipal, Udupi, India; ⁴Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, Telangana, India, Hyderabad, Telangana, India; ⁵Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Department of Medical Genetics, UDUPI, India

Background/Objectives: Variants in LMNA lead to collapse of nuclear systems network and underlie some disorders of premature aging, including Hutchinson-Gilford progeria syndrome.

Methods: We identified a de novo heterozygous nonsynonymous variant, c.784G>A (LMNA^E262K) in exon 4 of LMNA (NM_170707.4) in a sixteen-years old male by exome sequencing. We performed immunocytochemistry to see expression and localization of the mutant LMNA and immunoblotting to quantify the mutant LMNA in patient fibroblasts. To study the LMNA secondary structure, recombinant wild type and mutant were cloned and circular dichroism spectroscopy, atomic force microscopy, dynamic light scattering and isothermal titration calorimetry were performed. Computation tools and molecular dynamics simulation were used to assess the stability of LMNA^E262K.

Results: The proband had micrognathia, sparse scalp hair, eyebrows and eyelashes, narrow nasal bridge with broad nasal tip and dental crowding. His fibroblasts showed nuclear aggregates of the mutant LMNA with its mislocalization from the nuclear envelope. Structural destabilization of mutant region results in the clustering of multiple hydrophobic residues, making LMNA unstable and prone to aggregation. LMNA^E262K also disrupts the consensus binding site of E2-SUMO ligase UBE2I, further reducing the clearance of mutant LMNA in patient fibroblasts. Aggregates of LMNA^E262K temporally sequester HSPA1A1, PSMD8, MRE11 and KU80, resulting in impaired nuclear proteostasis and loss of DNA damage repair response in proband fibroblasts.

Conclusion: LMNA^E262K in the rod2 domain causes structure-function destabilization of LMNA, leading to nuclear proteotoxicity in premature aging.

References:.

Grants: India Alliance (IA/CRC/20/1/600002) funded this work.

Conflict of Interest: Shruti Pande: None declared, Debasish Kumar Ghosh: None declared, Jeevan Kumar: None declared, Dhanya Yesodharan: None declared, Sheela Nampoothiri: None declared, Periyasamy Radhakrishnan: None declared, Chilakala Gangi Reddy: None declared, Akash Ranjan: None declared, Katta Girisha Suma Genomics, Private Limited, Manipal (Director).

P05.032.A A novel SLC35D1 variant causing milder phenotype of Schneckenbecken dysplasia in a large pedigree

Leyla Ozer ¹, Suleyman Aktuna¹, Evrim Unsal¹, volkan baltaci¹

¹Mikrogen Genetik Tani Merkezi, Genetics, ANKARA, Turkey

Background/Objectives: SLC35D1 gene mutations causes Schneckenbecken dysplasia(SD) which is an rare autosomal recessive disorder and characterized by the snail-like pelvis, flattening of vertebral bodies, short and broad long bones with a dumbbell-like appearance, thoracic hypoplasia. Here we reported a family with novel SLC35D1 variant and milder phenotype of SD (1, 2).

Methods: The clinical features of 5 patients were represented in Table 1. Whole exome sequencing was done using the NextSeq500 Sequencer. The detected variant which was predicted as the causative variant was validated by Sanger sequencing in the proband and her parents. Sanger sequencing were studied for the other affected individuals in the family.

Results: Whole Exome sequencing of the proband revealed a homozygous missense variant of SLC35D1 gene; c.401T>C. A, p.Met134Thr. Affected sibling and her cousins with same phenotype have homozygous missense variant too. The parents and their healthy girl are heterozygous carriers.

Conclusion: Patients with mild phenotypic findings who carry SLC35D1mutation were reported for the first time in this study. This report will have significant consequences since it has the largest SD family with alive patients (ages ranged 4-31 years old) reported to date.

References: 1. Song Z. Roles of the nucleotide sugar transporters (SLC35 family) in health and disease. Molecular Aspects of Medicine. 2013; 34: 590–600.

2. Furuichi T et al. Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases. J Med Genet. 2009 ; 46(8): 562–568.

Grants: Table 1. Clinical features of the patients.

.

Conflict of Interest: None declared.

P05.033.B Mapping active regulatory signals at early embryonic stage of face development

Catia Attanasio ¹, Marion Leleu², Alexandre Reymond³

¹KU Leuven, Human Genetics, Leuven, Belgium; ²University of Lausanne, Bioinformatics Competence Center, Lausanne, Switzerland; ³University of Lausanne, Center for Integrative Genomics, Lausanne, Switzerland

Background/Objectives:.

While craniofacial malformations are among the most common congenital anomalies in humans at frequency 1/1600, their genetic causes remain largely unknown. Previous studies have shown that deletion or mutation of craniofacial enhancers modifies the activity of craniofacial gene(s) and affects face morphology. To reveal the complex regulatory networks that control craniofacial development and gain insights into the effect of non-coding variation, we mapped 3D interactions between craniofacial enhancers and genes.

Methods: We isolated the four facial prominences, i.e., mandible, maxillary, lateral and medial nasal prominences of E11.5 mouse embryo and performed Capture Hi-C to identify chromatin interactions at all gene promoters.

Results: We identified an average of 163’000 interactions per tissue, for a total of 327’459 unique loops. These are chiefly cis-interactions, which account for >98% of all detected interactions, with a median distance of interacting fragments of 280kb. Chromatin interactions between a gene and an intergenic space account for 77% of all detected interactions. Importantly, enrichment analyses of matched tissue and developmental stage epigenomics data (e.g. H3K27ac binding sites) show a significant enrichment of craniofacial regulatory sequences in our interacting fragments, supporting the biological significance of these craniofacial interactions’ maps. We recapitulate previously reported interactions, reveal new interesting regulatory landscape of craniofacial genes and link GWAS non-coding lead SNPs for craniofacial phenotypes to candidate target gene(s).

Conclusion: Our data provide a starting point to disentangle the gene regulatory signals controlling craniofacial development and point toward new candidate regions for determining the genetic origins of many craniofacial-associated disorders.

References:.

Grants: PRO-Femmes grant (UNIL, Switzerland).

Conflict of Interest: None declared.

P05.034.C Multiple epiphyseal dysplasia: A diagnostic challenge with genetic heterogeneity

Tuğba Nur Daşar ¹, ekim taskiran², gizem ürel-demir^1;3, beren karaosmanoglu², gozde imren², güney yılmaz⁴, Yasemin Alanay^1;5, Gulen Eda Utine¹, Koray Boduroğlu¹, Pelin Simsek-Kiper¹

¹hacettepe university, department of pediatrics, division of pediatric genetics, ankara, Turkey; ²hacettepe university, department of medical genetics, ankara, Turkey; ³mersin city research and training hospital, department of pediatrics, division of pediatric genetics, mersin, Turkey; ⁴hacettepe university, department of orthopedics, ankara, Turkey; ⁵acıbadem university, department of pediatrics, division of pediatric genetics, istanbul, Turkey

Background/Objectives: Multiple epiphyseal dysplasia (MED) is a rare genetic skeletal disorder with clinical and genetic heterogeneity. MED is caused by mutations in the genes encoding important cartilage extracellular matrix proteins, enzymes, and transporter proteins including COMP, MATN3, COL9A1, COL9A2, COL9A3, CANT1 and SLC26A2. Disproportionate short stature, joint pain, and early-onset osteoarthritis are the main clinical features (1). In this study we aimed to investigate the clinical and molecular findings along with natural course of the disease in a group of patients with MED.

Methods: The molecular etiology was investigated with Sanger sequencing and whole exome sequencing. The clinical findings of mutation positive patients were reviewed.

Results: A total of 36 patients with a clinical diagnosis of MED was evaluated and the genetic etiology was revealed in 20 (55.5%); 11 were male and 9 were female. COMP (n = 12, 60%), MATN3 (n = 6, 30%) and SLC26A2 (n = 2, 10%) mutations were detected in a decreasing order. The most frequent complaints for referral were difficulty in walking, fatigue and joint pain. Proportionate short stature was detected in %15 of patients. Orthopedic follow-up was required in most of the patients. In patients with SLC26A9 mutations characteristic findings of double layered patella and pes equinovarus were not present.

Conclusion: MED is genetically heterogenous yet with unidentified gene mutations in the etiology. The diagnosis should be considered even in the absence of characteristic clinical findings. Multidisciplinary follow-up is mandatory.

References: 1. Dennis EP, Greenhalgh-Maychell PL, Briggs MD. Multiple epiphyseal dysplasia and related disorders: Molecular genetics, disease mechanisms, and therapeutic avenues. Dev Dyn. 2021;250(3):345-359.

Grants:.

Conflict of Interest: None declared.

P05.035.D Further delineating the clinical spectrum of p.Arg444Cys variant in LRP5: a case report and a systematic review of the literature

Elena Luppi ^1;2, Guido Zavatta^2;3, Nicole Balducci⁴, Dario Cocciadiferro⁵, Uberto Pagotto^2;3, Marco seri^1;2

¹IRCCS Azienda Ospedaliero-Universitaria di Bologna, Medical Genetics Unit, Bologna, Italy; ²Alma Mater Studiorum University of Bologna, Department of Medical and Surgical Sciences (DIMEC), Bologna, Italy; ³IRCCS Azienda Ospedaliero-Universitaria di Bologna, Unit of Endocrinology and Prevention and Care of Diabetes, Bologna, Italy; ⁴IRCCS Azienda Ospedaliero-Universitaria di Bologna, Ophthalmology Unit, Bologna, Italy; ⁵Bambino Gesù Children’s Hospital, IRCCS, Translational Cytogenomics Research Unit, Rome, Italy

Background/Objectives: The LRP5 protein plays a key role in retinal vasculature development and helps regulate bone mineral density (BMD) homeostasis. Loss-of-function variants in LRP5 were associated with familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome and early-onset osteoporosis/osteopenia.

We describe the clinical and genetic features of a family with osteopenia as the presenting feature, displaying the p.Arg444Cys variant in LRP5.

Methods: Clinical and familial data were based on physical examination and medical records. Next-generation sequencing of an in-silico panel encompassing 20 genes correlated with bone fragility and detection of index mutation were performed with Twist Custom Panel (clinical exome-twist Bioscience) kit.

A systematic review of the p.Arg444Cys variant in LRP5, according to PRISMA guidelines, was performed.

Results: Our proband is a 36-year-old male who suffered from scoliosis and recurrent vertebral fractures even during bisphosphonate treatment. The genetic testing revealed the heterozygous variant c.1330C>T (p.Arg444Cys) in the LRP5 gene (NM_002335.4), segregating in his father and his 30-year-old sister. Both of them experienced less severe osteopenia and denied visual impairment, supporting the variant’s role in reduced BMD. Ophthalmic examination excluded signs of vitreoretinopathy in our proband and his father.

We systematically review 1384 records and we found four studies reporting the same variant. To our knowledge, the p.Arg444Cys variant was previously identified only in patients with FEVR with or without osteopenia.

Conclusion: We reported a patient displaying the p.Arg444Cys variant in LRP5 and familial low BMD without eye involvement, expanding the clinical spectrum of this variant. Further clinical and biochemical assessment of the other family carriers are ongoing.

References:.

Grants:.

Conflict of Interest: None declared.

P05.036.A Genetic basis of cleft lip and palate

Eleonore Pairet ^1;2, Peyman Ranji¹, Raphaël Helaers¹, Vera Lucia Gil-da-Silva-Lopes³, Benedicte Bayet⁴, Elin Malek Abrahimians⁴, Naima Deggouj⁴, Sara Castelein⁴, Nicole Revencu^2;4, Miikka Vikkula¹

¹De Duve Institute UCLouvain, Human Molecular Genetics, Woluwe-Saint-Lambert, Belgium; ²Cliniques universitaires Saint-Luc (UCLouvain), Center for Human Genetics, Bruxelles, Belgium; ³UNICAMP Universidade Estadual de Campinas, Medical Genetics, Campinas, Brazil; ⁴Cliniques universitaires Saint-Luc (UCLouvain), Centre labio-palatin Albert de Coninck, Bruxelles, Belgium

Background/Objectives: Cleft lip and/or palate (CL/P) is the most common cranio-facial malformation often divided into syndromic CL/P (syCL/P) and non-syndromic CL/P (nsCL/P). In general, patients with syCL/P follow Mendelian inheritance, whilst those with nsCL/P are thought to have a complex etiology.

Methods: We analyzed 81 a priori non-syndromic index CL/P patients from a continuously growing cohort of 1400 CL/P patients by whole exome sequencing (WES). We looked for Mendelian mutations using Highlander as well as copy number variations using ExomeDepth.

Results: We unraveled pathogenic or likely pathogenic variants in 12 families in COL2A1, CTNND1, TP63, CHD7, PHF8, IRF6, and GHRL3. We also identified, and validated by molecular karyotyping, a deletion in TP63 in 2 siblings.

Conclusion: We identified mutations in 16 % of index cases by WES, providing an accurate diagnosis as well as the possibility of genetic counseling. In some cases, we identified pathogenic variants in syCL/P genes in a priori nsCL/P cases, demonstrating that patients with CL/P without cardinal signs or familial history of a syndrome may still carry a mutation in a gene linked to syCL/P. We also identified a new phenotype in blepharocheilodontic syndrome 2: imperforate anus. These results show that WES is an important tool for identifying the genetic cause of CL/P. For the remaining patients for which we have not identified pathogenic variants in candidate genes, we will enlarge our research towards the rest of the genes in the human genome to identify new genes for clefts.

References:.

Grants: FNRS n°40000521.

Conflict of Interest: None declared.

P05.037.B Novel SDR9C7 mutation causes lamellar ichthyosis in a Spanish patient

Carlos Gutiérrez-Cerrajero^1;2;3, Nerea Gestoso-Uzal^1;2;3, Daniel Salete Granado^1;2, Janet Sotolongo-Ravelo ¹, David Diez-Castro^1;2, Ana-Belén Herrero^1;2;3, Ángela Hernández-Martín⁴, Rogelio González-Sarmiento^1;2;3

¹Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ²Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), Salamanca, Spain; ⁴Department of Dermatology, Niño Jesús Hospital, Madrid, Spain

Background/Objectives: Ichthyoses are a group of diseases characterized by dry, scaly skin and a disruption of skin barrier function(1). Lamellar ichthyosis (LI) is a severe variant (1) and SDR9C7 has recently been identified as one of the causal genes for LI. To date, four mutations in SDR9C7 have been linked to LI: two in Lebanese families in 2016, one in a Japanese woman in 2016 and one in a Pakistani family in 2017. Here we present a fifth mutation in a Spanish patient.

Methods: The patient was admitted to Hospital Niño Jesús in Madrid. Genomic DNA was extracted from peripheral blood by standard phenol/chloroform protocol, mutations were found using whole-exome sequencing and validated by Sanger sequencing. Candidate mutations were analysed using the predictors SIFT and Polyphen, as well as the databases ClinVar and Varsome.

Results: The patient shows ichthyosis with dark scales and sporadic superficial desquamation, hyperactivity and microcephaly. Whole exome sequencing revealed the patient was homozygous for mutation c.95G>A, p.Gly32Asp in SDR9C7 and Sanger sequencing validated it. The parents were heterozygous for this mutation. This mutation was not included in ClinVar, but SIFT, Polyphen and Varsome predicted its effect to be pathogenic or likely pathogenic.

Conclusion: We report the discovery of a novel mutation in SDR9C7 that causes LI. This is the first mutation discovered worldwide and the first in a European patient.

References: 1. V. Oji et al., J. Am. Acad. Dermatol. 63, 607–641 (2010).

Grants: This project was funded by FIS-FEDER PI20/01569.

Conflict of Interest: None declared.

P05.038.C Novel RIPK4 variants cause ectodermal dysplasia and alter cell-cell adhesion

Chiara De Luca ¹, Rosanna Monetta^1;1, Elisabetta Botti², Manuel Belli³, Maria Grazia Palmerini¹, Marco Salvatore⁴, Lucia Militti⁵, Arianna Di Daniele¹, Elena Cicchetti¹, Daniele Castiglia⁶, Francesco Brancati¹, Paola Fortugno⁷

¹University of L’Aquila, L’Aquila, Italy; ²University of Rome Tor Vergata, Rome, Italy; ³University of L’Aquila, L’Aquila, Italy; ⁴Istituto superiore di sanità, Rome, Italy; ⁵ASL1 Abruzzo, L’Aquila, Italy; ⁶Istituto dermopatia dell’Immacolata IRCCS, Rome, Italy; ⁷IRCCS San Raffaele Pisana, Rome, Italy

Background/Objectives: Ectodermal Dysplasia Syndactyly Syndrome (EDSS1) is caused by mutations in PVRL4 encoding nectin-4, a component of adherens junctions. EDSS1 shares cutaneous syndactyly (a mild form of pterygia) with other conditions such as Bartsocas-Papas syndrome caused by biallelic mutation in RIPK4, a key player in epidermal development, differentiation, and skin integrity. Here we report two siblings with a phenotype resembling EDSS1, with biallelic variants in RIPK4 and studied their functional effect.

Methods: Exome sequencing. Western blotting, autophosphorylation, proteasome inhibition and immunofluorescence analyses in patient’s primary keratinocytes and transfected HEK293 cell line. Immunofluorescence, haematoxylin and eosin staining and transmission electron microscopy on patient’s skin.

Results: Our siblings featured ectodermal dysplasia, plantar hyperkeratosis associated to syndactyly of hands and feet suggestive of EDSS1. After excluding PVRL4 variants, exome sequencing revealed biallelic likely pathogenic variants in RIPK4, inherited from heterozygous unaffected parents. Reverse phenotyping revealed that one brother was born with “closed eyes” (ankyloblepharon) and oral synechiae, surgically treated at birth, typically seen in RIPK4-related disorders. Functional studies showed that RIPK4 deficiency impaired cell adhesion organization downregulating PVRL4/nectin-4 expression through IRF6 transcription factor. Also, PKP1/DSG1/DSP altered expression or localization was detected and desmosomes showed abnormal morphology at electron microscopy.

Conclusion: This work further expands the clinical spectrum seen in RIPK4-pathies highlighting epithelial fusions as diagnostic handles for the disease. In adults, the phenotype coincides with EDSS1. Such clinical overlap is mirrored at functional level, as outlined by this newly uncovered RIPK4-IRF6-nectin-4 axis.

References: Online Mendelian Inheritance in Man, OMIM.

Grants: GR2013-02356227 and RC2020-2756828.

Conflict of Interest: Chiara De Luca full-time, Rosanna Monetta full-time, Elisabetta Botti full-time, Manuel Belli full-time, Maria Grazia Palmerini full-time, Marco Salvatore full-time, Lucia Militti: None declared, Arianna Di Daniele: None declared, Elena Cicchetti: None declared, Daniele Castiglia full-time, RC2020-2756828, Francesco Brancati full-time, GR2013-02356227, Paola Fortugno full-time.

P05.039.D A unique COL2A1 phenotype as a result of partial gene deletion

Sena Cetin ^1;2, Mustafa Gunes^1;2, Filiz Ozen², Elif Yilmaz Gulec^1;2

¹Istanbul Medeniyet University Medical School, Department of Medical Genetics, Istanbul, Turkey; ²Istanbul Goztepe Prof. Dr. Suleyman Yalcin City Hospital, Department of Medical Genetics, Istanbul, Turkey

Background/Objectives: Monoallelic COL2A1 (OMIM *120140) pathogenic variants cause various syndromes associated with several skeletal and ocular disorders such as achondrogenesis, chondrodysplasia, SED congenita, Spondyloperipheral dysplasia, Kniest dysplasia, Stickler syndrome type I, avascular necrosis of the femoral head.

Methods: A 24-year-old male patient referred with the complaint of short stature, skeletal deformities, arthralgia in weight bearing joints, easy fatiguability and high degree myopia. The patient and his skeletal survey were evaluated and multigene panel was performed and followed by multiplex ligation-dependent probe amplification (MLPA) assay.

Results: The patient had acro-mesomelic short stature with short and deformed forearms, legs, hands and feet. X-ray images showed bilateral thickened and short metatarsal and metacarpal bones, bilateral short ulnas, shortened and wide phalanxes. Pelvis MRI showed bilateral avascular necrosis of the femoral heads. Ophthalmologic examination revealed retinal detachment due to high degree myopia. Heterozygous deletion between exons 51-54 was detected in MLPA performed as a result of suspected CNVs detected in exon sequencing.

Conclusion: To date, the majority of variants reported in COL2A1 are single nucleotide changes. Only 2 cases with COL2A1 whole exon deletion have been reported, which have the phenotype of Stickler Syndrome Type 1(1). Our case is unique having a novel phenotype, which possess overlapping features of various COL2A1 related disorders with a monoallelic large deletion.

References: (1) Richards, Allan J et al. “Stickler syndrome and the vitreous phenotype: mutations in COL2A1 and COL11A1.” Human mutation vol. 31,6 (2010): E1461-71. https://doi.org/10.1002/humu.21257.

Grants:.

Conflict of Interest: None declared.

P05.040.A Sudden cardiac death - a new phenotypic aspect of PLACK syndrome?

Ceren Damla Durmaz^1;2, Aysel Ünal²

¹Hacettepe University, School of Medicine, Department of Medical Genetics, Ankara, Turkey; ²University of Health Sciences, Gazi Yaşargil Training and Research Hospital, Department of Medical Genetics, Diyarbakır, Turkey

Background/Objectives: PLACK syndrome (Peeling skin, Leukonychia, Acral punctate keratoses, Cheilitis and Knuckle pads syndrome, OMIM 616295) is an extremely rare genodermatosis caused by biallelic mutations in CAST. Here, we report a previously unreported CAST variant in a family with PLACK syndrome which may reveal an unexplored aspect of this syndrome for the first time: the sudden cardiac death (SCD).

Methods: Whole exome sequencing (WES) was employed to explore the molecular etiology of a dermatological phenotype comprised of punctate palmoplantar keratoderma, angular cheilitis and hyperkeratosis of the knees in two sisters (4 and 8 years old).

Results: A novel homozygous nonsense mutation (NM_001042440.5: c.1759C>T; p.Gln587Ter) in CAST was identified in both sisters, which segregated with the dermatological phenotype in family. During the follow-up, dilated cardiomyopathy became evident in the proband and resulted in SCD similar to her two affected brothers who died suddenly at 3 and 4 years. WES did not reveal any other pathogenic variants that may explain cardiomyopathy/sudden cardiac death.

Conclusion: CAST encodes calpastatin, an endogenous inhibitor of calpain. Calpain responds to increased Ca²⁺ in myocardial cells, leading to myocyte death by cleaving structural and functional proteins of myocytes during cardiac ischemia, which is inhibited by calpastatin. Although SCD/cardiomyopathy is not a known feature present in the seven reported families with PLACK syndrome, considering the function of CAST in myocardium, SCD/cardiomyopathy may be a hidden phenotypic feature of the syndrome. Whether this phenotype is specific to the variant identified here is yet to be explored.

References: PMID:24333421.

Grants:.

Conflict of Interest: None declared.

P05.041.B Identification of candidate mutations related to adolescent idiopathic scoliosis in the Caucasian population - focusing on post-zygotic alternations

Monika Horbacz ¹, Marek Rocławski², Marcin Ceynowa², Rafał Pankowski², Arkadiusz Piotrowski¹

¹Medical University of Gdansk, International Research Agenda 3P-Medicine Laboratory, Gdansk, Poland; ²Medical University of Gdansk, Department of Orthopedic Surgery, Gdansk, Poland

Background/Objectives: Adolescent idiopathic scoliosis is a multidimensional and multicausal spine deformity with frequency from 1-4 % in general population. Most studies focused on germline mutation analysis, omitting post-zygotic variation leading to pathological effects in organisms. Post-zygotic changes pop up in the human body de novo during a lifetime and usually is not inherited. “Benign” diseases research shows that a combination of germline and somatic variation that explore in the human body early in a lifetime may negatively impact and speed up the manifestation of the disease.

Methods: Whole-exome sequencing were performed to determine scoliosis-related genes in the Caucasian population of 32 adolescent patients with severe scoliosis, with a significant focus on mosaic variations. Types of biological material used: blood and articular processes.

Results: We identified recurrent missense germline and somatic variants located in OBSCN, NEB and other genes.

Conclusion: Mentioned genes encode proteins that are fundamental components in the assembly and functioning of vertebrate striated muscles. Germline and somatic variations coexistence across them might cause muscle weakness and correlate with scoliosis manifestation and its advanced level.

References: Cheng, J. C. et al. Nat. Rev. Dis. Prim. 1, (2015).

Forsberg, L. A. et al. Nat. Rev. Genet. 18, 128–142 (2017).

Mustjoki, S. et al. N. Engl. J. Med. 384, 2039–2052 (2021).

Murphy, S. et al. Expert Rev. Proteomics 16, 241–256 (2019).

Grants: Project POWR.03.05.00-00-z082/18 co-financed by the European Union through the European Social Fund under the Operational Programme Knowledge Education Development 2014–2020.

Conflict of Interest: None declared.

P05.042.C A case with spondyloenchondrodysplasia with immune dysregulation (SPENCDI) caused by a novel missense ACP5 mutation

nuray ozturk ¹, Dilek Uludağ Alkaya², Gokcen Karamik¹, banu nur¹, Beyhan Tüysüz², ercan mihci¹

¹Akdeniz University School of Medicine, Pediatric Genetics, Antalya, Turkey; ²İstanbul University Cerrahpaşa Faculty of Medicine, Pediatric Genetics, İstanbul, Turkey

Background/Objectives: Spondyloenchondrodysplasia with immune dysregulation (SPENCDI) is a rare autosomal recessive immunoosseous dysplasia, characterized by enchondromatous metaphyseal and vertebral lesions with immune dysfunction and neurologic involvement. SPENCDI is caused by biallelic mutations in the ACP5 gene which encodes tartrate-resistant acid phosphatase, a protein functioning in the type I interferon pathway. Here, we present a case with a novel variant in the ACP5 gene.

Methods: The patient is a 7-year-old male who initially presented with short stature and gait disturbance at the age of 5. He is the first child of consanguineous parents born at term. At the age of 2, he presented to hospital with toe walking, the neurological examination exhibit spasticity and hyperreflexia. Brain-Spinal MRI and EMG were normal. At the age of 5, his height was 95 cm (−3,2 SD), skeletal survey revealed metaphyseal irregularities and platyspondyly. Cranial CT revealed calcifications in the basal ganglia. He was hospitalized for autoimmune hemolytic anemia at age 7. Bone marrow aspiration, autoimmune and immunological screenings were normal.

Results: Whole-exome sequencing analysis displayed a homozygous missense variant of c.389G>T (p.Trp130Leu) in the ACP5 gene, later confirmed by sanger sequencing. It was predicted as a “pathogenic” change conforming ACMG criteria in VARSOME database and wasn’t reported in the literature. Parents were found to be heterozygous carriers of the variant.

Conclusion: SPENCDI is a clinically heterogeneous disease with distinctive skeletal findings and pleiotropic extra-osseous phenotype. We aimed to raise awareness of SPENCDI, which has multisystem involvement and a significant risk of morbidity and mortality. References:.

https://doi.org/10.1007/s10875-016-0252-y.

Grants: None.

Conflict of Interest: None declared.

P05.043.D Identification of effector cell types for orofacial clefting through integration of murine single-cell expression data and GWAS results

Anna Siewert ¹, Julia Welzenbach¹, Benedikt Reiz², Elisabeth Mangold¹, Henning Dickten², Kerstin Ludwig¹

¹Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Genomics, Bonn, Germany; ²FASTGenomics, Comma Soft AG, Bonn, Germany

Background/Objectives: Orofacial clefting (OFC) is among the most common human birth defects. Although about 60 risk loci have been identified to date, the functional consequences of the identified variants are still largely unknown, as are the cell types in which the candidate genes exhibit their effects. We here analyzed the gene expression patterns of OFC candidate genes in recently published single-cell RNA sequencing (scRNA-seq) data from embryonic mice.

Methods: We re-analyzed scRNA-seq data from the mouse face (~8,000 cells at E11.5)[1] and whole embryos (~1.4 million cells E9.5–E13.5)[2] using Seurat v4 and scCATCH. The analyses were reproducibly run on the FASTGenomics platform.

Results: We observed predominant expression of OFC candidate genes in epithelial cells (e.g. Irf6, Grhl3, Tfap2a), and in a combination of chondrocytes, osteoblasts and progenitor cells of connective tissue, jaw and teeth (e.g. Fgfr1, Fgf10, Mmp16). Furthermore, we found that Irf6, Grhl3 and Tfap2a are co-expressed in an epithelial cell sub-population and additional OFC candidate genes are specifically expressed in Irf6+ epithelial cells.

Conclusion: Epithelial cells are involved in processes of proliferation and patterning of the mesenchyme and are therefore one of the crucial cell types during craniofacial development. Our results further suggest a distinct epithelial cell sub-population in which OFC candidate genes are active during this developmental timeframe. We will follow this up by studying a more systematic enrichment of OFC candidate genes on a cell-type and single-cell level using single-cell disease relevance scores[3].

References: [1] Li et. al 2019.

[2] Cao et. al 2019.

[3] Zhang et. al 2021.

Grants:.

Conflict of Interest: Anna Siewert: None declared, Julia Welzenbach: None declared, Benedikt Reiz Comma Soft AG, Elisabeth Mangold: None declared, Henning Dickten Comma Soft AG, Kerstin Ludwig: None declared.

P05.044.A Exploring genotype-phenotype correlations, penetrance and expressivity of HOXD13 associated synpolydactyly in a cohort of 17 families with HOXD13 variants

Annika Gottschalk ¹, Henrike Sczakiel^1;2;3, Wiebke Hülsemann⁴, Sarina Schwartzmann¹, Angela Abad Perez¹, Malte Spielmann^5;6, Stefan Mundlos^1;2, Denise Horn¹, Martin Atta Mensah^1;7;7

¹Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institut für Medizinische Genetik und Humangenetik, Berlin, Germany; ²Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany; ³Berlin Institute of Health at Charité – Universitätsmedizin Berlin, BIH Biomedical Innovation Academy, BIH Charité Junior Clinician Scientist Program, Berlin, Germany; ⁴Kinderkrankenhaus Wilhelmstift, Handchirurgie, Hamburg, Germany; ⁵Universität zu Lübeck, Institut für Humangenetik Lübeck, Lübeck, Germany; ⁶Max Planck Institute for Molecular Genetics, Human Molecular Genomics Group, Berlin, Germany; ⁷Berlin Institute of Health at Charité – Universitätsmedizin Berlin, BIH Biomedical Innovation Academy, BIH Charité Digital Clinician Scientist Program, Berlin, Germany

Background/Objectives: The homeobox transcription factor HOXD13 is an important regulator of embryonic limb development. Mutations leading to haploinsufficiency of HOXD13 – typically expansions of a polyalanine repeat in exon 1 – cause synpolydactyly type 1. How these contribute to genotype-phenotype correlations, penetrance and expressivity of HOXD13 associated synpolydactyly remains mostly illusive. We present a large cohort of 43 affected individuals from 17 families with HOXD13 variants.

Methods: 41 patients with synpolydactyly suggestive of HOXD13 haploinsufficiency were selected for analysis of HOXD13 (NM_000523.4) by Sanger sequencing, microsatellite analysis and next generation sequencing.

Results: We identified 15 causative variants and 2 variants of uncertain significance in HOXD13. The most frequent variants (12/17) were repeat expansions of the alanine stretch (5 expansions by 8 and 7 by 7 alanines). Additionally, we identified 3 (likely) pathogenic variants (one frameshift, one stop-gain, one single amino acid deletion) and 2 missense variants of uncertain significance. Pedigree and/or segregation analysis of these index patients revealed a total of 43 affected individuals. The observed phenotypes ranged from unaffected carriers to severe osseous synpolydactyly with phenotypic heterogeneity occurring not only across but also within families and asymmetrically affected individuals. We could also corroborate a previously suggested genotype-phenotype correlation of longer alanine repeat expansions with more severe phenotypes.

Conclusion: In a large cohort of 43 affected individuals from 17 families with HOXD13–associated synpolydactyly we could identify variable penetrance and expressivity of HOXD13 variants and confirm a positive correlation of alanine repeat expansion length and phenotypic severity.

References:.

Grants:.

Conflict of Interest: None declared.

P05.045.B Incontinentia pigmenti female with the NEMOdel4-10 deletion in the IKBKG/NEMO gene in a mosaic form

Ezia Spinosa ¹, Michele Salvia¹, Carmela Casale¹, Alessandra Pescatore¹, Annalaura Torella^2;3, Giulio Piluso², Vincenzo Nigro^2;3, Vincenzo Piccolo⁴, Andrea Diociaiuti⁵, Maya El Hachem⁵, Matilde Valeria Ursini¹, Francesca Fusco¹

¹Institute of Genetics and Biophysics “Adriano Buzzati- Traverso” IGB-CNR, Naples, Italy; ²Dipartimento di Medicina di Precisione, Università della Campania “Luigi Vanvitelli”, Naples, Italy; ³Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; ⁴Dermatology Unit, University of Campania Luigi Vanvitelli, Naples, Italy; ⁵Dermatology Unit, Bambino Gesù. Children’s Hospital—IRCCS, Rome, Italy

Background/Objectives: Incontinentia pigmenti (IP; OMIM#308300) is an X-linked dominant disease, generally lethal in male, caused by mutations in IKBKG/NEMO gene, essential for NF-κB activation. The IP phenotype is characterized by typical skin lesions and by neuroectodermal defects that contribute to a wide variability of severity of disease.

Here we report the case of IP female with neurological and ocular impairment, carrying the recurrent deletion in NEMO/IKBKG gene as a somatic mutation. Moreover, the patient showed also a de novo pathogenic variant c.1708_1709del, p.Ser570fs*27in MED13L gene.

Methods: An IP female patient with severe form of IP, confirmed by skin biopsy, was analyzed in the IP locus on DNA from blood and the presence of NEMOdel4-10 was detected by long-range PCR and quantified by QPCR.

Results: The IP locus analysis revealed the presence of de novo NEMOdel4-10 deletion and excluded the presence of the risk alleles for IP (MER67dup, NEMOPdel). Somatic mosaicism was strongly suggested by quantitative analysis of the ratio of allele mutated versus wild-type allele in genomic DNA from blood. Consistent with somatic mosaicism, the sample of patient had lower ratios of mutant versus wild-type allele compared to the fully heterozygote IP female control.

Conclusion: Postzygotic genetic mosaics for the IKBKG/NEMO mutation are reported only in IP male, indeed the IP phenotype is almost entirely restricted to females heterozygous for IKBKG/NEMO gene mutation. This is the first report demonstrating mosaicism as a cause of IP in female. To note that the contribute of constitutive alteration in MED13L gene (NM_015335.5: c.1708_1709del) will be discussed.

References:.

Grants:.

Conflict of Interest: None declared.

P05.046.C Two new PRKG1 gene mutations among Polish patients with Marfan syndrome and related disorders

Maria Pilarska-Deltow ¹, Marzena Skrzypczak-Zielinska², Anna Junkiert-Czarnecka¹, Aneta Bąk¹, Marta Heise¹, Olga Haus¹

¹Department of Clinical Genetics, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland; ²Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland

Background/Objectives: Hereditary connective tissue disorders are a group of over two hundred diseases described so far, with Marfan syndrome (MFS) as the best known. MFS overlaps symptoms with such marfanoid syndromes as Loeys-Dietz and the vascular subtype of Ehlers-Danlos syndrome, making differential diagnosis extremely difficult. MFS and MFS-like syndromes are caused by damage to the connective tissue of different systems. One of the main features of MFS-like syndromes is ascending aorta dissection, and aortic aneurysm. According to current knowledge, PRKG1 gene is on the list of genes related to the pathogenesis of aneurysms.

Methods: NGS of 12 gene panel sequencing was performed for 105 Polish patients with suspicion of Marfan or a Marfan-like syndrome. Control group consisted of 100 people, healthy at the time of the examination, without family history of MFS or MFS-like syndromes.

Results: As the result of the analysis, two mutations in PRKG1 gene were detected. Both, c.1040C>G (p.Ser347Cys) and c.1075A>C (p.Lys359Gln) mutations were new, not registered in internet databases or reported in the literature. They also were absent in the control group. According to VarSome, c.1040C>G was classified as VUS, and c.1075A>C as pathogenic mutation. The patient with the first mutation was a 12-year-old boy with physical features of MFS. The 9-year-old girl with the second mutation had only excessive joint mobility. They both had no cardiovascular problems, possibly due to their young age.

Conclusion: The results of the study expands the mutational spectrum of PRKG1 and may help in prevention, early diagnosis and treatment/management of MFS and MFS-like syndromes.

References:.

Grants:.

Conflict of Interest: None declared.

P05.047.D Internal Skeletal Dysplasia Registry within the electronic database of Department of Clinical Genetics University Children’s Hospital in Belgrade - basis for a personalised medicine in the future

Marija Mijovic ¹, Goran Cuturilo^1;2, Jelena Ruml Stojanovic¹, Aleksandra Miletic¹, Brankica Bosankic¹, Hristina Petrovic¹

¹University Children’s Hospital, Department of Clinical Genetics, Belgrade, Serbia; ²Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background/Objectives: Skeletal dysplasia are heterogeneous group of genetic disorders affecting skeletal development. Progress in molecular genetic testing and up-to-date knowledge from pre-clinical trials have been leading to novel therapeutic approaches for genetic skeletal disorders despite the challenges of drug development in rare diseases. Timely diagnosis and patients’ information availability is crucial for early and proper application of new treatment options.

Methods: We systematically analyzed data from the internal electronic database of our genetic service in order to create an internal sub-register of patients with genetic skeletal disorders.

Results: In the past six years, 137 patients with suspected genetic skeletal disorders have been referred to the Department of Clinical Genetics, University Children’s Hospital in Belgrade. Next generation sequencing was performed for 64 patients with suspected heterogeneous skeletal dysplasia, among them there are confirmed cases of very rare or severe skeletal diseases. Also, single gene sequencing or specific mutation analysis was performed for 27 patients according to clinical suspicion. Other patients are clinically monitored or their proposed genetic testing is in progress. We categorized all patients according to the latest classification of genetic skeletal disorders. The FGFR3 chondrodysplasia group has 19 pediatric patients, with three familial cases of achondroplasia.

Conclusion: The Internal Skeletal Dysplasia Registry should allow for better monitoring of data and visibility results of genetic testing for these patients so that they are timely included specific treatments in the context of personalised medicine in the future.

References: Non applicable.

Grants: Non applicable.

Conflict of Interest: None declared.

P05.048.A A most unusual phenotype in a patient with a mosaic ASXL1 deletion

Destrez Alban¹, Guillaume Jedraszak², Severine Fritot³, Catherine Gondry⁴, Sophie Bryselbout⁵, Marie-Christine Plancq⁶, Cica Gbaguidi⁷, Bénédicte DEMEER ⁸

¹CHU Amiens Picardie / UPJV / Institut Faire Faces, Department of maxillo-facial surgery / UR 7516 CHIMERE, Amiens, France; ²CHU Amiens Picardie, Laboratory of Molecular Genetics, Amiens, France; ³CHU Amiens Picardie, Department of pediatric physical medicine and rehabilitation, Amiens, France; ⁴CHU Amiens Picardie, Department of Radiology, Amiens, France; ⁵CHU Amiens Picardie, Department of Ophthalmology, Amiens, France; ⁶CHU Amiens Picardie, Department of Pediatric orthopaedics, Amiens, France; ⁷CHU Amiens Picardie, Department of maxillo-facial surgery, Amiens, France; ⁸CHU Amiens Picardie / UPJV / Institut Faire Faces, Department of medical genetics / UR 7516 CHIMERE, Amiens, France

Background/Objectives: Bohring-Opitz syndrome (BOS), caused by loss of function mutation in ASXL1, is characterized by distinctive facial features and posture, growth failure, intellectual disability, and variable anomalies.

Methods: We report a patient carrying a mosaic ASXL1 deletion (arr[hg19]20q11.21(31,004,670-31,147,256)x1) found in approximately 30% of cells from both lesional facial tissue and blood.

Results: She is the first child of unrelated parents, with no familial history. Diagnosis of bilateral cleft of the lip and the palate (BCLP) was made on the 24GW ultrasound. She was born at 37GW with BW:2.680kg, BH:46cm, BHC:32.5cm. A right partial ablepharon and a temporal cutaneous band, 2 scalp cutaneous outgrowths and scalp defect, a left interrupted superior eyelid with irregular arched eyebrows and a pseudo temporal cutaneous band, altogether defining a left Tessier #2, #9 and right Tessier #3, #9 facial cleft, and a right microtia were diagnosed. She had hypoplasia of the right hand with metacarpophalangeal joint hyperextension contrasting with flessum of the proximal and distal interphalangeal joints. On her left-side she had foot hypoplasia, tarsal malformation, instep fatty hypertrophy, IV and V hypoplasia. She had normal psychomotor development. At age 6 years, when last seen, she had normal growth parameters and no intellectual disability. Left upper limb hyperplasia became more evident as she grew.

Conclusion: We consider the deletion as causal and the asymmetry due to the mosaicism. Nethertheless, because of most unusual phenotype, further molecular studies were discussed. No mosaic mutation of KRAS or FGFR1 was found on DNA from lesional tissue, WES studies are ongoing.

References:.

Grants:.

Conflict of Interest: None declared.

P05.049.B A progeroid syndrome with severe osteogenesis imperfecta segregates with an intronic TAPT1 homozygous variant that creates a knockout allele

nasrinsadat nabavizadeh ^1;2;3, Annkatrin Bressin⁴, PohHui Chia¹, Ricardo Moreno Traspas¹, Nathalie Escande-Beillard^1;3, Carine Bonnard¹, Zohreh Hojati², Scott Drutman⁵, Jean-Laurent Casanova^5;6;7, mohammad Shboul⁸, Andreas mayor⁴, Bruno Reversade^1;3;9

¹Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore., Singapore, Singapore; ²Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran., Isfahan, Iran; ³Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey., Istanbul, Turkey; ⁴Max Planck Institute for Molecular Genetics, Berlin, Germany., Berlin, Germany; ⁵St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, New York, United States; ⁶Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France., Paris, France; ⁷University of Paris, Imagine Institute, Paris, France., Paris, France; ⁸Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan., Irbid, Jordan; ⁹Institute of Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, Proteos, Singapore., singapore, Singapore

Background/Objectives: Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, contain structural, regulatory, and transcribed information that cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA sequencing a powerful companion diagnostic.

Methods: Here, we identified five patients with a recessive Osteogenesis Imperfecta (OI) syndrome characterized by bone defects and neonatal progeria. We integrated results obtained from homozygosity mapping, genome and RNA sequencing to find the causative gene.

Results: We delineated a non-coding TAPT1 mutation (c.1237-52G>A) that segregated with the disease. This private mutation, which is predicted to serve as an alternative splicing branchpoint, results in exon 12 skipping and creates a protein-null allele. Functional studies performed on patients’ fibroblasts support the notion that 1) TAPT1 resides in the ER/Golgi, 2) is not an essential receptor for human Cytomegalovirus (HCMV) and 3) controls pathways involved in collagen and extracellular matrix biology.

Conclusion: Overall, our work highlights the power of transcriptomic approaches in identifying genetic defects as well as in illuminating the molecular mechanisms and underlying dysregulated pathways in human diseases.

References:.

Grants: This work was supported by a Strategic Positioning Fund on Genetic Orphan Diseases (GODAFIT) and a Use-Inspired Basic Research (UIBR) grant from Agency for Science, Technology and Research (A*STAR) in Singapore to B.R. This work was also funded by the Max Planck Society (to A.M.) and the Deutsche Forschungsgemeinschaft (DFG, grant 418415292 to A. M.).

Conflict of Interest: nasrinsadat nabavizadeh: None declared, Annkatrin Bressin: None declared, PohHui Chia: None declared, Ricardo Moreno Traspas: None declared, Nathalie Escande-Beillard: None declared, Carine Bonnard: None declared, Zohreh Hojati: None declared, Scott Drutman: None declared, Jean-Laurent Casanova: None declared, mohammad Shboul: None declared, Andreas mayor: None declared, Bruno Reversade Principle investigator.

P06 Cardiovascular Disorders

P06.001.C Diagnostic yield of a NGS panel in a Brugada syndrome cohort

Eline Simons ¹, Ewa Sieliwonczyk¹, Maaike Bastiaansen¹, Ilyas Louarroudi¹, Bart Loeys¹, Maaike Alaerts¹

¹University of Antwerp, Center of Medical Genetics, Edegem, Belgium

Background/Objectives: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia disorder affecting 1/2000 individuals. Its diagnosis requires presence of a spontaneous or sodium channel blocker induced ST-segment elevation on an electrocardiogram (ECG). BrS patients are at risk for ventricular fibrillations which could lead to sudden cardiac death. In general, only for 25-30% of the patients a genetic diagnosis can be established in one of the BrS associated genes, of which 20-25% carry a variant in the SCN5A gene.

Methods: We collected clinical history, ECG parameters and genetic results of 294 BrS patients (61% male) screened with a diagnostic panel for inherited primary electrical disorders covering initially 51 and in a later version 60 genes.

Results: In total, 43.5% of patients carried a variant of uncertain significance (VUS, class 3; n = 102) or (likely) pathogenic variant (class 4 and 5; n = 26) following the ACMG guidelines. Most of the class 4/5 variants are found in the SCN5A gene (23/26), whereas the remainder were identified in KCNE1/LMNA/SCN2B. 43.9% of patients had a Shangai score above 3.5 (definite BrS) of which 14.7% carried a class 4/5 variant. Only 4.2% of patients with a Shangai score between 2 and 3 carried a (likely) pathogenic variant. Of the 22.4% of patients with a familial history, 20% carried a class 4/5 variant.

Conclusion: The overall diagnostic yield in our cohort is 8.8%, increasing to 15% in BrS patients with a definite diagnosis, or 20% in clear familial patients, which is slightly lower than reported in literature.

References:.

Grants: Research Foundation Flanders.

Conflict of Interest: None declared.

P06.002.D Clinical characteristics, genetic findings and arrhythmic outcomes of patients with catecholaminergic polymorphic ventricular tachycardia across the globe

Danny Radford ^1;2, Emma Coakley-Youngs^1;2, Meltem Altinsoy^2;3, sharen lee², elham mahmoudi^2;4, Levent pay⁵, Göksel Çinier⁶, pawel matusik^2;7, George Bazoukis^2;8, Sebastian Garcia-Zamora², Jeremy Man Ho Hui², Yan Hiu Athena Lee², Konstantinos Letsas⁹, Ziliang Chen^2;10, Kamalan Jeevaratnam¹¹, Adrian Baranchuk^2;12, Tong Liu^2;13, Gary Tse²

¹Kent and Medway Medical School, Canterbury, United Kingdom; ²Cardiovascular Analytics Group, Hong Kong, China; ³Diskapi Yildrim Beyazit Training and Research Hospital, Department of Cardiology, Istanbul, Turkey; ⁴Tehran University of Medical Sciences, Tehran, Iran; ⁵Dr Siyami Ersek Hospital Thoracic and Cardiovascular Surgery Training and Research Hospital, Department of Cardiology, Istanbul, Turkey; ⁶Dr Siyami Ersek Hospital Thoracic and Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey; ⁷Jagiellonian University Medical College, Krakow, Poland; ⁸University of Nicosia, Nicosia, Cyprus; ⁹Evangelismos General Hospital of Athens, Athens, Greece; ¹⁰Tianjin Institute of Cardiology, Tiajin, China; ¹¹University of Surrey, Guildford, United Kingdom; ¹²Queen’s University, Kingston, Canada; ¹³Second Hospital of Tianjin Medical University, Tianjin, China

Background/Objectives: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare cardiac ion channelopathy. This study examined the clinical characteristics, genetic basis and arrhythmic outcomes of CPVT patients globally.

Methods: PubMed was systematically searched for case reports or series on CPVT. Clinical characteristics, genetic findings and primary outcome of spontaneous ventricular tachycardia/ventricular fibrillation (VT/VF) were analysed.

Results: A total of 442 patients (mean presentation age: 15±12-years-old, 52% male) were included. On presentation, 368 patients (83%) were initially symptomatic and 214 (48%) had VT. PVCs were present in 356 patients (81%) and VT was present in 352 patients (80%). Genetic tests were performed on 257 (58%) patients with a yield of 98%. RyR2, CASQ2, TERCL and KCNJ2 mutations were found in 232(89%), 27(10%), 2 (0.8%) and 1 (0.4%) patients, respectively. Out of 336 patients, 302 (90%) were prescribed beta-blockers, 88 (29%) were prescribed flecainide, 82 (27%) were prescribed propranolol, 26 (9%) were prescribed atenolol, 24 (8%) were prescribed verapamil, 4 (1%) were prescribed propafenone. Out of 442, implantable-cardioverter defibrillator (ICD) were inserted for 120 (27%) patients and sympathectomy was performed on 32 (7%) of patients. On follow-up, 73 patients (38%) had VT/VF.

Conclusion: This is the first systematic review and meta-analysis of CPVT cases globally. Most patients had symptoms on initial presentation and approximately half had VT as the presenting complaint. RyR2 mutations accounts for the majority (89%) of the CPVT cases, followed by CASQ2 (10%), then TERCL and KCNJ2 mutations (1%). Most patients received beta-blocker therapy. 7% had sympathectomy and 27% had ICDs implanted.

References: None.

Grants: None.

Conflict of Interest: None declared.

P06.003.A Association of the genetic variation in the long non-coding RNA FENDRR with the risk of developing Hypertrophic Cardiomyopathy

Elías Cuesta Llavona ¹, Rebeca Lorca¹, Israel David Duarte Herrera¹, Belén Alonso¹, Sara Iglesias Álvarez¹, Jose Julian Rodriguez Reguero¹, Eliecer Coto¹, Juan Gómez¹

¹Central University Hospital of Asturias, Oviedo, Spain

Background/Objectives: Hypertrophic Cardiomyopathy (HCM) is the most common hereditary heart disease. However, in around 40-60% of cases no pathogenic variants are identified in the exome. Studies suggest that there are other genetic factors that could explain part of the risk of developing HCM. A group of candidate genes to be evaluated are those that encode lncRNAs. The aim of this study was to evaluate the possible association of lncRNAs with the risk of developing HCM.

Methods: We sequence a total of 238 index cases of HCM (58 ± 16 years old, 63% male) and 212 controls (70 ± 7 years old; 45% male) through a panel of 10 lncRNAs coding genes that have been associated with cardiovascular disease (H19, KCNQ1OT1, MHRT, CARMEN, FENDRR, TINCR, ANRIL, MIAT, PVT1, MALAT1) with semiconductor chips and the Ion GeneStudio S5 Sequencer (Ion Torrent). The Chi-square test were used to compare allelic frequencies in the polymorphisms identified.

Results: We observed that FENDRR rs39527 A> G, rs39529 G>C and rs40384 T>C polymorphisms were significantly associated with the risk of developing HCM in our cohort (0.006 patients vs 0.03 controls; p = 0.0274; OR: 0.2381; IC: 0.0660-0.8594).

Conclusion: In summary, this study identified the significant protective effect of the rare allele in the FENDRR rs29527, rs39529 and rs40384 polymorphisms on this disease in a Spanish population. These variants could involve a change in the structure of FENDRR that would modify the regulation of gene expression controlled by this lncRNA. However, the functional relevance of this change requires experimental validation.

References:.

Grants: FIS PI17/00648.

Conflict of Interest: None declared.

P06.005.C Pathogenic variants are distributed among different genes when comparing adult LQTS patients to infants

Alexander Moscu-Gregor ¹, Christoph Marschall¹, Sabine Lippert¹, Sebastian Eck¹, Konstanze Hörtnagel¹, Imma Rost¹

¹MVZ Martinsried GmbH, Medicover Group, Martinsried, Germany

Background/Objectives: Congenital long-QT-syndrome (LQTS) is a rare heart disease characterized by a prolonged ventricular repolarization leading to a QTc of more than 460 ms. Patients are usually between 5 and 18 years old when diagnosed. However, little is known about the genetic background of severe LQTS in the very young (especially neonates/infants). This raises the question which variants in which genes are responsible for the early manifestation.

Methods: Between January 2017 and December 2021, 1391 index patients with suspected LQTS were referred to our laboratory for genetic testing. All the genes for which there was at least limited evidence of association with LQTS were examined. The patients were sorted by age (neonate/infant/toddler/>4years), and it was determined which genes were affected in each age group.

Results: Of the 36 toddlers examined, 8 were confirmed genetically positive. All 8 patients carried variants exclusively in the KCNQ1 gene, indicating that this gene is overrepresented when compared to the 1271 older (>4years) patients (≈45% of positive cases). Additionally, pathogenic variants of one gene were detected in neonates or infants with severe LQTS only: CACNA1C.

Conclusion: In the very young patients (<4 years), the variants are distributed among different genes than in adults. This can be used for variant classification according to ACMG/ACGS guidelines. Criterion PP4, which refers to phenotype specificity, could be assigned to CACNA1C variants if a severe LQTS phenotype is present in a neonate/infant. Moreover, PP4 could be modified to a moderate or strong pathogenic criterion for KCNQ1 variants detected in toddlers.

References:.

Grants:.

Conflict of Interest: None declared.

P06.006.D Heart transcriptome profile of a novel transgenic mouse model for arrhythmogenic cardiomyopathy

Martina Calore ¹, Sofia Compagno², Sara Vencato², Robin Colpaert¹, Claudia Sacchetto¹, Alessandra Lorenzon², Tommaso Becchi², Nicola Facchinello³, Chiara Romualdi², Leon de WIndt¹, Paola Braghetta³, Libero Vitiello², Alessandra Rampazzo²

¹Maastricht University, Department of Molecular Genetics, Maastricht University, The Netherlands, Maastricht, Netherlands; ²Padova University, Department of Biology, University of Padova, Italy., Padova, Italy; ³Padova University, Department of Molecular Medicine, University of Padova, Italy, Padova, Italy

Background/Objectives: Arrhythmogenic cardiomyopathy (ACM) is one of the most commonly inherited cardiomyopathies, characterized by the progressive substitution of the myocardium with fibrofatty tissue (1). Clinically, ACM is characterized by ventricular arrhythmias, syncope, and sudden cardiac death and shows wide phenotypic heterogeneity (1). Of the known disease genes, desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are most commonly mutated (2). To study the pathogenic mechanisms of ACM, we generated a novel mouse model for the disease.

Methods: We generated transgenic mice overexpressing desmoglein-2 carrying the p.G100R mutation (TgG) found in an affected patient. To establish the transcriptomic ACM signature, we performed RNA seq on cardiac samples from 6 month-old TgG and control mice.

Results: TgG mice present several of the clinical features of ACM, such as fibrous replacement, and increased distance between cardiomyocyte membranes. Importantly though, we did not detect the same reductions of the canonical Wnt/b-catenin signalling pathway reported in other ACM models, indicating that additional pathways must be perturbed in TgG. Enrichment analysis and network construction identified upregulation of immune- and extracellular matrix-related processes, as well as epigenetic mechanisms, such as histone acetyltransferase activity. By contrast, downregulated processes included the MAPK and TGF-ß pathways.

Conclusion: Collectively, these findings identified numerous processes potentially altered in TgG mice that could pave the way for further studies focused on ACM pathogenic mechanisms.

References: (1) Thiene et al.,N Engl J Med (1988)318:129–1331988.

(2) Calore et al.,Cell Tissue Res (2015)360:491–500.

Grants: Hartstichting DCVA2017-2018ARENA-PRIME, Beat the beat donation.

Conflict of Interest: None declared.

P06.007.A Identification of rare genetic variants associated with stroke outcome

Estefanía Alcaide ^1;2;3;4, Núria Martínez-Gil^1;2;3;4, Georgia Escaramís^5;6, Uxue Lazcano⁷, Marina Mola-Caminal⁷, Cristòfol Vives-Bauza⁸, Jordi Jiménez⁷, Israel Fernández–Cadenas⁹, Susanna Balcells^1;2;3;4, Raquel Rabionet^1;2;3;4

¹Universitat de Barcelona, Genètica, Microbiologia i Estadística, Barcelona, Spain; ²Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain; ³Institut de Recerca Sant Joan de Déu (IRSJD), Barcelona, Spain; ⁴CIBERER, Barcelona, Spain; ⁵CIBERESP, Barcelona, Spain; ⁶Institut de Neurociències, Universitat de Barcelona, Barcelona, Spain; ⁷Hospital Del Mar, Servei de Neurologia, Barcelona, Spain; ⁸Universitat de les Illes Balears, Biologia, Palma de Mallorca, Spain; ⁹Hospital de la Santa Creu i de Sant Pau, Barcelona, Spain

Background/Objectives: Stroke is a cerebrovascular disease that may lead to an important adult disability. There is a large variability in the functional outcome after a stroke, which may be in part regulated by genetic factors. With the aim to further investigate the genetics of stroke outcome, we performed exome sequencing followed by targeted resequencing in a set of 702 patients.

Methods: A pilot study was performed with 90 exomes of extreme stroke recovery scores (modified Rankin Scale (mRS) at 90 days 0-1 vs 3-5) and target genes involved in functional outcome were selected. 702 samples were sequenced by targeted next-generation sequencing using a capture assay that included these targets along with selected regions based on previous GWAS results. Here, we performed continuous (mRS 0-6) and dichotomic (mRS 0-1 vs 3-5 and 0-1 vs 3-6) analyses with Bayesian-based rare variant association (BATI)¹ adjusting for multiple testing and selecting rare variants with a CADD score >20.

Results: These analyses highlighted coding rare variants in CNTN5 and VNN2 genes. Coding variants in VNN2, a protein that may act in cell adhesion and migration of neutrophils, were significantly enriched in cases with better outcome. In contrast, rare variants in CNTN5, a protein involved in synaptogenesis, are associated with a poor outcome.

Conclusion: CNTN5 and VNN2 appear to be linked to stroke outcome. Further functional experiments are needed to understand how mutations in these genes lead to differences in recovery.

References: ¹Susak et al., PLoS Comput Biol. 2021 17(2):e1007784.

Grants:.

“La Fundació de la Marató de Tv3” (Reg.70/307_Proj.201726).

Conflict of Interest: None declared.

P06.008.C Penetrance and disease expression of (likely) pathogenic variants associated with inherited cardiomyopathies in the general population

Mimount Bourfiss¹, Marion van Vugt ¹, Abdulrahman Alasiri¹, Bram Ruijsink^1;2, Jessica Van Setten¹, Amand Schmidt^1;3, Dennis Dooijes⁴, Esther Puyol-Antón², Birgitta Velthuis⁵, Peter van Tintelen⁴, Anneline te Riele^1;6, Annette Baas⁴, Folkert Asselbergs^3;4

¹University Medical Center Utrecht, Department of Cardiology, Utrecht, Netherlands; ²King’s College London, London, United Kingdom; ³University College London, London, United Kingdom; ⁴University Medical Center Utrecht, Department of Genetics, Utrecht, Netherlands; ⁵University Medical Center Utrecht, Department of Radiology, Utrecht, Netherlands; ⁶Netherlands Heart Institute, Utrecht, Netherlands

Background/Objectives: (Likely) pathogenic variants associated with arrhythmogenic cardiomyopathy (ACM), dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are recommended to be reported as secondary findings in genome sequencing studies. This provides opportunities for early diagnosis, but also fuels uncertainty in variant carriers (G+), since disease penetrance is incomplete. We assessed the prevalence and disease expression of G+ in the general population.

Methods: We calculated the prevalence of (likely) pathogenic variants associated with ACM, DCM and/or HCM extracted from two databases in the UK Biobank. Furthermore, we analysed the frequency of cardiomyopathy/heart failure diagnosis in individuals carrying these variants (G+). In undiagnosed individuals, we analysed early signs of disease expression.

Results: We found a prevalence of 1:578, 1:251 and 1:149 for (likely) pathogenic variants associated with ACM, DCM and HCM respectively. Compared to controls, cardiovascular mortality was higher in DCM G+, but similar in ACM and HCM G+. More specifically, cardiomyopathy or heart failure diagnosis were more frequent in DCM G+ and HCM G+, but comparable in ACM G+. In contrast, ACM G+ had more ventricular arrhythmias. Left ventricular ejection fraction was reduced in undiagnosed DCM G+ individuals.

Conclusion: In the general population, (likely) pathogenic variants associated with ACM, DCM or HCM are not uncommon. Although G+ have increased mortality and morbidity, disease expression in these carriers from the general population remains low (1.2-4%). Decisions on application of cascade screening and frequency of cardiological examination should be based on multiple factors, such as the gene, variant type and family history.

References:.

Grants:.

Conflict of Interest: Mimount Bourfiss: None declared, Marion van Vugt: None declared, Abdulrahman Alasiri: None declared, Bram Ruijsink: None declared, Jessica Van Setten: None declared, Amand Schmidt Servier and Pfizer funding for unrelated work, Dennis Dooijes: None declared, Esther Puyol-Antón: None declared, Birgitta Velthuis: None declared, Peter van Tintelen: None declared, Anneline te Riele: None declared, Annette Baas: None declared, Folkert Asselbergs: None declared.

P06.009.C Using genotyping and whole-exome sequencing data to improve genetic risk prediction in deep venous thrombosis

Valeria Lo Faro ¹, Therese Johansson^1;2, Åsa Johansson¹

¹Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala, Sweden; ²Womher, Uppsala University, Centre for Women’s Mental Health during the Reproductive Lifespan, Uppsala, Sweden

Background/Objectives: Deep venous thrombosis (DVT) is the formation of blood clots in the deep veins. Blood clots traveling to the lung can cause organ damage or sudden death. More than 60% of DVT risk is influenced by genetic factors, such as the Leiden mutation in F5 (FVL). Characterizing the genetic contribution and stratifying individuals based on their genetic makeup can favourably impact risk prediction.

Methods: We performed a genome-wide association study and constructed a polygenic risk score (PRS) in the 60% (N = 284,591) of the UK Biobank cohort. The remaining 40% (N = 198,362) was employed to evaluate the PRS, and to perform gene-based test on exome-sequencing data to investigate effects by rare variants.

Results: We identified and replicated a new variant (rs11604583) near TRIM51 gene, and a rare variant (rs187725533), associated with 2.2-fold higher risk of DVT, in CREB3L1 gene. The top PRS decile was associated with 3.4-fold risk of DVT, an effect that was still 2.3-fold, when excluding FVL carriers. Cumulative risk of DVT at the age of 70 years for FVL carriers in the top PRS decile is of 10%, contraposed to 5% for non-carriers.

Conclusion: We showed that common and rare variants influence DVT risk, and that the PRS improve risk prediction on top of FVL. This suggests that individuals classified with high PRS score could benefit from early genetic screening.

References: Stone et al. “Deep vein thrombosis: pathogenesis, diagnosis, and medical management.” Cardiovasc Diagn Ther (2017).

Grants: This work was funded by the Swedish Heart-Lung foundation (nr.20200687).

Conflict of Interest: None declared.

P06.010.D Clinical utility of genetic testing in pediatric patients with polymorphic ventricular tachycardia

Magdalena Pelc ¹, Dorota Jurkiewicz¹, Paweł Kowalski¹, Agnieszka Madej-Pilarczyk¹, Joanna Kosińska², Malgorzata Rydzanicz², Piotr Stawiński^2;3, Maria Posadowska⁴, Katarzyna Pręgowska⁴, Monika Brzezinska⁴, Elżbieta Ciara¹, Dorota Piekutowska-Abramczuk¹, Paulina Halat-Wolska¹, Beata Chałupczyńska¹, Marzena Gawlik¹, Dorota Siestrzykowska¹, Dorota Wicher¹, Krystyna Chrzanowska¹, Rafał Płoski², Katarzyna Bieganowska⁴

¹The Children’s Memorial Health Institute, Department of Medical Genetics, Warsaw, Poland; ²Warsaw Medical University, Department of Medical Genetics, Warsaw, Poland; ³Institute of Physiology and Pathology of Hearing, Department of Genetics, Warsaw, Poland; ⁴The Children’s Memorial Health Institute, Department of Cardiology, Warsaw, Poland

Background/Objectives: Primary electrical disorders affect the myocyte transmembrane ion channels and predispose to malignant arrhythmias, including polymorphic ventricular tachycardia (PVT), which is rare in children. Genetically determined channelopathies, such as catecholaminergic PVT (CPVT), long/short QT (LQTS/SQTS) and Brugada syndromes, may be the cause of these arrhythmias. They present with incomplete penetrance and variable expressivity.

Methods: Next generation sequencing (NGS) analysis of genes associated with inherited arrhythmia conditions was performed in 33 Polish patients with documented PVT.

Results: Sixteen patients had a clinically significant variant in: RYR2, KCNH2, KCNJ2, CALM1, SCN5A or 1p13.2 duplication encompassing KCND3. In one CPVT patient two novel biallelic RYR2 variants co-occurred, and in another with LQTS concomitance of RYR2 and KCNH2 defects implied digenic etiology. Additionally, in two probands a rare variant of unknown significance (VUS), favouring pathogenic, in MYBPC3 was noted. Five known VUS in KCNH2, SCN5A, SCN10A, TRPM4 were denoted possibly benign due to high frequency in an in-house Polish database.

Conclusion: Genetic testing has an essential role in PVT diagnosis, influencing risk stratification, preventive and therapeutic management and genetic counseling. NGS-based testing in PVT patients provides good diagnostic yield (~55%), however variant classification criteria should consider the unique characteristics of primary arrhythmias and population aspects. In PVT patients, concomitance of two or more pathogenic variants (biallelic/digenic) may occur, resulting in exacerbation of the phenotype, and phenotypic overlap may be observed in RYR2 or SCN5A-positive cases. Rarely, PVT may be associated with underlying cardiomyopathy.

References:.

Grants: Partially supported: CMHI-M31/18.

Conflict of Interest: None declared.

P06.012.B Family screening of relatives with vascular Ehlers-Danlos syndrome, an asset in preventing arterial events

Jean-Michaël Mazzella ¹, Boris Oehmichen¹, Clarisse Billon^1;2, Michael Frank¹, Xavier Jeunemaitre^1;2, Tristan Mirault^1;2

¹Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Centre de Référence des Maladies Vasculaires Rares, Paris, France; ²University of Paris, INSERM, U970, Paris, France

Background/Objectives: Vascular Ehlers-Danlos syndrome (vEDS) is a rare inherited disorder leading to mainly arterial complications, due to pathogenic COL3A1 variations. Ong et al (Lancet 2010) showed that the introduction of celiprolol significantly reduced arterial events. Screening of relatives allows specific and multidisciplinary management, but the benefit of celiprolol in those without arterial events remains uncertain. We wanted to evaluate the occurrence of arterial events during vEDS relatives follow-up.

Methods: All vEDS relatives diagnosed in our department since 2004 were included. We retrospectively analyzed several criteria (duration of follow-up, presence of arterial events, introduction and dose of celiprolol…).

Results: n = 72 relatives had at least one outpatient visit. Arterial events were found in n = 36 (50%), symptomatic (n = 20) or silent (n = 16). Median age was 44.5yrs vs. 23.0yrs for n = 36 without any arterial event (p < 10^-3). During follow-up, n = 7 (19%) had a first arterial event at 29yrs and were all treated with celiprolol. Duration of treatment was 3yrs at the onset of the first silent event and 10yrs for the first symptomatic event. Celiprolol was introduced in n = 27 (75%) after genetic diagnosis disclosure. Treated relatives had a follow-up duration about 4yrs vs. 2yrs for the untreated ones (p = 0.01).

Conclusion: These results confirm the importance of family screening of vEDS relatives. Despite the introduction of celiprolol, some relatives have presented an arterial event during follow-up. However, our data are probably too low to assess the occurrence of events. A study on probands without arterial events at first outpatient visit could confirm the benefit of celiprolol in all vEDS patients.

References:.

Grants:.

Conflict of Interest: None declared.

P06.014.D Cardiovascular and Connective Tissue Disorder features in FLNA-related PVNH patients: progress towards a refined delineation of this syndrome

Clarisse Billon ^1;2, Salma Adham³, Natalia Hernandez Poblete⁴, Anne Legrand^1;2, Michael Frank^1;2, Laurent Chiche⁵, Stephane Zuily⁶, Karelle Benistan⁷, Laurent Savale⁸, Khaoula Zaafrane Khachnaoui⁹, Anne-Claire Brehin¹⁰, Laurence Bal¹¹, Tiffany Busa¹², Melanie FRADIN¹³, Chloe Quelin¹³, Bertrand Chesneau¹⁴, Denis Wahl⁶, Patricia Fergelot⁴, Cyril GOIZET⁴, Tristan Mirault^1;2, Xavier Jeunemaitre^1;2, Juliette Albuisson^1;2;15

¹AP-HP, Hôpital Européen Georges Pompidou, Genetics Department, National Referral Center for Rare Vascular Diseases, Paris, France; ²2 Université de Paris, INSERM, U970 PARCC, Paris, France; ³University Hospital Center Saint Eloi Hospital, Vascular department, Montpellier, France; ⁴Chu Bordeaux - Site Pellegrin, Department of Medical Genetics, Bordeaux, France; ⁵University Hospitals Pitié Salpêtrière - Charles Foix, Department of Vascular and Endovascular Surgery, Tertiary Aortic Center, Paris, France; ⁶Hospital Center Regional And University De Nancy Hospital Central, Vascular Medicine Division and Regional Competence Center for Rare Vascular And Systemic Autoimmune Diseases, Nancy, France; ⁷Raymond Poincaré University Hospital - (AP-HP), Centre de Référence des Syndromes d’Ehlers-Danlos non Vasculaires, Garches, France; ⁸Bicetre Hospital, Service de Pneumologie, Le Kremlin-Bicêtre, France; ⁹Hospital L’archet, Medical Genetics Unit 2, Nice, France; ¹⁰Hospital Center University De Rouen, Normandy Center for Genomic and Personalized Medicine, Rouen, France; ¹¹Marseille University Hospital Timone, Regional reference center in Marfan and Related Syndroms, Aortic Center, Marseille, France; ¹²Marseille University Hospital Timone, Département de Génétique Médicale, Marseille, France; ¹³South Hospital, Service de Génétique Clinique, Centre de Référence Maladies Rares CLAD-Ouest, Rennes, France; ¹⁴Hospital Center University De Toulouse, Département de Génétique Médicale, Centre de Référence du syndrome de Marfan et des syndromes apparentés, Toulouse, France; ¹⁵Centre G.f Leclerc, Platform of Transfer in Cancer Biology, Dijon, France

Background/Objectives: FLNA Loss-of-Function (LoF) causes periventricular nodular heterotopia type 1 (PVNH1), an acknowledged cause of seizures of various types. Neurological symptoms are inconstant, and cardiovascular (CV) defects or connective tissue disorders (CTD) have regularly been associated. We aimed at refining the description of CV and CTD features in patients with FLNA LoF and depicting the multisystemic nature of this condition.

Methods: We retrospectively evaluated FLNA variants and clinical presentations in FLNA LoF patient with at least one CV or CTD feature, from three cohorts: ten patients from the French Reference Center for Rare Vascular Diseases, 23 patients from the national reference diagnostic lab for filaminopathies-A, and 59 patients from literature review.

Results: Half of patients did not present neurological symptoms. Most patients presented a syndromic association combining CV and CTD features. CV anomalies, mostly aortic aneurysm and/or dilation were present in 75% of patients. CTD features were present in 75%. Variants analysis demonstrated an enrichment of coding variants in the CH1 domain of FLNA protein.

Conclusion: In FLNA LoF patients, the absence of seizures should not be overlooked. When considering a diagnosis of PVNH1, the assessment for CV and CTD anomalies is of major interest as they represent interlinked features. We recommend systematic study of FLNA within CTD genes panels, regardless of the presence of neurological symptoms.

References:.

Grants:.

Conflict of Interest: None declared.

P06.015.A Genome-wide epistasis for cardiovascular severity in Marfan study design: patient organization driven research

Lotte Van Den Heuvel ^1;2;3, Josephina (Jeannette) Meester^1;2, Silke Peeters^1;2, romain alderweireldt⁴, Aline Verstraeten^1;2, Paul Coucke³, Bart Loeys^1;2

¹University of Antwerp, Antwerpen, Belgium; ²Antwerp University Hospital, Edegem, Belgium; ³Ghent University, Ghent, Belgium; ⁴Foundation 101 Genomes, Brussel, Belgium

Background/Objectives: Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with manifestations in the ocular, skeletal and cardiovascular system. Morbidity and mortality are mostly determined by aortic disease. Although mutations in FBN1 are the well-established genetic cause of MFS, there is a poor correlation with regards to phenotypical outcome, especially cardiovascular. Wide intra- and interfamilial phenotypical variability is observed, but the underlying mechanisms remain largely elusive. Consequently, the identification of genetic variation that modifies these effects will add important novel insights.

Methods: A worldwide collaborative project driven by researchers and a Belgian patient organization, ‘Foundation 101 Genomes’ (F101G), was established to maximize the number of patients to study the genetic basis of phenotypical variability. RNA-sequencing will be integrated with WGS to reveal MFS aortopathy genetic modifiers, which will be validated using CRISPR/Cas9 in IPSC-VSMC models.

Results: Our research institutions already gathered DNA and PBMCs of 35 patients carrying the most common FBN1 missense variant (p.Ile2585Thr;c.7754T>C). International collaborations yield at least 200 patients carrying this specific variant. Together with F101G, we created a website to guide patients to participate in our research.

Conclusion: Despite the large number of patients already included, more patients are needed to identify genetic modifiers for MFS aortopathy. Understanding how mother nature by itself modifies the outcome of the primary FBN1 mutation will individualize current treatment protocols to deliver true precision medicine and offer promising new leads to novel therapeutic strategies.

References:.

Grants:.

Conflict of Interest: Lotte Van Den Heuvel Fonds voor wetenschappelijk onderzoek, Josephina (Jeannette) Meester: None declared, Silke Peeters: None declared, romain alderweireldt: None declared, Aline Verstraeten: None declared, Paul Coucke: None declared, Bart Loeys Fonds voor wetenschappelijk onderzoek.

P06.016.B Casq2 deletion: a zebrafish model of CPVT

Ewa Sieliwonczyk ¹, Bert Vandendriessche¹, Charlotte Claes¹, Evy Mayeur², Maaike Alaerts¹, Bart Loeys¹, Dorien Schepers¹

¹Center of Medical Genetics, Faculty of Medicine and Health Sciences, University of Antwerp and Antwerp University Hospital, Edegem, Belgium; ²Laboratory for Molecular, Cellular and Network Excitability, University of Antwerp, Antwerp, Belgium

Background/Objectives: Autosomal recessive mutations in CASQ2 are the second most common genetic defect identified in catecholaminergic polymorphic ventricular tachycardia (CPVT), one of the most lethal inherited cardiac arrhythmias. Recent findings suggest an autosomal dominant inheritance is also possible for CASQ2.(1) We developed a zebrafish knockout model, which will be used to further explore this hypothesis.

Methods: The casq2 knockout was generated on the background of a transgenic zebrafish line expressing cardiac dual voltage and calcium reporters (Ace2N-mNeon and R-GECO) with CRISPR/Cas9. Loss of expression was confirmed by RT-qPCR. The voltage and calcium signals were measured with a Leica SP8 light sheet microscope at 3 days post-fertilization, after overnight exposure to the adenylate cyclase activator forskolin.

Results: The casq2 knockout line contains a 5 base pair deletion in exon 2. Casq2-/- embryos showed a decreased heart rate at baseline compared to wildtype. Delayed afterdepolarizations (DADs) induced by forskolin were observed significantly more often in casq2-/- embryos (table 1).

Table 1:

	DADs	No DADs	p-value
Casq2-/- 5µM Forskolin	8 (57%)	6 (43%)	0.018 (Fisher’s exact test)
Casq2+/+ 5µM Forskolin	2 (12%)	15 (88%)

Conclusion: The casq2 knockout is the first zebrafish model of the classical CPVT genes. Similar to the human phenotype, we observe bradycardia at rest and a sensitivity to DADs upon stimulation. This model will provide a promising opportunity for further testing of CASQ2 inheritance patterns.

References: (1) Ng K, et al. Circulation, 2020. 142(10):932. PMID:32693635.

Grants: Fund for Scientific Research, Flanders (FWO) and the Antwerp University Research Fund (BOF).

Conflict of Interest: None declared.

P06.017.C A trans-ancestry Mendelian Randomisation study to estimate the causal effects of cardiometabolic factors on coronary artery disease in British Pakistanis and Bangladeshis

Diana Dunca ¹, Qinqin Huang², Neneh Sallah³, Hilary Martin⁴, Thomas Lumbers³, Karoline Kuchenbäcker⁵

¹University College London, UCL Genetics Institute, London, United Kingdom; ²Wellcome Sanger Institute, Department of Human Genetics, Cambridge, United Kingdom; ³University College London, Institute of Health Informatics, London, United Kingdom; ²Wellcome Sanger Institute, Department of Human Genetics, Cambridge, United Kingdom; ⁵University College London, Division of Psychiatry, London, United Kingdom

Background/Objectives: British people with South-Asian ancestry have a higher risk of coronary artery disease (CAD) than other ancestry groups. Statistical power can be a limiting factor when extending Mendelian Randomisation (MR) analyses to non-European populations, because ancestry-matched GWAS for risk factors (RFs) of interest might not be sufficiently large.

Methods: We compared different strategies for trans-ancestry MR to assess the effect of cardiometabolic RFs (BMI, triglycerides, HDL-cholesterol, LDL-cholesterol, systolic and diastolic blood pressure) on the risk of CAD in 22,000 British Pakistani and Bangladeshi (BPB) individuals from the Genes&Health cohort. We used an ancestry-matched sample to derive instruments in a two-sample MR of CAD in Genes&Health, with summary statistics for RFs from the BPB group in the UK Biobank. However, insufficient number of genome-wide significant instruments were identified in the UK Biobank BPB population. Therefore, we used a less stringent p-value threshold (p < 5x10-5) for selecting instruments, incorporating results from large European GWASs, and using a subset of loci with evidence of transferability.

Results: We found that most of the associations were not significant in the ancestry-matched MR. We found a risk increasing effect for LDL-cholesterol and risk decreasing effect for HDL-cholesterol when using the variants from the large European GWAS as instruments, and also for the subset of loci that were transferable. The associations for BMI with CAD were significant only for transferable loci.

Conclusion: Incorporating findings from European GWAS can increase power for MR in other ancestry groups. We demonstrated the importance of considering transferability of RF loci to ensure casual inference.

References:.

Grants: No.206194,No.116074,MR/S003754/1.

Conflict of Interest: None declared.

P06.018.D Whole exome/genome sequencing joint analysis in a family with oligogenic familial hypercholesterolemia

Youmna Ghaleb ^1;2, Sandy Elbitar^1;2, Anne Philippi³, Petra El Khoury^1;2, Yara Azar^1;2;4, Miangaly Andrianirina¹, Alexia Loste^1;4, Yara Abou-khalil^1;2;4, Gaël Nicolas^4;5, Marie Le Borgne^1;4, Philippe Moulin^6;7, Mathilde Di Filippo^7;8, Sybil Charriere^6;7, Michel Farnier⁹, Cécile Yelnik^10;11, Valérie Carreau¹², Jean Ferrières¹³, Jean-Michel Lecerf¹⁴, Alexa Derksen^15;16;17, Geneviève Bernard^15;17;18, Marie-Soleil Gauthier¹⁶, Benoit Coulombe^16;19, Lütjohann Dieter²⁰, Bertrand Fin²¹, anne boland²¹, robert olaso²¹, Jean-François Deleuze^21;22, Jean-Pierre Rabes^1;23, Catherine Boileau^1;4;24, Marianne Abifadel^1;2, Mathilde VARRET^1;4

¹INSERM, Laboratory for Vascular Translational Science (LVTS), F-75018 Paris, France, Paris, France; ²Laboratory of Biochemistry and Molecular Therapeutics (LBTM), Faculty of Pharmacy, Pôle Technologie-Santé (PTS), Saint-Joseph University, Beirut, Lebanon, Beirut, Lebanon; ³Université de Paris, Institut Cochin, INSERM U1016, CNRS UMR-8104, Paris, France, Paris, France; ⁴Université de Paris and Université Sorbonne Paris Nord, France, Paris, France; ⁵INSERM U1149, CNRS ERL 8252, Centre de Recherche sur l’inflammation, Paris, France;, Paris, France; ⁶Hospices Civils de Lyon, Louis Pradel Cardiovascular Hospital, Department of Endocrinology, nutrition and metabolic diseases, Bron, France; ⁷CarMen Laboratory, INSERM U1060, INRAE U1397, Université Lyon 1, Oullins, France;, Lyon, France; ⁸Department of Biochemistry and Molecular Biology, Bron, France; ⁹EA 7460 Physiopathologie et Epidémiologie Cérébro-Cardiovasculaires (PEC2), Université de Bourgogne-Franche Comté, Dijon, France;, Dijon, France; ¹⁰Département de Médecine Interne et Immunologie Clinique Centre de Référence des Maladies Auto-immunes Systémiques Rares du Nord et Nord-Ouest de France (CeRAINO) CHU de Lille, Lille, France;, Lille, France; ¹¹U1167 Risk Factors and Molecular Determinants of Aging-Related Diseases Inserm CHU de Lille Univ. Lille, Lille, France, Lille, France; ¹²Department of Endocrinology and Prevention of Cardiovascular Disease, Institute of Cardio Metabolism and Nutrition (ICAN), La Pitié-Salpêtrière Hospital, AP-HP, Paris, France;, Paris, France; ¹³Department of Cardiology and INSERM UMR 1295, Toulouse Rangueil University Hospital, Toulouse, France, Toulouse, France; ¹⁴Nutrition Department, Institut Pasteur de Lille, 59019 Lille Cedex, France;, Lille, France; ¹⁵Child Health and Human Development Program, Research Institute of the McGill University Health Centre, Montréal, Québec, Canada, Montréal, Canada; ¹⁶Translational Proteomics Laboratory, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada, Montréal, Canada; ¹⁷Department of Neurology and Neurosurgery, McGill University, Montréal, Québec, Canada, Montréal, Canada; ¹⁸Division of Medical Genetics, Department of Specialized Medicine, McGill University Health Centre, Montréal, Québec, Canada, Montréal, Canada; ¹⁹Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Canada, Montréal, Canada; ²⁰Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany, Bonn, Germany; ²¹Université Paris-Saclay, CEA, Centre National de Recherche en Génomique Humaine, 91057, Evry, France and Laboratory of Excellence GENMED (Medical Genomics), Evry, France; ²²Centre d’Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France, Paris, France; ²³Ambroise Paré University Hospital, APHP., Department of Biochemistry and Molecular Genetics, Paris, France; ²⁴Genetic Department, AP-HP, Hôpital Bichat, F-75018 Paris, France., Paris, France

Background/Objectives: Autosomal Dominant Hypercholesterolemia (ADH) is a genetic disorder caused by pathogenic variants in LDLR, APOB, PCSK9 and APOE genes. We sought to identify a new candidate gene responsible of the ADH phenotype in patients with no pathogenic variants in known ADH causing genes.

Methods: We performed linkage analysis, whole exome and whole genome sequencing in one French family, with affected and non-affected members presenting a high ADH polygenic risk score (wPRS). We also performed functional studies in HEK293T cells of the four LRP6 mutants.

Results: Linkage analysis, whole exome and whole genome sequencing in one French family, with affected and non-affected members presenting a high ADH polygenic risk score (wPRS) allowed us to identify p.(Pro398Ala) in CYP7A1, p.(Val1382Phe) in LRP6 and p.(Ser202His) in LDLRAP1. Six other variants were identified in 6 from 160 unrelated ADH probands: p.(Ala13Val) and p.(Aps347Asn) in CYP7A1, p.(Tyr972Cys), p.(Thr1479Ile) and p.(Ser1612Phe) in LRP6 and p.(Ser202LeufsTer19) in LDLRAP1. All these six probands presented a moderate wPRS. Serum analysis of carriers of p.(Pro398Ala) variant in CYP7A1 showed no differences in bile acids synthesis when compared to non-carriers serums. Functional studies in HEK293T cells of the four LRP6 mutants showed contradictory results. None of the family members heterozygous carriers of the LDLRAP1 p.(Ser202His) variant alone presented ADH.

Conclusion: Altogether, each variant alone does not seem to contribute sufficiently to the elevation of LDL-C and it is the oligogenic combination of two or three variants that is necessary to reveal the ADH phenotype.

References:.

Grants:.

Conflict of Interest: None declared.

P06.019.A Contribution of SCN5A copy number variations to the genetic diagnosis of Brugada syndrome

Laura Cazón ¹, Luis De la Higuera Romero¹, Marlene Perez Barbeito¹, Rosalía Peteiro¹, Iria Gómez Diaz¹, Paula Rebolo¹, Paula Velez¹, Maria Sanchez¹, Anahi Sanluis Verdes¹, Guillermo Smith Ramos¹, Emilia Maneiro¹, Xusto Fernandez¹, Almudena Amor¹, María Valverde¹, Soledad García Hernández^1;2, Ivonne Cárdenas Reyes¹, Martin Ortiz Genga¹, Juan Pablo Ochoa³

¹Health in Code, Scientific Committee, A Coruña, Spain; ²Hospital Universitario Clínico San Cecilio, Cardiology, Granada, Spain; ³Health in Code, Cardiology director scientific committee, A Coruña, Spain

Background/Objectives: Loss-of-function variants in SCN5A are identified in 15-25% of Brugada syndrome (BrS) cases. Most of them are single-nucleotide variants, small insertions / deletions, and splicing errors, whereas information on copy number variations (CNVs) is still limited. The objective of this study is to determine the contribution of CNVs to the genetic diagnosis of BrS.

Methods: A total of 977 consecutive unrelated probands with suspected BrS were genotyped by next-generation-sequencing (NGS) using a panel of genes that included SCN5A. Filtering and classification of NGS data was performed using a custom pipeline; CNVs were detected using a read depth approach and confirmed by orthogonal molecular techniques.

Results: Actionable variants in SCN5A were identified in 128 probands (13%): 83 were pathogenic/ likely pathogenic and 47 variants of unknown significance but favoring pathogenic. CNV analysis could be performed in 888 (90.9%) of the probands and revealed six carriers of CNVs in SCN5A (table 1).

Gender	Age	SCN5A CNV	Confirmation technique
Male	13	630kb deletion (8 whole genes, including SCN5A)	SNP-array
Female	43	143kb deletion (SCN5A exons 1-16 and SCN10A exons 15-27)	SNP-array
Male	Unknown	Deletion exon 4	Sanger sequencing
Male	35	Deletion exons 8-10	MLPA
Male	40	Deletion exons 13-27	Sanger sequencing
Female	61	Deletion exon 22	MLPA

Conclusion: SCN5A CNVs were detected in 0.6% of the BrS probands, but they represented 7.2% of pathogenic / likely pathogenic variations identified in our cohort. This result emphasizes the importance of a complete genetic study including CNVs in the diagnosis of BrS.

References:.

Grants:.

Conflict of Interest: Laura Cazón Full time, Luis De la Higuera Romero Full time, Marlene Perez Barbeito Full time, Rosalía Peteiro Full time, Iria Gómez Diaz Full time, Paula Rebolo Full time, Paula Velez Full time, Maria Sanchez Full time, Anahi Sanluis Verdes Full time, Guillermo Smith Ramos Full time, Emilia Maneiro Full time, Xusto Fernandez Full time, Almudena Amor Full time, María Valverde Full time, Soledad García Hernández Full time, Ivonne Cárdenas Reyes Full time, Martin Ortiz Genga Full time, Juan Pablo Ochoa Full time.

P06.020.B Spontaneous coronary artery dissection: role of the genetic background in pathogenesis and management

Marta Casula¹, lucia trevisan ^2;3, Fabio Gotta³, Daniela Marchetti⁴, laura pezzoli⁴, antonio zingarelli³, Paola Mandich^2;3, Maria Iascone⁴

¹Università degli Studi di Genova, genoa, Italy; ²Università degli Studi di Genova, dinogmi, Genova, Italy; ³IRCCS Ospedale Policlinico San Martino, Genova, Italy; ⁴ASST Papa Giovanni XXIII, Laboratorio di Genetica Medica, Bergamo, Italy

Background/Objectives: Spontaneous coronary artery dissection (SCAD) is a rare, non-atherosclerotic disease of the coronary vascular tunics, frequently starting as an acute coronary syndrome. Recent genetic studies on SCAD have identified a possible correlation with inherited connective tissue disorders and a possible predisposition linked to CGG repeat expansion in FMR1 gene. This study explored rare genetic factors that may contribute to the pathogenetic mechanisms underlying SCAD.

Methods: After the SCAD event between January 2010 and February 2021, fourteen patients referred to the Cardiology Department of IRCCS Policlinico San Martino underwent clinical genetic evaluation, whole-exome sequencing analyses (trio or singleton), and FRAXA analyses. This study was approved by local Ethical Committee.

Results: Although none of the 14 patients enrolled showed clinical features associated with connective tissue disorders, in the 21% of our cohort, we identified variants in genes involved in the formation and in the integrity of connective tissue. We also detected an intermediate allele in the FMR1 gene in one patient and a de novo variant in a new candidate gene in another one.

Conclusion: Despite the small number of patients analysed, we identified several genetic risk factors for SCAD and a promising novel candidate gene. Although further studies are needed, this work could contribute to give insight into the pathogenetic pathways of this rare condition.

References: Tassanakijpanick N. et al, Cardiovascular problems in the Fragile X premutation; Front Genet. 2020.

Amrani-Midoun A. et al, Recent advances on the genetics of spontaneous coronary artery dissection; CircGenomPrecisMed 2021.

Grants: This project was not supported by any grant.

Conflict of Interest: None declared.

P06.021.C Polygenic risk scores predict overweight and obesity in the Dutch population

Bahar Sedaghati-khayat ¹, Linda Broer¹, Annemieke Verkerk¹, André Uitterlinden^1;2, Joyce Van Meurs^1;2, Jeroen van Rooij¹

¹Erasmus MC, Department of Internal medicine, Rotterdam, Netherlands; ²Erasmus MC, Department of Epidemiology, Rotterdam, Netherlands

Background/Objectives: Obesity, the fifth leading risk of global deaths, has grown to epidemic proportions. Its etiology is largely unknown but it has a substantial hereditary component. Global GWAS have identified 941 genetic variants influencing body-mass index explaining 6% of heritability. This study combined these variants into a polygenic risk score (PRS) to assess obesity-risk in a Dutch Caucasian population cohort.

Methods: The PRS was tested in 11,209 participants of the Rotterdam Study (mean±SD age = 65.7±10.2 years) to predict BMI as a continuous and categorical outcome (under-weight, normal-weight, overweight, obese, and morbid-obese). We evaluated the risk conveyed by PRS as a linear instrument (per 1 SD) and categorical (highest 10% vs. middle 50% of the PRS distribution).

Results: The PRS was associated with BMI (beta-estimate = 0.9 [95% confidence interval 0.7-1.1];p < 1 × 10⁻¹⁶). One SD increase of the PRS significantly increased the risk of being underweight, over-weight, obese and morbid-obese by 0.8, 1.3, 1.8 and 2.2 fold times, respectively. Similarly, the top 10% of the population with the highest BMI-PRS showed increased risks of 1.1, 1.5, 2.6 and 4.5 for these BMI categories, respectively. The risk increased exponentially in the PRS distribution tails, up to 8.2 of the top 1% PRS for morbid-obese vs. normal-weight.

Conclusion: These results confirm that the PRS significantly impacts BMI in a Dutch Caucasian elderly population. Identifying the biological pathways affected by an individuals’ genetic background could aid in targeted and personalized intervention strategies long before the onset of obesity. BMI-PRS utility is being investigated as part of the Genotyping on all patients (GOALL) project.

References:.

Grants:.

Conflict of Interest: None declared.

P06.022.D Novel loss of function KCNA5 pathogenic variants in pulmonary arterial hypertension

Natalia Gallego ^1;2;3, Alba Vera-Zambrano^4;5;6, Mauro Lago-Docampo^7;8, Juan Felipe Franco-González⁹, Daniel Morales-Cano^10;11, Alejandro Cruz-Utrilla^12;13, Marta Villegas^4;14, Pilar Escribano-Subias^12;13, Jair Tenorio^1;2;3, Francisco Perez Vizcaino^4;14, Diana Valverde Pérez^7;8, Teresa González^5;6, Angel Cogolludo^4;14

¹Institute of Medical and Molecular Genetics (INGEMM)-IdiPAZ, Madrid, Spain; ²CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain; ³ITHACA, European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability, Hospital Universitario La Paz, Madrid, Spain; ⁴Department of Pharmacology and Toxicology, School of Medicine, University Complutense of Madrid, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain; ⁵Department of Biochemistry, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain; ⁶Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, Madrid, Spain; ⁷CINBIO, Universidad de Vigo, Vigo, Spain; ⁸Instituto de Investigación Sanitaria Galicia Sur (IIS Galicia Sur), SERGAS-UVIGO, Vigo, Spain; ⁹Department of Structural and Chemical Biology, Centre for Biological Research Margarita Salas, CIB-CSIC, Madrid, Spain; ¹⁰Atherosclerosis Research Unit, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; ¹¹Experimental Pathology of Atherosclerosis Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain; ¹²CIBER Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; ¹³Unidad Multidisciplinar de Hipertensión Pulmonar, Servicio de Cardiología, Hospital Universitario 12 de Octubre, Madrid, Spain; ¹⁴Ciber Enfermedades Respiratorias (CIBERES), Madrid, Spain

Background/Objectives: Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Genetic variants in KCNA5 have been found in PAH patients. However, their functional consequences and potential impact on the disease is largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients.

Methods: Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC).

Results: KCNA5 variants (namely, p.Arg184Pro and p.Gly384Arg) resulted in a loss of potassium channel function as assessed by electrophysiological and molecular modelling analysis. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affect channel activity, both variants were associated with a clear impairment in a key PASMC process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analysed was around 1 %.

Conclusion: Our data indicates that some KCNA5 mutations present in PAH patients have critical consequences for channel function. This encourages the idea that KCNA5 pathogenic variants may be a developing or contributing factor for PAH.

References:.

Grants: PI18/01233, FCHP unrestricted grant.

Conflict of Interest: None declared.

P06.023.A Genetic testing outcomes in a cohort of 21,159 children with heart disease

Robert Nussbaum ¹, Flavia Facio², Ana Morales², Asia Mitchell², John Garcia³, Dianalee McKnight², Tom Callis⁴, Chad Moretz⁵, Matteo Vatta⁶, Swaroop Aradhya²

¹Invitae, San Francisco, United States; ²Invitae, Medical Affairs, San Francisco, United States; ³Invitae, Systems Development, San Francisco, United States; ⁴Invitae, CNP US Sales, San Francisco, United States; ⁵Invitae, Health Economics and Outcomes Research, San Francisco, United States; ⁶Invitae, Clinical Operations, San Francisco, United States

Background/Objectives: Pediatric heart conditions affect 1/77 US children. Guidelines recommend cardiogenetic testing in children and at-risk relatives. We investigated outcomes in a large pediatric cohort undergoing genetic testing for a wide range of cardiogenetic conditions.

Methods: Probands <18 years had next generation sequencing and deletion/duplication analysis for cardiogenetic conditions (up to 224 genes). A positive result was defined as a single pathogenic/likely pathogenic (P/LP) variant in a gene associated with an autosomal dominant or X-linked disorder, or two P/LP variants in a gene associated with a recessive disorder. Outcomes were compared to cardiogenetic testing results of 61,368 adults by t-tests with multiple comparison correction.

Results: 21,159 probands were tested (median = 10.03 years, SD = 5.75) and positive result were reported in 16.6%. Cascade testing was pursued in 3.9% of families (mean 3.48 relatives/family; 59.1% aged <10 years). Positive results were more frequent in children than in adults (16.6% vs 15.3%), and were particularly enriched in genes associated with syndromic disorders relative to non-syndromic ones (41.4% vs 29.7%). Families with probands aged <10 had cascade testing more often in comparison to those with older probands (4.6%; p = 0.0069).

Conclusion: One in six children referred for cardiogenetic testing received a positive result, highlighting opportunities for management. Cascade testing was pursued more often than in families with probands <10 years in comparison to those with adult probands. These findings provide rationale for family-based care to identify patients who can benefit from genetics-guided interventions.

References:.

Grants:.

Conflict of Interest: Robert Nussbaum: None declared, Flavia Facio Invitae, Ana Morales Invitae, Asia Mitchell Invitae, John Garcia Invitae, Dianalee McKnight Invitae, Tom Callis Invitae, Chad Moretz Invitae, Matteo Vatta Invitae, Swaroop Aradhya Invitae.

P06.024.B Identification of novel loss-of-function genes associated with carotid intima-media thickness

Abdulrahman Alasiri ^1;2, Marion van Vugt¹, Jessica Van Setten¹, Folkert Asselbergs^1;3;4, Sander van der Laan^1;5

¹University Medical Center Utrecht, Department of Cardiology, Utrecht, Netherlands; ²King Abdullah International Medical Research Center, Medical Genomics Research Department, Riyadh, Saudi Arabia; ³University College London, Health Data Research UK and Institute of Health Informatics, Utrecht, Netherlands; ⁴University College London, Institute of Cardiovascular Science, Utrecht, Netherlands; ⁵University Medical Center Utrecht, Central Diagnostic Laboratory, Utrecht, Netherlands

Background/Objectives: Ultrasound measurement of the carotid intima-media thickness (cIMT) of the carotid artery is frequently employed as a biomarker for subclinical atherosclerosis and the extent of vascular remodelling. Large-scale genetic studies identified common genetic risk factors for cIMT. Here, we aimed to identify loss-of-function (LoF) genes that are inactive in one or two copies (i.e. full knockouts) associated with cIMT.

Methods: We used LoFTK (https://github.com/CirculatoryHealth/LoFTK) to annotate LoF genes from UK Biobank exomes and UCC-SMART imputed genotypes. We performed LoF-wide association studies (LoFWAS) in both cohorts and conducted a meta-analysis to identify genes associated with cIMT. We explored tissue- and cell-specific expression in carotid atherosclerotic plaques.

Results: We identified four genes associated with cIMT in our meta-analysis. Three of these genes (PPP1R21, HSD3B2 and ULK2) were inactive in one copy, while one gene (KIR3DL1) was a full knockout. Single-cell RNA sequencing revealed cellular subtype-specific expression patterns of PPP1R21, ULK2, and KIR3DL1 in carotid plaques.

Conclusion: We identified 4 novel genes associated with cIMT. Our study provides insights into LoF genes and cIMT that underpin the genetic and biological mechanisms of atherosclerosis.

References:.

Grants:.

Conflict of Interest: None declared.

P06.025.C A novel rare pathogenic variant in TLN1 in a family with systemic capillary leak syndrome

naama elefant ¹, Shira Yanovsky-Dagan², danit Oz-Levi³, Racheli Sion-Sarid⁴, Doron Lancet⁵, Vardiella Meiner¹, Ben Goult⁶, Vasso Kostourou⁷, Tamar Harel^1;2

¹Hadassah Medical Center, Department of clinical genetics, Jerusalem, Israel; ²Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel; ³My Heritage, or-yehudah, Israel; ⁴Edith Wolfason Medical Center, Holon, Israel; ⁵Weizmann Institue of Science, Department of Molecular Genetics, rehovot, Israel; ⁶University of Kent, School of bioscience, Canterbury, Israel; ⁷Biomedical Sciences Research Center “Alexander Fleming”, Institute of Immunology, athens, Greece

Background/Objectives: Systemic capillary leak syndrome (SCLS) is a rare life threatening disorder that presents with episodes of severe hypotension, hypoalbuminemia, and hemoconcentration due to profound vascular leak. The condition was first described by Clarkson in 1960, yet its etiology remains unknown. Current hypotheses include abnormal endothelial cell response to normal stimulation, or normal endothelial cell response to dysregulated or excessive systemic signaling or inflammation. We describe three individuals with SCLS from an extended pedigree suggestive of AD inheritance with incomplete penetrance.

Methods: We conducted exome sequencing analysis on peripheral blood of two family members, followed by Sanger sequencing for segregation in additional family members. cDNA analysis from blood and fibroblast samples was used to determine the consequence of a variant of interest.

Results: A rare novel heterozygous variant in the TLN1 gene (c.7188+2T>C) was identified in all three family members with SCLS. This variant causes in-frame skipping of exon54 and is predicted to affect the c-terminal actin binding domain in the Talin-1 protein (domain ABS3).

Conclusion: Talin-1 (TLN1) has a key role in cell adhesions via the linkage of integrins to the actin cytoskeleton and by activation of integrins. Studies have shown that Talin-1 regulates VE-Cadherin localization, which plays an important role in endothelial cell barrier function. Based on our findings we suggest that pathogenic variants in TLN1 underlie SCLS. Future studies are warranted to further investigate the mechanism of the disease and to explore possible therapeutic options.

References:.

Grants:.

Conflict of Interest: None declared.

P06.026.D Phenotypic and genetic factors modifying the risk of developing cardiomyopathy symptoms for PLN-p.Arg14del carriers

Esteban Lopera-Maya ¹, Shuang Li¹, Remco de Brower¹, Ilja Nolte², Justin van Breen¹, Jan Jongbloed³, Morris Swertz^1;4, Harold Snieder², Lude Franke¹, R.A. de Boer³, Cisca Wijmenga¹, Patrick Deelen¹, Paul van der Zwaag¹, Serena Sanna^1;5

¹University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands; ²University of Groningen, University Medical Center Groningen, Department of Epidemiology, Groningen, Netherlands; ³University of Groningen, University Medical Center Groningen, Department of Cardiology, Groningen, Netherlands; ⁴University of Groningen, University Medical Center Groningen, Genomics Coordination Center, Groningen, Netherlands; ⁵National Research Council (CNR), Institute for Genetic and Biomedical Research (IRGB), Cagliari, Italy

Background/Objectives: The phospholamban (PLN) p.Arg14del Dutch founder variant has been associated with dilated and arrhythmogenic cardiomyopathy.¹ Some carriers show life-threatening symptoms, while others remain asymptomatic or show only mild symptoms at old age. To understand the mechanisms behind this incomplete penetrance, we aimed to identify protective factors in asymptomatic PLN-Arg14del carriers in a large population cohort.

Methods: We identified 74 (0.2%) carriers in 36,339 genotyped individuals of the Dutch Lifelines cohort², of which 48 were asymptomatic according to their ECG and questionnaires. We interrogated 38 quantitative measurements and 97 polygenic scores (PGS) of cardio-metabolic traits, performed a GWAS, and a rare variant burden analysis in 82 risk loci. We used the non-standard approach of comparing asymptomatic carriers to asymptomatic non-carriers, and confirmed the results by comparing each group to symptomatic carriers.

Results: Compared to the asymptomatic non-carriers, the asymptomatic carriers showed lower QRS (p = 0.002), an effect that replicated in another Lifelines subset (N = 20,221) and in the ACM patient registry (N = 592). Furthermore, symptomatic carriers showed a higher correlation between PGS_PR and PR (p = 0.022) and between PGS_QRS and QRS (p = 1.9x10^-5). These results indicate symptomatic PLN-Arg14del carriers have an increased sensitivity for common genetic variation affecting cardiac rhythm.

Conclusion: We have used a population cohort to identify protective factors for PLN-Arg14del carriers. These results can improve risk prediction models for cardiac outcomes and guide future studies on genetic diseases with incomplete penetrance.

References: ¹van der Zwaag et al, Eur J Heart Fail 2012.

²Scholtens et al, Int J Epidemiol 2015.

Grants: UMCG-HAP-2017-2 324.

Conflict of Interest: None declared.

P06.027.A NGS analysis in a patient with conduction disorders and dilated cardiomyopathy: a case report of R222Q SCN5A variant, benefiting from mutation specific target therapy

Martina Callegari ¹, Francesca Bianchi², Valeria Bracciamà¹, Giulia Margherita Brach del Prever¹, Angelo Corso Faini¹, Silvia Kalantari¹, Tiziana Vaisitti¹, Stefano Grossi², Antonio Amoroso¹, Silvia Deaglio¹

¹Transplant Regional Center-Piedmont, Immunogenetics and Transplant Biology, AOU Città della Salute e della Scienza, Department of Medical Sciences, University of Turin, Torino, Italy; ²Division of Cardiology, Mauriziano Umberto I Hospital, Turin, Italy

Background/Objectives: Mutations in SCN5A, which encodes the alpha-subunit of cardiac sodium channel Nav1.5., are reported non only in patients with Brugada Syndrome but also in those with dilated cardiomyopathy (DCM). In these patients, cardiac dilation is preceded by conduction system abnormalities. Genetic diagnosis in these cases can determine therapeutic choices with consequences on patient’s quality of life and survival.

Methods: We performed Next Generation Sequencing (NGS) analysis as part of the routine work-out of a 53-year-old woman with a history of ventricular extrasystoles (burden > 50%) since the age of 20, syncopal sinus pauses with required PM implantation, atrial fibrillation and hypokinetic cardiomyopathy with dilation of left ventricle and atrium and mild left ventricular ejection fraction reduction. Due to worsening of NYHA class, several ablation procedures targeting the frequent ventricular arrhythmia were performed, with later recurrence. Genetic analysis focused on genes associated with DCM and arrhythmogenic cardiomyopathy.

Results: We identified a heterozygous pathogenic variant (p.Arg222Gln) in SCN5A, previously reported to be responsive to sodium channel blockers. Hydroquinidine treatment led to normalization of ventricular cell potentials. Patient showed almost complete regression of arrhythmic manifestations and NYHA class improvement.

Conclusion: Identification of R222Q in SCN5A was fundamental for the patient’s prognosis. This paradigmatic case underlines the importance of NGS analysis for optimal therapeutic management of patients with conduction disorders and DCM. For these patients, genetic studies should be considered integral components of the diagnostic phase.

References: Mann SA et al, JACC 2012.

Grants: Ministero dell’Istruzione, Progetto Strategico di Eccellenza Dipartimentale #D15D18000410001.

Conflict of Interest: None declared.

P06.028.B Yield of 3 years of diagnostic testing of TNNI3K in Arrhythmia and Cardiomyopathy Patients

Karolina Andrzejczyk ¹, Caroline Pham¹, Kaylin Palm¹, Leander Beekman¹, Alexa Vermeer², Janneke Nijman², Imke Christiaans³, Doris Škorić-Milosavljević², Ronald Lekkane Dit Deprez², Philip Jansen², Niels Vos², Svitlana Podliesna¹, Connie R. Bezzina¹, Elisabeth Lodder^1;2

¹Amsterdam University Medical Center, Clinical and Experimental Cardiology, Amsterdam, Netherlands; ²Amsterdam University Medical Center, Human Genetics, Amsterdam, Netherlands; ³University Medical Center Groningen, Clinical Genetics, Groningen, Netherlands

Background/Objectives: Genetic variants in TNNI3K have been associated with supraventricular arrhythmias (SVTs), conduction disease (CCD), and dilated cardiomyopathy (DCM) in a limited number of papers thus far. The yield in diagnostic screening of this gene in cardiac diseases has not yet been evaluated, we here aim to fill this gap. As pathogenic variants in TNNI3K have been shown to affect the level of autophosphorylation, we evaluated this for identified variants.

Methods: We collected and analysed clinical data of individuals with variants in TNNI3K. These variants were identified by (i) systematic clinical genetic screening of gene panels in patients with cardiomyopathies and/or arrhythmias between 08-2018 and 12-2021 at the Amsterdam UMC, and (ii) collaboration with multiple centres in the Netherlands. Functional in vitro studies were performed to assess the effects of the identified variants on TNNI3K auto-phosphorylation.

Results: We identified 19 rare coding variants in TNNI3K in 24 probands. Among these two novel likely pathogenic variants (p.H592T and p.I512T) were found in eight families. Individuals harbouring these variants demonstrated SVTs, CCD, DCM, and/or out-of-hospital cardiac arrest. Functional studies revealed significantly increased auto-phosphorylation levels in both TNNI3K variants.

Conclusion: Screening of rare variants in TNNI3K adds to the diagnostic yield for cardiac diseases. However, considering the current lack of validation of the functional tests, co-segregation analysis is paramount for the classification of identified variants.

References: The Diverse Roles of TNNI3K in Cardiac Disease and Potential for Treatment. Doi: Caroline Pham, Noelia Muñoz-Martín, Elisabeth Lodder.

Grants: Netherlands Organisation for Scientific Research (VIDI-91718361).

Conflict of Interest: None declared.

P06.029.C Common rearrangements of the LDLR gene in the Czech population likely arise from one mutational event

Katerina Konecna ¹, Lukas Tichy¹, Tomas Freiberger²

¹University Hospital Brno, Centre of Molecular Biology and Genetics, Brno, Czech Republic; ²Center of Cardiovascular and Transplant Surgery, Brno, Czech Republic

Background/Objectives: Mutations in the low-density lipoprotein receptor gene (LDLR) are the most common cause of familial hypercholesterolemia in the Czech Republic. Out of all Czech probands with an LDLR mutation, nearly 10% are carriers of a deletion or duplication spanning whole exons. We characterized the breakpoints of 8 large rearrangements of the LDLR gene. One of the goals was to analyze the breakpoint of all probands carrying a specific rearrangement to verify the hypothesis that all probands of each rearrangement have identical breakpoints inherited from a common ancestor.

Methods: The breakpoint sequence was determined by PCR amplification and Sanger sequencing.

Results: We sequenced the breakpoint of 8 rearrangements of the LDLR gene, including the four most common rearrangements in Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1-4 probands). For all analyzed rearrangements, all Czech probands with a specific rearrangement shared identical breakpoint position and sequence, suggesting shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 5 out of 8 breakpoints, there was high homology (>75%) between the two Alu repeats in which the break occurred.

Conclusion: Most common rearrangements of the LDLR gene in the Czech population likely arise from one mutational event. Alu elements likely played a role in generation of the majority of rearrangements within the LDLR gene, but not all.

References:.

Grants: Supported by the Ministry of Health, Czech Republic, grant number NU20-02-00261.

Conflict of Interest: None declared.

P06.030.D Characterising the phenotype and outcomes of cascade-tested individuals carrying a pathogenic sarcomere-variant

Wajeeh Chaudhry ¹, Catherine McWilliam¹, Anna-Maria Choy², David Goudie¹, jonathan berg¹

¹School of Medicine - University of Dundee, Division of Population Health and Genomics, Dundee, United Kingdom; ²School of Medicine - University of Dundee, Division of Molecular and Clinical Medicine, Dundee, United Kingdom

Background/Objectives: Cascade testing of relatives of individuals with pathogenic genetic variants causing hypertrophic cardiomyopathy is recommended. Little is known of the clinical outcomes in cascade-identified individuals. We quantified clinical endpoints in such individuals.

Methods: All individuals reviewed by Genetics in NHS Tayside for a personal or family history of hypertrophic cardiomyopathy between January 2010 and December 2018 were included. Casenote review included genetic testing, echocardiography and clinical outcomes.

Results: 282 individuals from 64 families were included with a total follow-up of 2493 patient years (mean: 8.8±6.3). Of 200 patients under review; 121 were probands (mean age: 65.6 ± 13.9) and 79 cascade-identified (mean age: 48.3 ± 17.0). Causative genes were: MYBPC3 (n = 46);MYH7 (n = 12);TNNT2 (n = 2);TNNI3(n = 1);PKP2 (n = 1);CSRP3 (n = 1);GLA (n = 1).

Baseline asymmetrical myocardial hypertrophy was recorded in 45.6% of cascade-identified individuals. The mean age-adjusted interventricular septal size for cascade-identified individuals (13.8mm;95% CI:12.6-15.0) was lower than that of probands (19.8mm,95%CI:18.8-20.7; p < 0.001) but higher than the control group (10.5mm,95% CI:9.1-11.9; p < 0.001). Age-adjusted multivariant event analysis demonstrated decreased risk of adverse cardiac events in cascade-identified patients compared to probands (hazard ratio 0.27;95% CI:0.15-0.49; p < 0.001), and increased compared to controls (hazard ratio:0.26;95% CI:0.09-0.71; p = 0.009). The lifetime incidence of atrial fibrillation, ventricular tachycardia, heart failure and acute coronary syndromes are higher in proband patients versus cascade-tested carrier patients (p < 0.05).

Conclusion: Although cascade-tested individuals carrying pathogenic sarcomere gene variants exhibit a milder phenotype and decreased risk of major adverse cardiac events compared to probands, these individuals still have a high rate of complication justifying their identification and follow-up.

References:.

Grants:.

Conflict of Interest: None declared.

P06.031.A Prenatal Long QT syndrome associated with homozygous KCNH2 pathogenic variants

Ana Grangeia ^1;2, Márcia Baixia³, Sofia Granja Da Silva⁴, Susana Guimarães⁵, Carla Ramalho^6;7;8, Raquel Silva³, Sérgio Castedo^1;2;3, Renata Oliveira¹

¹São João Universitary Hospital Center, Genetics, Porto, Portugal; ²Faculdade de Medicina da Universidade do Porto - FMUP, Porto, Portugal; ³IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal; ⁴São João Universitary Hospital Center, Pediatric Cardiology, Porto, Portugal; ⁵São João Universitary Hospital Center, Anatomic Pathology, Porto, Portugal; ⁶São João Universitary Hospital Center, Obstetrics, Porto, Portugal; ²Faculdade de Medicina da Universidade do Porto - FMUP, Porto, Portugal; ⁸I3s, Instituto de Investigação e Inovação em Saúde, Porto, Portugal, Porto, Portugal

Background/Objectives: Long QT syndrome type 2 is an inherited autosomal dominant disorder usually caused by heterozygous pathogenic variants in the KCNH2 gene. However, homozygous KCNH2 variants have been proposed to cause a more severe cardiac phenotype associated with QT prolongation and functional atrioventricular conduction disturbances before and immediately after birth. Only three families with homozygous KCNH2 variants have been reported so far.

Methods:.

Results: We report an asymptomatic Portuguese consanguineous couple, whose first child, presented in the prenatal period chaotic foetal rhythm (periods of tachycardia and complete atrioventricular block) and hydrops. She was born at 33 weeks of gestation requiring a pacemaker implantation at day one of life. Postnatal echocardiogram showed left ventricular non-compaction cardiomyopathy. She died at 18 months of age from sudden death. Dilated cardiomyopathy NGS panel study did not identify any pathogenic variant. The second child was a healthy female, now 3 years old. During their third gestation, foetal hydrops was detected associated with global left ventricular dysfunction and cardiomegaly. The pregnancy was terminated at 24 weeks of gestation. Necropsy confirmed foetal hydrops and dilated cardiomyopathy. Considering a potential disease recurrence, a comprehensive cardiomyopathy NGS panel was performed in the index case and a homozygous frameshift pathogenic variant [c.785del, p.(Gly262Alafs*98)] was identified in the KCNH2. The same variant was found in the foetus (homozygous) and in the mother (heterozygous). Genetic screening of the father and healthy sister is ongoing.

Conclusion: This report illustrates the severe cardiac phenotype, of prenatal onset, associated with a homozygous KCNH2 pathogenic variant.

References:.

Grants:.

Conflict of Interest: None declared.

P06.033.C Mutations in the GTPBP3 are associated with hypertrophic cardiomyopathy with rapid progression to burn out phase complicated by severe systolic dysfunction and ventricular tachycardia

Petya Angelova ¹, Vasil Velchev², Nikolay Stoyanov², Slavena Atemin^1;3, Vanyo Mitev¹, Albena Todorova¹

¹Medical University Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ²University Hospital “St. Anna”, Department of Cardiology, Sofia, Bulgaria; ³Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria

Background/Objectives: About 100 genes have been associated with cardiomyopathies with genotype-phenotype correlations often hard to establish. Genetic testing may help to confirm the genetic diagnosis and assess the risk of inheritance in the family.

Methods: A 26-year old Caucasian male with hypertrophic cardiomyopathy (HCM) and suspected Wolff-Parkinson-White syndrome was referred for genetic testing by his cardiologist. Whole-exome sequencing (WES) was performed, followed by Sanger sequencing segregation analysis in the family.

Results: The targeted PRKAG2 gene screening turned out to be negative. WES revealed the following variants: c.247G>C (p.Ala83Pro) and c.1265C>T (p.Thr422Met) in the GTPBP3 gene in heterozygous state, as well as c.752C>T (p.Thr251Ile) and c.1760C>T (p.Pro587Leu) in the POLG gene. Family segregation analysis showed that the patient’s mother is a carrier of the c.247G> C variant and the patient’s paternal grandmother is a carrier of the variant c.1265C> T in the GTPBP3 gene. The findings of the family segregation analysis are in accordance with an autosomal recessive model of inheritance of the disease. Both variants in the POLG gene are found paternally inherited in the patient’s healthy half-brother, thus are not considered disease-causing.

Conclusion: WES led to the detection of two heterozygous variants in the GTPBP3 gene. Variants in this gene have been reported in patients with HCM, associated with combined oxidative phosphorylation deficiency 23. These heterozygous variants represent the probable cause of the observed clinical symptoms in the patient.

References:.

Grants: The financial support of Medical University Sofia, Grant № D-125/2021 is gratefully acknowledged.

Conflict of Interest: None declared.

P06.034.D Novel ALPK3 variants cause infantile and late onset hypertrophic cardiomyopathy in a single family

Tomer Poleg ¹, ofek freund¹, Matan M. Jean¹, Nadav Agam¹, Amit Safran¹, Vadim Dolgin¹, Marina Eskin-Shwartz^1;2, Ohad Shmuel Birk^1;2

¹The Morris Kahn Laboratory of Human Genetics, National Institute for Biotechnology in the Negev and Faculty of Health Sciences, Ben-Gurion University of the Negev., Beer Sheva, Israel; ²Genetics institute, Soroka Medical Center, Ben-Gurion University., Beer Sheva, Israel

Background/Objectives: Cardiomyopathies are clinically heterogeneous disorders that impair heart function and are the leading cause of cardiovascular morbidity and mortality in both children and adults. Of these, hypertrophic cardiomyopathy (HCM) characterized by increased left ventricular wall thickness and dilated cardiomyopathy (DCM) displaying ventricular dilatation, are most common. Alpha-protein kinase 3 (ALPK3) plays an essential role in sarcomere organization. Its mutations have been recently reported to be causative of the recessively inherited severe pediatric-onset DCM and HCM, accompanied by skeletal involvement and facial dysmorphism, as well as of the dominantly inherited adult-onset cardiomyopathy. We have ascertained kindred with multiple family members affected by infantile or late onset cardiomyopathy and aimed to elucidate the genetic basis of cardiomyopathy in the affected individuals.

Methods: The family members underwent clinical assessment and genotyping using whole-exome sequencing and Sanger sequencing.

Results: Compound heterozygosity for two novel variants in ALPK3 has been identified in two identical twin sisters, with severe infantile onset HCM and facial dysmorphism. Both their father and grandfather died by sudden death at young age, without specific diagnosis. Their uncle was diagnosed with adult-onset cardiomyopathy at the age of 56, and has been found to be heterozygous for the truncating ALPK3 variant, identified in both of his nephews.

Conclusion: We report two novel variants in ALPK3 in a kindred with individuals affected by both infantile onset and adult onset cardiomyopathy, and provide additional evidence for the reported phenotypic spectrum of ALPK3-related cardiac disease.

References:.

Grants: The Morris Kahn Family Foundation.

Conflict of Interest: None declared.

P06.035.A Multidisciplinary and standardised post-mortem genetic analysis in a representative Czech cohort of sudden cardiac death (SCD) victims, together with genetic screening of their living relatives, yields high diagnostic yield in cases with positive family history and renders primary prevention of SD in affected families

Pavel Votypka¹, Petra Peldova¹, Patricia Norambuena², Stepanka Pohlova-Kucerova³, Terezia Tavacova⁴, Milan Macek Sr¹, Milan Macek ¹, Jan Janousek⁴, Josef Kautzner², Alice Krebsová²

¹Charles University 2nd Faculty of Medicine and Motol University Hospital, Department of Biology and Medical Genetics, Prague, Czech Republic; ²Institute for Clinical and Experimental Medicine, Department of Cardiology, Prague, Czech Republic; ³Charles University Faculty of Medicine Hradec Kralove, Department of Forensic Medicine, Hradec Kralove, Czech Republic; ⁴Charles University 2nd Faculty of Medicine and Motol University Hospital, Children Heart Centre, Prague, Czech Republic

Background/Objectives: Post mortem genetic analysis in SCD, together with the cardiologic examination of victim´s relatives, represents a multidisciplinary approach for its prevention. We assessed the molecular aetiology of SCD in a representative cohort and evaluated how its results facilitate prevention of SCD.

Methods: A total of 115 SCD cases (34 females/81 males; av. age 34.1 years; period 2016-2021) was ascertained. Following genetic consultation and cardiological examination of victims´ living relatives 100 candidate genes in 106/115 cases on the MiSeq platform (Illumina) followed by SOPHiA GENETICS DDM bioinformatics (Switzerland) were examined. Class IV-V variants were validated by Sanger DNA sequencing and segregation analyses.

Results: In total 20 sudden arrhythmic death (SADS), 22 -sudden unexplained (infant) death (SUD/SUID), 12- hereditary cardiomyopathy (HCM) and 14 - dilated cardiomyopathy/left ventricular noncompaction (DCM/LVNC), 22 - arrhythmogenic right ventricular cardiomyopathy (ARVC), 7 – sudden infant death (SIDS) and 11 - acute aortic dissection were diagnosed. Positive family history (pFH) of cardiac disease or SCD was identified in 27/115 (23.4 %; median age 41.0 years) of victims, whereas in cases without pFH their median age was significantly lower – 30.0 years. Molecular aetiology (i.e. presence of Class 4 -5 variants) was detected in a total 21/106 cases (19.8%) in RYR2, KCNH2, KCNQ1, SCN5A, FLNC, GLA, TTN, TNNT2, RBM 20, MYPN, MYBPC3, FHL1, TGFBR1 and COL3A1. Diagnostic yield in cases with pFA reached 13/27 cases (48.1 %).

Conclusion: We demonstrate the utility of post mortem cardiogenetic examination in individuals older than 40 years of age.

References:.

Grants: NV18-02-00237 and the Czech SCD Research Consortium.

Conflict of Interest: None declared.

P06.036.B Molecular analysis of cardiomyopathies using next generation sequencing technologies in Slovak patients

Jarmila Bernasovská ¹, Iveta Boronova¹, Michaela Zigová¹, Eva Petrejčíková¹

¹University of Presov, Institute of Biology, Presova, Slovakia

Background/Objectives: The aim of the study was to analyze a panel of genes associated with dilated or hypertrophic cardiomyopathy based on previously published results in order to identify the subjects at risk.

Methods: The method of next-generation sequencing by IlluminaHiSeq 2500 platform was used to detect sequence variants in 28 individuals diagnosed with dilated or hypertrophic cardiomyopathy. Detected variants were filtered and the functional impact of amino acid changes was predicted by computational programs.

Results: DNA samples of the 28 patients were analyzed by whole exome sequencing. We identified six nonsynonymous variants that were shown to be pathogenic in all used prediction softwares: rs3744998 (EPG5), rs11551768 (MGME1), rs148374985 (MURC), rs78461695 (PLEC), rs17158558 (RET) and rs2295190 (SYNE1). Two of the analyzed sequence variants had minor allele frequency (MAF)<0.01: rs148374985 (MURC), rs34580776 (MYBPC3).

Conclusion: Our data support the potential role of the detected variants in pathogenesis of dilated or hypertrophic cardiomyopathy; however, the possibility that these variants might not be true disease-causing variants but are susceptibility alleles that require additional mutations or injury to cause the clinical phenotype of disease must be considered.

References: Rocarati,M.,Latronico,M.V.G.,Musumeci,M. et al.: Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac miosin finding protein genes in Italian patients with hypertrophic cardiomyopathy. J Cell Physiol, 226,2894-2900, 2011.

Yamada T, Nomura S. Recent Findings Related to Cardiomyopathy and Genetics. Int J Mol Sci. 2021 Nov 20;22(22):12522. https://doi.org/10.3390/ijms222212522. PMID: 34830403; PMCID: PMC8623065.

Grants: KEGA 032PU-4/2021.

Conflict of Interest: None declared.

P06.037.C Aortic rupture force in mice modelling hereditary aortic diseases

Nicolo Dubacher^1;2, Kaori Sugiyama^3;4, Jeffrey Smith⁵, Vanessa Nussbaumer¹, Sylvan Caspar¹, Marc Schönholzer ¹, Janine Meienberg¹, Hiromi Yanagisawa³, Mary Sheppard^6;7, Gabor Matyas^1;8

¹Center for Cardiovascular Genetics and Gene Diagnostics, Swiss Foundation for People with Rare Diseases, Schlieren-Zurich, Switzerland; ²Translational Cardiovascular Technologies, Department of Health Sciences, ETH Zurich, Zurich, Switzerland; ³Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan; ⁴Institute For Advanced Research of Biosystem Dynamics, Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; ⁵Saha Cardiovascular Research Center, University of Kentucky, Lexington, United States; ⁶Department of Family and Community Medicine, University of Kentucky, Lexington, United States; ⁷Saha Aortic Center, University of Kentucky, Lexington, United States; ⁸Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland

Background/Objectives: Individuals suffering from hereditary aortic diseases (ADs) are at increased risk for aortic dissections and ruptures. We established an objective approach to measure the rupture force of the murine thoracic aorta, thereby explaining the outcomes of clinical studies and assessing an added value of old drugs in vascular Ehlers-Danlos syndrome (vEDS). Here, we applied our approach to six additional mouse AD models.

Methods: We used two mouse models for Marfan syndrome (MFS) as well as one smooth-muscle-cell-specific Efemp2 knockout (SMKO) and three CRISPR/Cas9-engineered knock-in models (Ltbp1, Mfap4, and Timp1). Moreover, one mouse MFS model was subjected to 4-week-long losartan treatment previously shown to reduce aneurysm growth. As previously described, 1.5-mm-long sections of the murine thoracic aorta were mounted on a tissue puller and uniaxially stretched until rupture.

Results: The aortic rupture force was significantly lower in both MFS and SMKO models, but mice with knock-in mutations in the genes Ltbp1, Mfap4, and Timp1 did not present with an impaired aortic integrity. As expected, the losartan treatment of MFS-modelling mice led to the reduction of aneurysm formation, which, surprisingly, had no impact on the aortic rupture force.

Conclusion: We show for the first time that our read-out system is able to characterize the aortic biomechanical integrity of mice modelling not only vEDS but also related ADs. Furthermore, aneurysm progression alone may not be a sufficient read-out for aortic rupture, as blood-pressure-lowering therapies preventing aortic aneurysms might still not strengthen the weakened aortic wall. These results may contribute to better medical therapies of hereditary ADs.

References:.

Grants:.

Conflict of Interest: None declared.

P06.038.D Functional characterization of iPSC-derived cardiomyocytes from Brugada syndrome patients of a genetically unresolved family reveals distinct underlying mechanisms

Dogan Akdeniz ¹, Aleksandra Nijak¹, Eline Simons¹, Bert Vandendriessche¹, Dieter Van de Sande¹, Erik Fransen¹, Ewa Sieliwonczyk¹, Dorien Schepers¹, Dirk Snyders¹, Emeline Van Craenenbroeck², Johan Saenen², Alain Labro³, Maaike Alaerts¹, Bart Loeys¹

¹University of Antwerp, Antwerp, Belgium; ²Antwerp University Hospital, Edegem, Belgium; ³Ghent University, Ghent, Belgium

Background/Objectives: Brugada syndrome (BrS) is an inherited cardiac arrhythmia characterized by a specific ECG pattern of ST-segment elevation, predisposition to ventricular fibrillation and sudden cardiac death. Over 20 genes have been associated with the disorder, however roughly 70% of patients remains without a genetic diagnosis.

Methods: In a large family with six mutation-negative BrS patients, we performed a SNP-based linkage analysis and whole-genome sequencing (WGS) on three patients. From two patients, one unaffected relative and an unrelated healthy control individual iPSC-derived cardiomyocytes (iPSC-CMs) were created. Molecular and electrophysiological characterization of the iPSC-CMs was performed using qPCR, immunocytochemistry, patch-clamping and calcium imaging.

Results: We detected significant linkage with a chromosome 2 locus (LOD-score 3.16). In the WGS-data, no interesting shared candidate variants were identified, also not in the linked locus. Studying the iPSC-CM models, one patient showed significantly reduced sodium current density, a positive shift in sodium channel voltage dependence of activation and reduced action potential amplitude and upstroke velocity. The second patient showed changes in calcium transient duration and rise time, while in both patients arrhythmia-like events occurred during the calcium recordings.

Conclusion: Characterization of iPSC-CMs of two patients from a BrS family showing significant linkage on chromosome 2, showed one patient with a sodium current loss-of-function phenotype consistent with previously reported BrS phenotypes, while the other one only showed calcium handling abnormalities. These results suggest different underlying disease mechanisms within one family, underscoring the complex nature of BrS.

References:.

Grants: Research Foundation Flanders, University of Antwerp BOF, ERC.

Conflict of Interest: None declared.

P06.039.A WES and its application for diagnostic purposes in hypertrophic cardiomyopathy

Albena Todorova ¹, Petya Angelova¹, Slavena Atemin^1;2, Nikolay Stoyanov³, Tihomir Todorov², Mila Sleptsova², Krasimira Hristova⁴, Mariana Gospodinova⁵, Vanyo Mitev¹, Vasil Velchev³

¹Medical University of Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ²Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria; ³University Hospital “St. Anna”, Department of Cardiology, Sofia, Bulgaria; ⁴Medical Center for Cardiovascular Disorders, Sofia, Bulgaria; ⁵University Hospital “St. Ivan Rilski”, Sofia, Bulgaria

Background/Objectives: Hypertrophic cardiomyopathy (HCM) is a common genetically heterogeneous disorder with an autosomal dominant inheritance and incomplete penetrance. More than 50 genes are associated with HCM and it is the most common cause of cardiac death due to cardiac failure. Genetic testing is strongly recommended for patients with clinically suspected HCM due to echocardiography/ECG data, family history for the disease and/or anamnestic data. The phenotype-genotype correlations are difficult to interpret: patients with the same genetic variant could express different clinical presentation, even in the same family.

Methods: A group of 20 HCM patients was screened for pathogenic variants by use of WES (Whole Exome Sequencing). The analysis included a panel of 242 genes associated with cardiomyopathy. The WES data is interpreted by GenesearchNGS software.

Results: The genetic diagnosis was clarified in 9 cases. Pathogenic variants were detected in the following genes: MYBPC3 - 4 variants; TNNI3; LAMP2; RBM20; MYLK2 and JPH2. The suspected clinical diagnosis of Danon disease was genetically confirmed by mutation in the LAMP2 gene. Severe hypertrophic and restrictive cardiomyopathy is provoked by TNNI3 mutation. The gene RBM20 seems to be a promising candidate gene for HCM.

Conclusion: The application of WES for diagnostic purposes in HCM cases turned out to be very useful in order to study the molecular bases of cardiomyopathies. In total 45% of our cases are genetically diagnosed. Moreover, the affected families are offered adequate genetic counselling and prenatal diagnostics.

References:.

Grants: The financial support of Medical University-Sofia, Grant № D-125/2021 is gratefully acknowledged.

Conflict of Interest: None declared.

P06.040.B APOE molecular spectrum in a French cohort with primary dyslipidemia

Yara Abou Khalil^1;2;3, Oriane Marmontel^4;5, Olivier Blureau⁶, Catherine Boileau^1;2;7, Marianne Abi-Fadel^1;3, Mathilde Di Filippo^4;5, Alain Carrié⁶, Jean-Pierre Rabes^8;9, Mathilde VARRET ^1;2

¹INSERM, Laboratory for Vascular Translational Science (LVTS), F-75018 Paris, France, Paris, France; ²Université de Paris and Université Sorbonne Paris Nord, Paris, France; ³Laboratory of Biochemistry and Molecular Therapeutics (LBTM), Faculty of Pharmacy, Pôle Tech-nologies-Santé (PTS), Saint-Joseph University, Beirut, Lebanon; ⁴CarMen Laboratory, INSERM U1060, INRAE U1397, Université Lyon 1, Oullins, France; ⁵Hospices Civils de Lyon, Department of Biochemistry and Molecular Biology, bron, France; ⁶INSERM UMRS 1166, Faculty of Medicine Pitié-Salpêtrière, Sorbonne University, Paris, France; ⁷Genetic Department, AP-HP, Hôpital Bichat, F-75018 Paris, Paris, France; ⁸Department of Biochemistry and Molecular Genetics, Ambroise Paré University Hospital, APHP, Boulogne Billancourt, France; ⁹UFR Simone Veil-Santé, University of Versailles-Saint-Quentin-en-Yvelines, Paris-Saclay University, Montigny-le-Bretonneux, France

Background/Objectives: Primary hypercholesterolemia is characterized by elevated LDL-cholesterol (LDL-C) levels isolated in case of autosomal dominant hypercholesterolemia (ADH) or associated with elevated triglycerides levels in case of familial combined hyperlipidemia (FCHL), both leading to cardiovascular diseases (CVD). Along with LDLR-APOB-PCSK9, rare APOE gene variants were reported in ADH and FCHL. We explored the APOE molecular spectrum in a French ADH/FCHL cohort of 5,743 unrelated probands.

Methods: NGS was performed on coding DNA sequence and flanking introns (exon padding+/- 30 bp) of the LDLR, PCSK9, APOB and APOE genes and on the 12 SNPs of the polygenic score..

Results: The LDLR, PCSK9, APOB and APOE sequencing revealed 76 carriers of a rare APOE variant, without a mutation in the first three genes. Among the 31 variants 5 were described in ADH/FCHL: p.Leu167del, p.Leu46Pro, p.Arg163Cys, p.Arg269Gly and p.Gly145Asp. Twelve novel missense, five synonymous, two intronic and seven variants in regulatory regions were also identified. Sixteen variants were predicted in silico pathogenic or likely pathogenic, and their carriers had a significantly lower polygenic risk score than carriers of predicted benign variants. We did not observe any correlation between the LDL-C levels and the polygenic risk score which is in favour of a major effect of APOE potentially pathogenic variants. The p.Leu167del carriers were associated with a more severe phenotype and our data suggest that APOE variant carriers are better responders to statins than carriers of a LDLR mutation.

Conclusion: Altogether, we show that the APOE variants account for a significant part of ADH and FCHL.

References:.

Grants:.

Conflict of Interest: None declared.

P06.041.C Discovery of rare variants associated with resting heart rate

Julia Ramírez ¹, Stefan van Duijvenboden¹, Lahiru Thomas Sooriyabandara², Andrew Tinker¹, Patricia Munroe¹

¹Queen Mary University of London, William Harvey Research Institute, London, United Kingdom; ²University of Nottingham, Nottingham, United Kingdom

Background/Objectives: Resting heart rate (RHR) is associated with cardiovascular disease. Genome-wide association studies (GWAS) have identified common variants at 437 loci, but their biological processes are not fully characterised. Identification of rare variants may help to pinpoint candidate genes.

Methods: We first performed a rare variant GWAS (RV-GWAS) for RHR in 388,223 individuals filtering by allele count > 10. Next, whole exome sequencing (WES) analysis in 161,539 individuals was done to explore consistency of findings and discover coding variants. All participants were of European ancestry from UK Biobank.

Results: The RV-GWAS identified 29 rare variants (9 at known RHR loci). At novel loci, we observe three missense variants in genes AKTIP, TBX5 and DBH. AKTIP encodes an AKT interacting protein, and a knockout mouse model has shown heart abnormalities. The rare variants at TBX5 and DBH are identical to those reported for blood pressure traits. The WES analysis identified 6 rare variants. Three variants overlapped the RV-GWAS and three variants were novel. We observe a missense variant in HIGD1B, a gene with no prior associations with cardiovascular phenotypes. At a second novel locus, a knockout mouse model of TBC1D32 demonstrates several cardiovascular abnormalities.

Conclusion: We identified 35 rare variants associated with RHR across the RV-GWAS and WES, highlighting potential novel candidate genes. Ongoing studies are focused on gene-based testing and validation.

References: (1) Mensah-Kane et al. Front Genet 18; 12:569323 (2021); (2) Lee et al. AJHG 95, 1, 5-23 (2014); (3) Silvestri V et al. Cancer 123, 210-8 (2017).

Grants: MRC grant MR/N025083/1, European Union-NextGenerationEU.

Conflict of Interest: None declared.

P06.042.D Cardiac Arrhythmia Syndrome with ST-Segment Depression

Enrica Marchionni ¹, Ruggiero Mango², Valentina Ferradini³, Francesca Di Lorenzo¹, Giovanni Parlapiano¹, Giuseppe Novelli^1;3, Federica Sangiuolo^1;3

¹Medical Genetics Unit, Tor Vergata University Hospital, Department of Oncohematology, Rome, Italy; ²Cardiology Unit, Department of Emergency and Critical Care,Tor Vergata University Hospital, Rome, Italy; ³Laboratory of Molecular Genetics, Department of Biomedicine and Prevention, Tor Vergata University Hospital, Rome, Italy

Background/Objectives: We report a large three-generation Italian family presenting with a peculiar ECG pattern and sudden cardiac death (SCD) in several individuals.

Methods: The proband was referred at 59 y.o. after a resuscitated cardiac arrest. She presented with recurrent atrial fibrillation and an abnormal ECG pattern, showing a concave-upward ST-segment depression in leads I, II, aVL, aVF and V2-6. Coronary angiographic studies were normal. She soon after died of SCD as well as her younger sister and several other family members, including her son, father, paternal grandmother, two uncles and three cousins. The proband’s older sister and her son were clinically screened and presented similar ECGs patterns.

Results: Firstly, genomic DNA was extracted from peripheral blood of the proband and Next Generation Sequencing (NGS) analysis was performed through a targeted panel of 61 genes associated with channellopathies and cardiomyopathies. NGS analysis failed to evidence the underlying genetic cause in the proband. Secondly, genomic DNA was extracted from peripheral blood of the proband’s older sister, her affected son and the healthy father. Trio-based Exome Sequencing (ES) was performed but failed to evidence the underlying genetic cause.

Conclusion: The intriguing ECG pattern of the present family fulfilled the proposed clinical criteria of ST-segment depression syndrome recently observed in five other families and segregating as an autosomal dominant trait¹. Extensive examination of the NGS data revealed no coding variants segregating with the disease in any of the families reported to date. Genome sequencing is ongoing to reveal the likely underlying genetic cause.

References: ¹PMID:30380381.

Grants: None.

Conflict of Interest: None declared.

P06.043.A The genetic landscape and clinical implication of pediatric Moyamoya angiopathy in a multiethnic cohort

Paolo Zanoni ¹, Katharina Steindl¹, Heinrich Sticht², Beatrice Oneda¹, Pascal Joset¹, Ivan Ivanovski¹, Anselm Horn², Elena Cabello¹, Alessandra Baumer¹, Julia Laube¹, Markus Zweier¹, Anita Rauch^1;3;4, Nadia Khan⁵

¹University of Zurich, Institute of Medical Genetics, Schlieren-Zurich, Switzerland; ²Friedrich-Alexander University Erlangen-Nürnberg, Institute of Biochemistry, Erlangen, Germany; ³University of Zurich, Zurich Center for Integrative Human Physiology, Zurich, Switzerland; ⁴University of Zurich and ETH Zurich, Neuroscience Center Zurich, Zurich, Switzerland; ⁵University Children’s Hospital Zurich, Moyamoya Center, Zurich, Switzerland

Background/Objectives: To perform a comprehensive genotype-phenotype analysis in paediatric Moyamoya Angiopathy (MMA)^1,2 patients with focus on clinical implications.

Methods: We performed molecular karyotyping, exome sequencing and automated structural assessment of missense variants on a series of 88 paediatric MMA patients and correlated genetic, angiographic and clinical findings.

Results: The two largest subgroups consisted of RNF213 and neurofibromatosis patients. While deleterious RNF213 variants were associated with a severe MMA clinical course with early symptom onset, frequent posterior cerebral artery involvement and higher stroke rates in multiple territories, NF1 patients had a similar infarct burden compared to non-NF1 individuals and were often diagnosed incidentally during routine MRIs. Additionally, we found that MMA-associated RNF213 variants have lower predicted functional impact compared to those associated with aortic disease. We also raise the question of MMA as a feature of recurrent as well as rare chromosomal imbalances and further support the association of MMA with STAT3 deficiency.

Conclusion: We provide a comprehensive characterization at the genetic and clinical level of a large exclusively pediatric MMA population. Due to the clinical differences we found across genetic subgroups we propose genetic testing for risk stratification as part of the routine assessment of pediatric MMA patients.

References: 1. Scott RM, Smith ER. Moyamoya Disease and Moyamoya Syndrome. N Engl J Med. 2009;360(12):1226-1237.

2. Kim SK, Cho BK, Phi JH, et al. Pediatric moyamoya disease: An analysis of 410 consecutive cases. Ann Neurol. 2010;68(1):92-101.

Grants: This project was in part supported by the UZH Research Priority Program Itinerare (A.R.).

Conflict of Interest: None declared.

P06.044.B Prevalence and clinical consequences of multiple pathogenic variants in dilated cardiomyopathy

Sophie Stroeks¹, Ida Lunde^2;3, Debby Hellebrekers⁴, Godelieve Claes⁴, Ingrid Krapels⁴, Hiroko Wakimoto², Els Vanhoutte⁴, Arthur van den Wijngaard⁴, Michiel Henkens¹, Anne Raafs¹, Maurits Sikking¹, Jos Broers⁵, Miranda Nabben^1;4;5, Elizabeth Jones^1;6, Stephane Heymans^1;6, Han Brunner^4;7, Job Verdonschot ^1;4

¹Maastricht University, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, Netherlands; ²Harvard Medical School, Genetics, Boston, United States; ³Akershus University Hospital, Diagnostics and Technology, Oslo, Norway; ⁴Maastricht University Medical Center, Clinical Genetics, Maastricht, Netherlands; ⁵Maastricht University, Genetics and Cell Biology, Maastricht, Netherlands; ⁶KU Leuven, Cardiovascular Sciences, Leuven, Belgium; ⁷Radboud University Medical Center, Human Genetics, Nijmegen, Netherlands

Background/Objectives: Dilated cardiomyopathy (DCM) was considered a monogenetic disease which can be caused by over 50 genes. Evidence suggests that the combination of multiple pathogenic variants leads to greater disease severity and earlier onset. So far, not much is known about the prevalence and disease course of multiple pathogenic variants in patients with DCM. To gain insight in these knowledge gaps, we 1) systematically collected clinical information of a well-characterized DCM cohort and 2) created a mouse model.

Methods: Complete cardiac pheno- and genotyping was performed in 854 consecutive DCM patients. Patients were followed for an average of 54 months. Endomyocardial biopsies were taken from patients for RNA-sequencing. Compound heterozygous digenic (LMNA/TTNΔA), monogenic (LMNA/WT) and WT/WT mice were created and phenotypically followed over time. Heart tissue was used for RNA-sequencing.

Results: 131 (likely) pathogenic (LP/P) variants were found in 854 consecutive tested DCM patients (15.4%). Three of the 131 patients had a second LP/P variant (2.3%). These three patients had a comparable disease onset, disease severity, and clinical course compared to DCM patients with one LP/P. The LMNA/TTNΔA mice also had no functional differences compared to the LMNA/WT mice after 40 weeks of follow-up, although RNA-sequencing suggests increased cardiac stress and sarcomere insufficiency in the LMNA/TTNΔA mice.

Conclusion: 2.3% of DCM patients with one LP/P also have a second LP/P in a different gene. Although the second LP/P does not seem to influence the disease course of DCM in patients and mice, the finding of a second LP/P can be important for their relatives.

References:.

Grants:.

Conflict of Interest: None declared.

P06.045.C Specific de novo variants in RNF213 cause a monogenic early-onset multisystemic disease ranging from childhood stroke to Leigh-like syndrome

Theresa Brunet ^1;2, Victoria Lieftüchter³, Dominic Lenz⁴, Philipp Peters³, Robert Kopajtich², Benedikt Zott^5;6;7, Hanna Zimmermann⁸, Irina Hüning⁹, Diana Ballhausen¹⁰, Christian Staufner⁴, Alyssa Bianzano⁴, Joanne Hughes¹¹, Robert Taylor^12;13, Robert McFarland^12;13, Tamara Žigman¹⁴, Danijela Petković Ramadža¹⁴, Rohlfs Meino³, Ute Hehr¹⁵, Neal Sondheimer^16;17;18, Stacy Hewson¹⁶, NIKOLAOS MARINAKIS¹⁹, Konstantina Kosma¹⁹, Jan Traeger-Synodinos¹⁹, Holger Prokisch^1;2, Thomas Meitinger¹, Ingo Borggräfe^20;21, Juliane Spiegler²², Ivo Baric¹⁴, Marco Paolini²³, Lucia Gerstl²⁰, Matias Wagner^1;2;20

¹Technical University of Munich, School of Medicine, Institute of Human Genetics, Munich, Germany., Munich, Germany; ²Institute of Neurogenomics, Helmholtz Zentrum München, Munich, Germany., Munich, Germany; ³Department of Pediatrics, Dr. von Hauner Children’s Hospital, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany., Munich, Germany; ⁴Centre of Child and Adolescent Medicine, Department of Pediatric Neurology and Metabolic Medicine, Heidelberg University Hospital, Heidelberg, Germany; ⁵Institute of Neuroscience, Technical University of Munich, Munich, Germany; ⁶Munich Cluster for Systems Neurology, Technical University of Munich, Munich, Germany; ⁷Department of Neuroradiology, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany; ⁸Institute of Neuroradiology, University Hospital, LMU Munich, Munich, Germany; ⁹Institute of Human Genetics, University of Lübeck, Lübeck, Germany, Lübeck, Germany; ¹⁰Pediatric Metabolic Unit, Pediatrics, Woman-Mother-Child Department, University of Lausanne and University Hospital of Lausanne, Lausanne, Switzerland; ¹¹National Centre for Inherited Metabolic Disorders, Children’s Health Ireland at Temple Street, Dublin, Ireland., Dublin, Ireland; ¹²Wellcome Centre for Mitochondrial Research, Translational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne United Kingdom., Newcastle, United Kingdom; ¹³NHS Highly Specialised Services for Rare Mitochondrial Disorders, Royal Victoria Infirmary, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK., Newcastle, United Kingdom; ¹⁴Department of Paediatrics, University Hospital Center Zagreb and University of Zagreb School of Medicine, Zagreb, Croatia., Zagreb, Croatia; ¹⁵Center for Human Genetics, and Department of Human Genetics, University of Regensburg, Regensburg, Germany., Regensburg, Germany; ¹⁶Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada., Torono, Canada; ¹⁷Program in Genetics and Genome Biology Program, Sick Kids Research Institute, Toronto, ON, Canada., Toronto, Canada; ¹⁸Departments of Pediatrics and Molecular Genetics, University of Toronto, Toronto, ON, Canada., Toronto, Canada; ¹⁹Laboratory of Medical Genetics, St. Sophia’s Children’s Hospital, National and Kapodistrian University of Athens, Athens, Greece., Athens, Greece; ²⁰Department of Pediatric Neurology, Developmental Medicine and Social Pediatrics, University of Munich, 80337 Munich, Germany., Munich, Germany; ²¹Epilepsy Center, University of Munich, 80337 Munich, Germany., Munich, Germany; ²²Department of Pediatrics, University Hospital of Würzburg, 97080 Würzburg, Germany., Würzburg, Germany; ²³Department of Radiology, University Hospital, LMU Munich, Munich, Germany., Munich, Germany

Background/Objectives: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as key susceptibility gene for Moyamoya disease (MMD). To date, only single case descriptions have also implicated an association of specific variants in RNF213 with an early-onset form of MMD with full penetrance. We aimed to systematically elucidate the clinical spectrum associated with de novo variants in RNF213 and to evaluate genotype-phenotype correlations.

Methods: Patients were identified through reanalysis of exome sequencing data of an unselected cohort from a single tertiary care center. Additional patients were ascertained via Genematcher and ClinVar. Phenotypic characterization including cMRI analyses and segregation analysis were performed.

Results: Nine individuals from eight unrelated families with heterozygous (8 de novo, 1 inherited) missense variants in RNF213 clustering within or around the RING domain were identified. 6/9 individuals had ischemic stroke between the age of birth and 23 years, five of which showed MRI alterations suggestive of Moyamoya disease. In contrast, 2 individuals had bilateral basal ganglia T2 hyperintensities and high CSF lactate. Additionally, five individuals had recurrent episodes of elevated liver enzymes and three cases had cardiomyopathy. Secondary structural epileptic encephalopathy was frequent (6/9) requiring antiepileptic medication.

Conclusion: De novo missense variants in RNF213 clustering in the E3 RING or a region distal to it lead to a monogenic syndrome with two clinical presentations. Most patients presented with neonatal or childhood onset stroke and Moyamoya alterations whereas a second group had a Leigh-like phenotype. Hereby, we establish RNF213 as a Mendelian Disease Gene with symptoms beyond MMD.

References:.

Grants:.

Conflict of Interest: None declared.

P06.048.B The challenging choice of gene panel size: our experience with hypertrophic and dilated cardiomyopathy

Sebastian Suchet ¹, Philippe Meyer¹, Eva Hammar Bouveret¹, Maurice Beghetti¹, Frédéric Masclaux¹, Michel Guipponi¹, Julie Wacker¹, Agathe Py¹, Judith Schwaiger², Marc Abramowicz¹, Jean-Louis Blouin¹, Christiane Gruner², Siv Fokstuen¹

¹University Hospitals of Geneva, Geneva, Switzerland; ²University Hospital Zurich, Zurich, Switzerland

Background/Objectives: Implementation of next generation sequencing (NGS) has led to a rapid expansion in the number of genes included in diagnostic genetic testing for cardiomyopathies. This tendency is changing with the evidence-based assessment of genes conducted by ClinGen. Our aim was to evaluate the mutation detection rate and the involved genes in patients with isolated hypertrophic or dilated cardiomyopathy (HCM and DCM) according to the gene panel size.

Methods: Our NGS approach consist of targeted exome sequencing. Before September 2019, we have used in-house gene panels including 65/78 cardiomyopathy genes. Since October 2019, we switched to Genomics England PanelAPP, including 144/140 high-evidence based genes causative for the different subtypes of cardiomyopathy.

Results: Between 2015 and 2021, a total of 135 patients with non-syndromic HCM or DCM underwent genetic testing. Our mutation detection rate was 49 % (36/74) with the in-house panels, 38% (23/61) with the PanelAPP panel and 44 % (59/135) overall. Comparing the two panel sizes we didn’t see any difference in the genes found with causative variants. All the genes with pathogenic or likely pathogenic variants identified with the large PanelAPP panel were already included in our smaller in-house panels.

Conclusion: Our results confirm the lack of major clinical benefit of large panels in isolated HCM or DCM and support the recommendations of the 2021 European Society of Cardiology heart failure guidelines, which suggest to use small evidence-based panels and to consider additional large panel only if there is a clear family history or a specific phenotype.

References:.

Grants:.

Conflict of Interest: None declared.

P07 Metabolic and Mitochondrial Disorders

P07.001.C Loss of centrosomal gene ALMS1 alters cell metabolism through the TGF-β pathway

Brais Bea Mascato ¹, Diana Valverde Pérez¹

¹University of Vigo, Vigo, Spain

Background/Objectives: Alström syndrome (AS) is a rare autosomal recessive disease that is associated with mutations in the ALMS1 centrosomal gene. The main manifestations of this syndrome are retinitis pigmentosa, obesity and type 2 diabetes mellitus. The depletion of the ALMS1 gene has been associated with the alteration of different processes regulated through the primary cilium, such as the Notch signalling pathway or TGF-β. Despite this, little is known about which cellular processes associated with the TGF-β pathway can be altered in the absence of ALMS1.

Methods: In this study we analyse the gene expression profile by RNA-seq of a cell line hTERT-BJ-5ta deficient in ALMS1, after stimulating the TGF-β pathway. We also performed a LFQ proteomic analysis by LC-MS/MS under the same conditions, to integrate the level of gene and protein expression. Finally, we validate the data obtained by western blot and fluorescence microscopy.

Results: RNA-seq data showed an enrichment mainly associated with TGF-β coordinated pathways such as PI3K/AKT, MAPKs or p53. Proteomic analysis showed an association between ALMS1 deficiency and alterations in cell metabolism and the lumen of intracellular organelles such as the endoplasmic reticulum. Observing the overlap of both datasets reinforced the relationship of ALMS1 with the endoplasmic reticulum and lipid-dependent signalling pathways. Finally, an over-activation of the AKT pathway was observed in the absence of ALMS1.

Conclusion: ALMS1 deficiency disrupted cross-signalling between the TGF-β pathway and other dependent pathways in immortalised fibroblasts. Furthermore, altered cross-signalling has implications for cellular metabolism and leads to over-activation of the AKT pathway.

References:.

Grants:.

Conflict of Interest: None declared.

P07.002.D ASAH1-related disorders: Expanding phenotypic spectrum requires updated thinking on diagnostic testing

Kathleen Crosby ¹, Alexander Solyom²

¹Aceragen, Durham, United States; ²Aceragen, Basel, Switzerland

Background/Objectives: Pathogenic variants in the ASAH1 gene cause acid ceramidase deficiency, which causes a spectrum of phenotypes. Farber disease, with typical symptoms including joint disease, subcutaneous nodules, and dysphonia, along with other symptoms like osteolysis; and SMA-PME (spinal muscular atrophy with progressive myoclonic epilepsy).

Methods: Data including range of symptoms, age at symptom onset, time to diagnosis and outcome were assessed. Sources included recent literature and the Observational and Cross-Sectional Cohort Study of the Natural History and Phenotypic Spectrum of Farber Disease (NCT03233841). This systematic clinical study of patients with Farber disease included prospective evaluations, with 45 patients enrolled.

Results: 266 cases of ASAH1-related disorders were reviewed for phenotypic classification. 129 cases were classified as severe Farber disease with onset of symptoms and death before the age of 4 years; 66 cases as moderate to attenuated Farber disease with symptom onset in childhood and survival ranging from late childhood to the 6th decade; and 17 cases as unclassified Farber disease. 46 cases were classified as SMA-PME. Additional patients included 3 cases of late-onset SMA without epilepsy, one case of PME without SMA, 2 cases of SMA who later developed Farber disease symptoms, and 2 cases with both SMA-PME and Farber disease symptoms in childhood.

Conclusion: ASAH1-related disorders including Farber disease, SMA-PME, or variations of these phenotypes, are likely underdiagnosed. The broad phenotypic spectrum highlights the need for increased clinical suspicion and expanded inclusion of the ASAH1 gene in genetic testing for individuals presenting without textbook symptoms of acid ceramidase deficiency.

References:.

Grants:.

Conflict of Interest: Kathleen Crosby Aceragen, Aceragen, Alexander Solyom Aceragen, Aceragen.

P07.003.A Use of whole genome sequencing to determine the genetic basis of suspected mitochondrial disorders

Katherine Schon ¹, Rita Horvath², Wei Wei², Claudia Calabrese², Arianna Tucci³, Kristina Ibañez³, Thiloka Ratnaike², Robert Pitceathly⁴, Enrico Bugiardini⁴, Ros Quinlivan⁴, Michael Hanna⁴, Emma Clement⁵, Emma Ashton⁶, John Sayer⁷, Paul Brennan⁸, Dragana Josifova⁹, Louise Izatt⁹, Carl Fratter¹⁰, Victoria Nesbitt¹⁰, Timothy Barrett¹¹, Dominic McMullan¹¹, Audrey Smith¹², Charulata Deshpande¹², Sarah Smithson¹³, Richard Festenstein¹⁴, Natalie Canham¹⁵, Mark Caulfield³, Henry Houlden⁴, Shamima Rahman¹⁶, Patrick Chinnery¹

¹University of Cambridge, Department of Clinical Neurosciences, Cambridge, United Kingdom; ¹University of Cambridge, Department of Clinical Neurosciences, Cambridge, United Kingdom; ³Queen Mary University London, William Harvey Research Institute, London, United Kingdom; ⁴University College London, Queen Square Institute of Neurology, London, United Kingdom; ⁵Great Ormond Street Hospital for Children NHS Foundation Trust, Department of Clinical Genetics and Genomic Medicine, London, United Kingdom; ⁶Great Ormond Street Hospital for Children NHS Foundation Trust, NHS North Thames Genomic Laboratory Hub, London, United Kingdom; ⁷Newcastle University, Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle, United Kingdom; ⁸Newcastle Hospitals NHS Foundation Trust, Northern Genetics Service, Newcastle, United Kingdom; ⁹Guy’s Hospital, Department of Clinical Genetics, London, United Kingdom; ¹⁰Oxford University Hospitals NHS Foundation Trust, NHS Highly Specialised Services for Rare Mitochondrial, Oxford, United Kingdom; ¹¹Birmingham Women’s and Children’s NHS Foundation Trust, West Midlands, Oxford and Wessex Genomics Laboratory Hub, Birmingham, United Kingdom; ¹²Manchester Centre for Genomic Medicine, Manchester, United Kingdom; ¹³University Hospitals Bristol, Department of Clinical Genetics, Bristol, United Kingdom; ¹⁴Imperial College, Department of Brain Sciences, London, United Kingdom; ¹⁵Liverpool Women’s Hospital, Liverpool Centre for Genomic Medicine, Liverpool, United Kingdom; ¹⁶Great Ormond Street Hospital for Children NHS Foundation Trust, Metabolic Unit, London, United Kingdom

Background/Objectives: Diagnosis of mitochondrial disorders is challenging because they are clinically and genetically heterogeneous. WES has been effective for diagnosing mitochondrial disorders and discovering new disease genes. However, it is not possible to make a diagnosis in ~40%. WGS can diagnose pathogenic variants affecting the mtDNA and the nuclear genome, so it has the potential to make diagnoses in more families and shorten the ‘diagnostic odyssey’.

Methods: 319 families with suspected mitochondrial disorders were referred to the 100,000 Genomes Project after excluding common genetic causes. Human Phenotype Ontology terms were recorded, median 7 per participant. Short read WGS was analysed. Variants were prioritised using phenotype-based gene panels, Exomiser and comparison to ClinVar. mtDNA variants were called using an in-house pipeline. Copy number variants and short tandem repeats for thirteen neurological disorders were analysed.

Results: A definite or probable genetic diagnosis was identified in 98 families (31%), with an additional 6 possible diagnoses (2%). In total, 95 different genes were implicated. 37.5% of families had a mitochondrial diagnosis and 62.5% had a non-mitochondrial diagnosis. Diagnostic yield was higher in trios/quads (62/148) compared to singletons (23/102) (p = 0.005) and in children (64/143) compared to adults (46/202) (p < 0.001).

Conclusion: WGS is a useful diagnostic test in patients with suspected mitochondrial disorders yielding a diagnosis in a further 31% after excluding common causes. The majority of diagnoses were non-mitochondrial disorders, including developmental disorders with intellectual disability, epileptic encephalopathies, metabolic disorders, cardiomyopathies and leukodystrophies. These would have been missed using a targeted approach, with some having specific treatments.

References: BMJ 2021;375;e066288.

Grants:.

Conflict of Interest: Katherine Schon Addenbrooke’s Charitable Trust. Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1)., Rita Horvath Wellcome Trust Investigator (109915/Z/15/Z).

Medical Research Council (UK) (MR/N025431/1).

European Research Council,.

Newton Fund (UK/Turkey, MR/N027302/1).

Evelyn Trust.

Lily Foundation, Wei Wei: None declared, Claudia Calabrese: None declared, Arianna Tucci: None declared, Kristina Ibañez: None declared, Thiloka Ratnaike: None declared, Robert Pitceathly Medical Research Council Clinician Scientist Fellowship (MR/S002065/1).

Medical Research Council strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) (MR/S005021/1), Enrico Bugiardini: None declared, Ros Quinlivan: None declared, Michael Hanna Medical Research Council strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) (MR/S005021/1), Emma Clement: None declared, Emma Ashton: None declared, John Sayer: None declared, Paul Brennan: None declared, Dragana Josifova: None declared, Louise Izatt: None declared, Carl Fratter: None declared, Victoria Nesbitt: None declared, Timothy Barrett: None declared, Dominic McMullan: None declared, Audrey Smith: None declared, Charulata Deshpande: None declared, Sarah Smithson: None declared, Richard Festenstein: None declared, Natalie Canham: None declared, Mark Caulfield: None declared, Henry Houlden MRC, Wellcome Trust, Shamima Rahman BioMedical Insights - Payment to institution.

Neurovive - Payment to institution.

Partners4Access - Payment to institution.

Pfizer- Payment to institution.

Epirium - Consultancy fee.

Stealth Biotherapeutics - Payment to institution.

Taysha Gene Therapies, Inc. - Consultancy fee.

Modis Therapeutics, Inc., a wholly.

owned subsidiary of Zogenix, Inc. - Consultancy fee.

Pretzel Therapeutics, Inc. - Consultancy fee.

Access Infinity - Payment to institution, Editor, Journal of Inherited Metabolic Disease.

Senior Editor, Annals of Human Genetics.

Special Adviser to HFEA, Patrick Chinnery PFC is a Wellcome Trust Principal Research Fellow (212219/Z/18/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/9), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), the Leverhulme Trust (RPG-2018-408), an MRC research grant (MR/S035699/1), an Alzheimer’s Society Project Grant (AS-PG-18b-022) and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge.

P07.004.B Genome-wide expression analysis in Fabry disease human podocyte cell line

Sarah Snanoudj ¹, celine derambure², Céline Lesueur¹, Lénaïg Donval³, Stéphane Marret³, Soumeya Bekri¹, Abdellah Tebani¹

¹Department of Metabolic Biochemistry, UNIROUEN, INSERM U1245, CHU Rouen, Normandie University, Rouen, France; ²Normandie University, UNIROUEN, IRIB, Inserm U1245, Normandy Center for Genomic and Personalized Medicine, Rouen, France; ³Department of Neonatal Pediatrics, Intensive Care and Neuropediatrics, UNIROUEN, INSERM U1245, CHU Rouen, Normandie University, Rouen, France

Background/Objectives: Fabry disease (FD) is an X-linked lysosomal disease due to alpha-galactosidase A deficiency. This enzyme is involved in glycosphingolipid metabolism and its alteration leads to cellular dysfunction and microvascular pathology. Different organs are involved with kidney as a main organ target. The rise of omics sciences has prompted a paradigm shift, in both research and medicine. These approaches may open new insights into the pathophysiology of multifactorial complex diseases. We performed genome-wide expression analysis in an FD human podocyte model.

Methods: RNAseq-based genome-wide expression analysis was done on human immortalized alpha-galactosidase A deficient podocytes generated using CRISPR/Cas9 technology, and control podocytes. Differential expression analysis was performed using DESeq2 package.

Results: Two hundred and forty-seven genes were differentially expressed, with 111 genes overexpressed and 136 under-expressed in FD compared to controls. Genes known to be involved in angiogenesis (ITGB3), autophagy (TIA1, SMG1) and oxidative stress (CBR3, BLVRA) were among these genes. The STRIPAK complex, related to autophagy, and the NADP/NADPH pathway (oxidative stress) were among the most altered pathways. The downregulated genes included MOB1A linked to the Hippo pathway which participates in kidney podocytes homeostasis maintenance.

Conclusion: These preliminary results unveil Fabry disease-related transcriptomic expression patterns. Further characterization of these disrupted cellular pathways could enable deeper understanding of FD pathophysiology.

References:.

Grants: None.

Conflict of Interest: Sarah Snanoudj University Hospital of Rouen, University of Rouen Normandy, celine derambure University of Rouen Normandie, Céline Lesueur University Hospital of Rouen, Lénaïg Donval: None declared, Stéphane Marret University Hospital of Rouen, University of Rouen Normandie, Soumeya Bekri University Hospital of Rouen, University of Rouen Normandy, Abdellah Tebani University Hospital of Rouen, University of Rouen Normandy.

P07.005.C Rapid exome sequencing for children with severe acute encephalopathy, a case series

Clair Habib ¹, Tamar Paperna¹, Rinat Zaid¹, Josef Ben Ari², Sarit Ravid³, Galit Tal⁴, Karin Weiss¹, Tova Hershkovits¹

¹Rambam Health Care Campus, The Genetics Institute, Haifa, Israel; ²Rambam Health Care Campus, Pediatric Intensive Care Unit, Haifa, Israel; ³Rambam Health Care Campus, Pediatric Neurology department, Haifa, Israel; ⁴Rambam Health Care Campus, Metabolic Clinic, Haifa, Israel

Background/Objectives: Increasingly, next generation sequencing (NGS) is becoming an invaluable tool in the diagnosis of acute neurological disorders where a monogenic etiology is not suspected such as acute encephalopathy\encephalitis.

Here, we describe a brief series of pediatric patients hospitalized in the pediatric intensive unit. All presented with severe acute encephalopathy initially suspected to be of infectious or inflammatory origin, but subsequently diagnosed with a monogenic disorder.

Methods: Rapid exome sequencing was performed during the initial hospitalization of three unrelated patients. Data analysis and initial report was performed in-house in a few hours. All patients were of Muslim Arab descent with a history of consanguinity, previously healthy ranging from 1.5-3 years. One patient presenting with acute necrotizing encephalopathy (ANEC) had a sister who presented with ANEC one year prior.

Results: Exome sequencing was diagnostic in all three cases. One patient had a homozygous pathogenic variant in MOCS2, c.3G>A p.(Met1Ile) associated with late onset Molybdenum cofactor deficiency B. A second patient harbored a homozygous likely pathogenic variant in NDUFS8 c.441G>C p.(Met147Ile). Surprisingly, the initial work-up was not suggestive of this disorder. Finally, a likely pathogenic homozygous missense variant c.359T>C p.(Ile120Thr) in the DBR1 gene was identified in the patient presenting with ANEC which segregated as expected in the family.

Conclusion: This case series demonstrates use of rapid exome sequencing is shifting the paradigm of diagnostics even in critical care situations and should be considered early on in children with acute encephalopathy. Timely diagnosis can direct initial treatment as well as informing decisions regarding long term care.

References:.

Grants:.

Conflict of Interest: None declared.

P07.006.D Analysis of the mitochondrial 13513G>A mutation: A case report of five Hungarian patients

Vera Várhegyi ¹, Fruzsina Szabo¹, Zoltan Grosz¹, Viktor Molnár¹, Noemi Agnes Varga¹, Petra Zsidegh², Agnes Herczegfalvi³, Viktória Szabó⁴, Anita Maasz⁵, Judit Bene⁵, Kinga Hadzsiev⁵, Aniko Gal¹, Maria Judit Molnar¹

¹Institute of Genomic Medicine and Rare Disorders Semmelweis University, Budapest, Hungary; ²Semmelweis University, 1st Department of Pediatrics, Budapest, Hungary; ³Semmelweis University, 2nd Department of Pediatrics, Budapest, Hungary; ⁴Semmelweis University, Department of Ophthalmology, Budapest, Hungary; ⁵University of Pécs, Department of Medical Genetics, Pécs, Hungary

Background/Objectives: Pathogenic mutations in the mitochondrial DNA (mtDNA) underlying primary mitochondrial disease result in a wide variety of clinical phenotypes. The m.13513 G>A (p.D355N) mutation in the MT-ND5 gene has been reported in the literature in the background of Leigh syndrome, MELAS, and MELAS / LHON overlap syndrome. In the present study, we sought to answer the question about the proportion of this mtDNA alteration that occurs among the Hungarian population.

Methods: In this study, 537 patients were examined for the m.13513 G>A mutation by bidirectional sequencing. DNA isolation was performed in 390 cases from blood and in 147 cases from postmitotic tissue (muscle, urinary squamous cell).

Results: Among the studied patients, the mtDNA G13513A mutation was detected in five cases. The heteroplasmy ratio was in 50 - >95%. Clinical symptoms ranged from a broad spectrum of phenotypes such as Leigh syndrome, ataxia, strabismus, neuropathy, visual impairment, and renal failure. In four cases, the disease was associated with early onset Leigh syndrome, while in one patient, clinical symptoms manifested in adulthood with multisystemic involvement, with severe visual and renal impairment and hypoacusis.

Conclusion: The m.13513 G>A mtDNA alteration was present in about 1% of the total investigated cohort, while in the cases examined from postmitotic tissues this proportion was found to be 2.88%. Based on the above, we recommend a more comprehensive study of the m.13513 G>A mutation from postmitotic tissue.

References:.

Grants: Hungarian Brain Research Program, NKFIH_FK_132812, NKFIH_139010, Semmelweis University Startup grants, János Bolyai Research Scholarship, UNKP-21-5 Research Scholarship.

Conflict of Interest: Vera Várhegyi part-time, Fruzsina Szabo full time, Zoltan Grosz full time, Viktor Molnár full time, Noemi Agnes Varga full time, Petra Zsidegh full time, Agnes Herczegfalvi full time, Viktória Szabó full time, Anita Maasz full time, Judit Bene full time, Kinga Hadzsiev full time, Aniko Gal full time, Maria Judit Molnar full time.

P07.007.A Severity of ATAD3A-related pontocerebellar hypoplasia correlates with severity of mutations

Martina Skopkova ¹, Vibhuti Rambani¹, Hana Stufkova², Viktor Stranecky³, Katarina Brennerova⁴, Miriam Kolníková⁵, Michaela Pietrzykova⁶, Hana Hansikova², Daniela Gasperikova¹

¹Institute of Experimental Endocrinology, Biomedical Research Center SAS, Department of Metabolic Disorders, Bratislava, Slovakia; ²First Faculty of Medicine, Charles University and General University Hospital in Prague, Laboratory for Study of Mitochondrial Disorders, Department of Paediatrics and Inherited Metabolic Disorders, Prague, Czech Republic; ³First Faculty of Medicine, Charles University and General University Hospital in Prague, Research Unit for Rare Diseases, Department of Paediatrics and Inherited Metabolic Disorders, Prague, Czech Republic; ⁴Medical Faculty of Comenius University and National Institute of Children’s Diseases, Department of Paediatrics, Bratislava, Slovakia; ⁵Medical Faculty of Comenius University and National Institute of Children’s Diseases, Department of Paediatric Neurology, Bratislava, Slovakia; ⁶Medical Faculty of Comenius University and University Hospital in Bratislava, Department of Clinical Genetics, Institute of Medical Biology, Genetics and Clinical Genetics, Bratislava, Slovakia

Background/Objectives: Diagnostics of ATAD3A-related disorders is challenging from two reasons. Firstly, the ATAD3 locus contains three paralogous genes, making it difficult target for both sequencing and CNV analysis. Secondly, the clinical picture and severity are heterogeneous, ranging from recessive neonatal-lethal pontocerebellar hypoplasia through milder dominant Harel-Yoon syndrome to, again, neonatal-lethal but dominant cardiomyopathy. Fourteen different recessive variants in 17 families have been described with so far.

Methods: We report two families, each with two affected children. Patients and their parents were analysed using WES followed by CNV analysis (ExomeDepth). Variants were verified using Sanger sequencing and long-range PCR. Enzymological and biochemical studies were performed in muscle and cultivated fibroblasts of one proband.

Results: Decreased activity of complex IV and decreased levels of nuclear-encoded subunits of respiratory chain confirmed mitochondriopathy in the Family 1 proband. Compound heterozygous p.Leu77Val and exon 3-4 deletion in the ATAD3A gene were found in all four affected members of both families. The p.Leu77Val variant had previously been described as mild by functional studies (Yap et al., 2021), exon 3-4 deletion is considered loss-of-function with severe impact. This novel combination of high- and mild-impact variants resulted in strikingly homogenous phenotype in our patients - less severe and with longer life-span than in case of bi-allelic loss-of function variants.

Conclusion: Clinical picture and severity of ATAD3A-related disorders are dependent on the type of mutation and correlate with predicted severity of variants and their combinations.

References: Yap et al., Genome Med 2021.

Grants: APVV-17-0296, AZV MZCR NV19-07-00136.

Conflict of Interest: None declared.

P07.008.B Unraveling a case of Niemann-Pick disease using long read sequencing and adaptive sampling

Deniz Karadurmus ¹, Sylvain Brohée¹, Sandrine Mary¹, Valérie Benoit¹, Anne Destree¹, Dominique Roland¹, Pascale Hilbert¹

¹Institut de Pathologie et Génétique, Gosselies, Belgium

Background/Objectives: These last years, Oxford Nanopore Technology (ONT) emerged as a promising actor for structural variants identification and repetitive regions analysis by long read sequencing. Moreover, ONT recently added the adaptive sampling option on its sequencing devices, i.e. a software-controlled enrichment method which allows to target genomic regions of interest without any specific wet-lab enrichment procedure. This method has been shown to identify structural variants not detected by conventional genetic testing (1). Therefore, we used this approach to analyze the unsolved case of a patient affected by the autosomal recessive Niemann-Pick disease, but in whom only one pathogenic mutation was previously identified in NPC1 gene despite extensive genetic testing.

Methods: Adaptive sampling of ONT was performed using 1.5 µg of DNA, on the basis of a custom bed file targeting NPC1 and surrounding genes.

Results: We reached a mean fold-enrichment of 7 on targeted region, along with mean depth coverage of 25.5X. Our result suggested an inversion of 70kb, involving NPC1 and its adjacent gene ANKRD29. This was then confirmed by PCR and Sanger sequencing. Moreover, segregation study showed that this inversion was in trans with the known point mutation, confirming Niemann-Pick diagnostic at molecular level.

Conclusion: The adaptive sampling option proposed by ONT appears extremely flexible and allows easy case-by-case adjustment and analysis. While still perfectible, we believe that this approach could be a valuable help in daily practice of genetics experts, in conjunction with classic genetic testing.

References: 1. Miller et al., PMID: 34216551.

Grants: Institut de Pathologie et Génétique Research Funds.

Conflict of Interest: None declared.

P07.009.C Follow-up of family members of children with mtDNA-associated Leigh syndrome

Celia Azevedo Soares ^1;2, Margarida Paiva Coelho^3;4, Anabela Bandeira^3;4, Rute Martins⁴, Rosa Ribeiro⁴, Natalia Tkachenko¹, Ana Maria Fortuna^1;2, Esmeralda Martins^2;3;4

¹Centro Hospitalar Universitário do Porto, Serviço de Genética Médica, Porto, Portugal; ²Instituto de Ciências Biomédicas Abel Salazar/Universidade do Porto, Unit for Multidisciplinary Research in Biomedicine, Porto, Portugal; ³Centro Hospitalar Universitário do Porto, Serviço de Pediatria Médica, Porto, Portugal; ⁴Centro Hospitalar Universitário do Porto, Centro de Referência de Doenças Hereditárias do Metabolismo, Porto, Portugal

Background/Objectives: Leigh syndrome (LS) is a genetic neurometabolic disorder characterized by central nervous system degeneration. Some patients have missense pathogenic variants in mtDNA inherited from the matrilineal lineage. Disease´s expressivity is dependent on the heteroplasmy level at a given tissue. Given this variable expressivity, we reviewed the features of family members of a cohort of patients with mtDNA-associated LS (MALS) in their matrilineal lineage. With this revision, we aim to understand the necessity of a structured follow-up for extended family members.

Methods: We reviewed five families with children with MALS syndrome followed at Centro Hospitalar e Universitário do Porto.

Results: All mothers were asymptomatic at the time of their children´s diagnosis, but two mothers initiated potentiality mitochondrial-related complains after their child diagnosis. The mother of a child with MALS associated to m.10191T>C variant, presented headaches and abnormal subcortical white-matter T2 MRI signal, suggestive of adult onset-LS, with exclusion of autoimmunity and infections. The familial variant was not detected in this woman peripheral blood. A mother of a child with MALS associated to m.8993T>G variant, showed a heteroplasmy level of 75%, and presented complains of headaches and fatigue. Her MRI showed left cerebellar cortico-subcortical hypodense area, suggestive of previous ischemic event. In this second family, two family members by the matrilineal lineage present complains suggestive of neuropathy, ataxia, and retinitis pigmentosa.

Conclusion: Follow-up guidelines should be established for MALS family members at risk and genetic counseling should be offered to all family members in the matrilineal lineage of an index case.

References:.

Grants:.

Conflict of Interest: None declared.

P07.010.D ATAD3 knockout model in zebrafish

Shlomit Ezer ^1;2, Nathan Ronin^3;4, Shahar Rotem-Bamberger^3;4, Shira Yanovsky-Dagan¹, Adi Inbal^3;4, Tamar Harel¹

¹Hadassah Medical Center, Department of Genetics, Jerusalem, Israel; ²Hebrew University, Faculty of Medicine, Jerusalem, Israel; ³Hebrew University of Jerusalem, Department of Medical Neurobiology, Jerusalem, Israel; ⁴The Institute for Medical Research Israel-Canada, Jerusalem, Israel

Background/Objectives: ATAD3A encodes a mitochondrial expressed protein which spans the inner and outer mitochondrial membranes. It is involved in mitochondrial dynamics, mtDNA maintenance, cholesterol metabolism, ER-mitochondria interaction and more. ATAD3A pathogenic variants have been shown to cause distinct neurological diseases in humans. We aimed to generate an atad3-null line in zebrafish, in order to further characterize the gene and its role in health and disease and to test possible remedies.

Methods: Using CRISPR-Cas9 genome editing, we created two lines of heterozygous fish with frameshift variants which can be bred to produce atad3-null embryos. We examined the phenotype and mitochondrial content of the mutant embryos, and compared the transcriptome of wild-type and mutant embryos at 3 days post-fertilization (dpf) via RNAseq. Results were validated by quantitative real-time PCR at 3dpf and 5dpf.

Results: Atad3-null embryos demonstrated microcephaly, small eyes, pericardial edema and thinning of the musculature, closely correlating with the human disease. Mitochondrial content was reduced. Transcriptome analysis revealed an expected decline in most mitochondrial pathways in the mutant embryos. In addition, we witnessed an unexpected global upregulation of cytosolic tRNA synthetases, presumably secondary to ER stress.

Conclusion: Zebrafish atad3-null embryos can be used as a reliable model of human ATAD3A-associated disorders. ER stress signals seem to have a role in the pathogenesis, and blocking ER stress by small compounds may serve as a potential therapeutic pathway.

References: Harel, T., et al. (2016). Am J Hum Genet 99, 831-845.

Grants: ISF 1663/17 to TH.

Conflict of Interest: None declared.

P07.011.A Impact of GCSH-deficiency: a protein at the crossroad of one-carbon metabolism and cellular respiration

Laura Arribas ¹, Mike Swanson², Elsebet Østergaard³, Cristina Dallabona⁴, Kostas Tsiakas⁵, Maja Hempel⁶, Cecile Acquaviva⁷, Joseph Farris⁸, Nicholas Stence⁹, Martina Magistrati⁴, Stefanos Koutsoukos¹⁰, Elaine Spector², Kathryn Kronquist², Mette Christensen³, Helena Karstensen³, Rene G. Feichtinger¹¹, Melanie Achleitner¹¹, Lawrence Merritt², Belén Pérez¹, Magdalena Ugarte¹, Stephanie Grunewald¹², Anthony Riela¹³, Natalia Julve¹⁴, Jean-baptiste Arnoux¹⁵, Hans A. Mayr¹¹, Kastouri Haldar⁸, René Santer⁵, Allan Lund³, Claudia Donnini⁴, Pilar Rodríguez¹, Johan Van Hove²

¹Universidad Autónoma Madrid, Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa, CBM-CSIC, Departamento de Biología Molecular, Madrid, Spain; ²University of Colorado, Department of Pediatrics, Aurora, Colorado, United States; ³Rigshospitalet - Copenhagen University Hospital, Centre for Inherited Metabolic Diseases, Department of Clinical Genetics, Copenhagen, Denmark; ⁴University of Parma, Department of Chemistry, Life Sciences and Environmental Sustainability, Parma, Italy; ⁵Department of Pediatrics, University Medical Center Hamburg Eppendorf, Hamburg, Germany; ⁶Insitute of human genetics, University Medical Center Hamburg Eppendorf, Hamburg, Germany; ⁷CHU de Lyon, Services Maladies Héréditaires du Métabolisme, Centre de Biologie Est, Lyon, France; ⁸University of Notre Dame, Boler-Parseghian Center for Rare and Neglected Disease, and Department of Biological Sciences, Notre Dame, Indiana, United States; ⁹University of Colorado, Radiology Department, Aurora, Colorado, United States; ¹⁰University of Colorado, Department of Pediatrics, Section of Clinical Genetics and Metabolism, Aurora, United States; ¹¹University Children’s Hospital, Paracelsus Medical University, Salzburg, Austria; ¹²Great Ormond Street Hospital, Institute for Child Health, Department of Metabolic Medicine, London, United Kingdom; ¹³Texas Child Neurology, Piano, TX, United States; ¹⁴IMED Valencia Hospital, Department of Pediatrics, Valencia, Spain; ¹⁵Necker Enfants Malades Hospital, Centre de Reference des Maladies Hereditaires du Metabolisme, Paris, France

Background/Objectives: Post-translational lipoylation of the 2-ketoacid dehydrogenases (2KDH) is essential in the bioenergetics metabolism of eukaryotes. The GCSH-protein, lipoylated subunit of the glycine cleavage enzyme, has been proposed as the only acceptor of lipoate for transferring to other 2KDH in mammals but, this dual function remains unproven. We present an extensive analysis of the nucleotide variations identified in a cohort of six patients with genetic confirmation of bi-allelic changes in the GCSH gene.

Methods: The patient’s phenotyping included clinical record evaluation, biochemical analysis and brain MRIs. Genetic analysis was based on exome sequencing. Pathogenicity of nucleotide changes was assessed by in silico modelling and functional analysis in patient’s cells, knock-down models and purified recombinant proteins.

Results: The clinical presentations ranged from very severe early presentation with a fatal outcome to late milder courses. Patients had increased glycine levels in plasma and cerebrospinal fluid and mostly normal lactate. Genetic analysis identified four missense changes, one nonsense and two genomic rearrangements. Functional studies showed decreases in GCSH-protein amount, lipoylation status and mitochondrial respiration either in patient’s cells or after overexpressing missense GCSH-proteins in two GCSH knock-down models generated in COS7 and S. cerevisiae. Purified recombinant GCSH protein studies resembled those results.

Conclusion: We unravel the final step in post-translational lipoylation in mammals and describe a cohort of patients with pathogenic GCSH variants responsible for a new variant-form of NKH combining deficient GCS complex and bioenergetics defects.

References:.

Grants: Partially granted for the Spanish Ministerio de Ciencia e Innovación/Fondo Europeo de Desarrollo Regional (AEI/FEDER UE) PI19/01155.

Conflict of Interest: None declared.

P07.013.C Biallelic variants in PYROXD2 cause a severe infantile metabolic disorder affecting mitochondrial function

Nicole Van Bergen^1;2, Daniella Hock³, Lucy Spencer¹, Sean Massey¹, Tegan Stait¹, Zornitza Stark^2;4;5, Sebastian Lunke^2;6, Ain Roesley⁴, Heidi Peters^2;7, Joy Lee^2;7, Anna Le Fevre ⁴, Oliver Heath⁷, Cristina Mignone⁸, Joseph Yang^2;9;10, Monique Ryan¹¹, Colleen D’Arcy¹², Margot Nash¹³, Sile Smith¹⁴, Nikeisha Caruana^3;15, David Thorburn^1;2;4, David Stroud^1;3, Sue White^2;4, John Christodoulou^1;2;16, Natasha J Brown^2;4

¹Murdoch Children’s Research Institute, Brain and Mitochondrial Research Group, Parkville, Australia; ²University of Melbourne, Department of Paediatrics, Parkville, Australia; ³University of Melbourne, Department of Biochemistry and Pharmacology and Bio21 Molecular Science and Biotechnology Institute, Parkville, Australia; ⁴Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Parkville, Australia; ⁵Australian Genomics Health Alliance, Parkville, Australia; ⁶University of Melbourne, Department of Pathology, Parkville, Australia; ⁷Royal Children’s Hospital, Department of Metabolic Medicine, Parkville, Australia; ⁸Royal Children’s Hospital, Medical Imaging Department, Parkville, Australia; ⁹Royal Children’s Hospital, Department of Neurosurgery, Neuroscience Advanced Clinical Imaging Service, Parkville, Australia; ¹⁰Murdoch Children’s Research Institute, Developmental Imaging, Parkville, Australia; ¹¹Royal Children’s Hospital, Neurology Department, Parkville, Australia; ¹²Royal Children’s Hospital, Anatomical Pathology Department, Parkville, Australia; ¹³Royal Children’s Hospital, General Medicine, Parkville, Australia; ¹⁴Royal Children’s Hospital, Paediatric Intensive Care Unit, Parkville, Australia; ¹⁵Victoria University, Institute for Health and Sport, Footscray, Australia; ¹⁶University of Sydney, Discipline of Child and Adolescent Health, Camperdown, Australia

Background/Objectives: Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2) is a mitochondrial inner membrane/matrix-residing protein reported to regulate mitochondrial function. Little is known about the clinical importance or precise biological function of PYROXD2. We report biallelic variants in PYROXD2 in a patient with suspected mitochondrial disease.

Methods: A male infant presented with acute unresponsive episodes and extreme metabolic acidosis. He developed progressive neurological deterioration on a background of postnatal-onset poor growth and microcephaly. He died at 6.5 months age. Magnetic resonance brain imaging showed changes resembling Leigh syndrome. The proband and his unaffected parents underwent genome sequencing and RNA-seq analysis. Functional assays were conducted to assess mitochondrial function using patient fibroblasts. Proteomic differences between the proband and control fibroblasts were investigated with high-resolution tandem mass spectrometry and quantitative protein analysis.

Results: No causative variants were found through standard trio analysis of known disease-causing genes, including analysis of the mitochondrial genome. A genome-wide analysis identified compound heterozygous variants in PYROXD2.

Functional assays revealed increased mitochondrial superoxide levels and a heightened sensitivity to culturing in galactose media, a known mitochondrial stressor, indicating impaired mitochondrial activity in fibroblasts.

Proteomic results demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect.

Conclusion: Our findings support the critical role of PYROXD2 in human cells, suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction and are the likely cause of the proband’s clinical presentation.

References:.

Grants: Australian Government GHFM76747.

Conflict of Interest: None declared.

P07.014.D MITODIAG: A French network of diagnostic laboratories for mitochondrial diseases

Cécile Rouzier ^1;2, Emmanuelle Pion³, Céline Bris⁴, Patrizia Bonneau^4;5, Valérie Desquiret^4;5, Jean-Paul BONNEFONT⁶, Giulia Barcia⁶, Alessandra Pennisi⁶, Julie Steffann⁶, Pauline Gaignard⁷, Elise Lebigot⁷, Samira Ait-el-mkadem Saadi^1;2, Sylvie Bannwarth^1;2, Konstantina Fragaki^1;2, Benoit Rucheton⁸, Bruno Francou¹, Marie-Laure Martin Négrier⁹, Aurélien Trimouille⁹, Cecile Acquaviva¹⁰, Cécile Pagan¹⁰, Anne-Sophie Lèbre¹¹, Gaelle Hardy¹², Stéphane Allouche¹³, Pascal Reynier^4;5, Mireille Cossee¹⁴, Annamaria Molon¹⁵, Shahram Attarian¹⁶, Véronique Paquis-Flucklinger^1;2, Vincent Procaccio⁴

¹Service de génétique médicale, Centre de référence des maladies mitochondriales, CHU Nice, Nice, France; ²Université Cote d’Azur, CNRS, INSERM, IRCAN, Nice, France; ³Filnemus, laboratoire de génétique moléculaire, CHU Montpellier, Montpellier, France; ⁴Service de génétique, Institut de Biologie en santé, Centre National de référence Maladies Neurodégénératives et Mitochondriales, CHU Angers, Angers, France; ⁵Service de biochimie, Institut de Biologie en santé, Centre National de référence Maladies Neurodégénératives et Mitochondriales, CHU Angers, Angers, France; ⁶Fédération de génétique médicale, Service de génétique moléculaire du GH Necker-enfants malades, Hôpital Necker-Enfants Malades, Paris, France; ⁷Laboratoire de Biochimie, Pôle BPP, CHU Paris Sud, Hôpital Bicêtre-le Kremlin Bicêtre, Paris, France; ⁸Pôle de biologie et pathologie, CHU Bordeaux, Bordeaux, France; ⁹Unité fonctionnelle d’histologie moléculaire, Service de pathologie, CHU Bordeaux-GU Pellegrin, Bordeaux, France; ¹⁰Service de biochimie et biologie moléculaire Grand Est, UM Maladies Héréditaires du Métabolisme, Centre de biologie et pathologie Est, CHU Lyon HCL, GH Est, Lyon, France; ¹¹Laboratoire de génétique, Hématologie et Immunologie, CHU Reims, Reims, France; ¹²Laboratoire de génétique moléculaire: maladies héréditaires et oncologie, Service de biochimie, biologie moléculaire et toxicologie environnementale, CHU Grenoble et des Alpes, Institut de biologie et pathologie, Grenoble, France; ¹³Service de biochimie, Pôle Biologie, Pharmacie et Hygiène, CHU Caen, Hôpital de la Côte de Nacre, Caen, France; ¹⁴Laboratoire de Génétique Moléculaire, CHU Montpellier, PhyMedExp, Université de Montpellier, INSERM, CNRS, Montpellier, France; ¹⁵Filnemus, Assistance Publique Hôpitaux Marseille, Marseille, France; ¹⁶Service de Neurologie, FILNEMUS, Hôpital La Timone, CHU, Marseille, France

Background/Objectives: Mitochondrial diseases (MD) are characterized by a huge heterogeneity which poses significant diagnostic challenges for clinicians and around 50% of patients are still undiagnosed. Since 2000, the French Network of Mitochondrial Diseases Diagnostic Laboratories, called MITODIAG, has been created and works in close collaboration with the two National Reference Centers, CARAMMEL and CALISSON, and the Neuromuscular rare disease network FILNEMUS, in order to improve the diagnosis and health care for patients with MD.

Methods: We describe here the organization of the MITODIAG network, the evolution of genetic diagnosis in MD in France these last years and the interactions with national platforms of genome core sequencing facilities named AURAGEN and SEQOIA.

We also report the first clinical and genetic description of a cohort of more than 400 patients tested by NGS, in whom a diagnosis could be confirmed by the identification of pathogenic variants in nuclearly-encoded genes.

Results: 3/4 of the patients are children under 18 with often early and severe phenotypes, of mostly autosomal recessive inheritance. In these patients, 30% of pathogenic variants are located in complex I genes or genes involved in mitochondrial translation. In adults, diseases are more heterogeneous, from moderate to very severe, mainly with neuromuscular symptoms due to pathogenic variants in mtDNA maintenance machinery.

Conclusion: These data will be implemented in the MITOMATCHER database in order to improve phenotype-genotype correlations and facilitate development of therapeutic strategies. Collaborations between MITODIAG and FILNEMUS will also facilitate patient follow-up in diagnostic wandering and will facilitate the identification of diagnosis for these patients.

References:.

Grants:.

Conflict of Interest: None declared.

P07.015.A Interpreting the pathogenicity of genetic variants in rare diseases: lessons from Fabry disease

Dominique P. Germain ^1;2, Thierry Levade^3;4, Eric Hachulla⁵, Bertrand Knebelmann⁶, Didier LACOMBE⁷, Vanessa Leguy-Seguin⁸, Karine NGUYEN PHONG⁹, Esther Noel¹⁰, Jean-Pierre Rabes^2;11

¹French Referral Centre for Fabry Disease, AP-HP University Paris Saclay, Medical Genetics, Garches, France; ²University of Versailles-Saint-Quentin-en-Yvelines, Medical Genetics, Montigny le Bretonneux, France; ³INSERM UMR1037, Cancer Research Center of Toulouse (CRCT) and Paul Sabatier University, Toulouse, France; ⁴Clinical Biochemistry Laboratory, Reference Center for Inherited Metabolic Diseases, Federative Institute of Biology, University Hospital of Toulouse, Toulouse, France; ⁵Claude Huriez Hospital, University of Lille, Internal Medicine and Clinical Immunology, Lille, France; ⁶AP-HP, Necker Enfants Malades Hospital, University of Paris, Nephrology-Dialysis Department, Paris, France; ⁷University Hospital of Bordeaux and INSERM U1211, Medical Genetics, Bordeaux, France; ⁸François Mitterrand Hospital, Dijon University Hospital, Internal Medicine and Clinical Immunology, Dijon, France; ⁹APHM, Timone Children Hospital, Medical Genetics, Marseille, France; ¹⁰Strasbourg University Hospital, Internal Medicine, Strasbourg, France; ¹¹Ambroise Paré University Hospital, APHP. Université Paris-Saclay, Biochemistry and Molecular Genetics, Boulogne-Billancourt, France

Background/Objectives: Fabry disease (FD) is an X-linked genetic disease due to pathogenic variants in GLA (Gene ID: 2717). Over 1,000 GLA variants were identified, a significant number through screening protocols in newborns and at-risk populations more susceptible to disclose variants of unknown significance (VUS). This, together with the non-specificity of symptoms, challenges physicians at the time of diagnosis. Here, a panel of FD specialists convened to study how expertise compares with the traditional approach in interpreting variants.

Methods: Several highly controversial GLA VUS (p.Ser126Gly, p.Ala143Thr, p.Asp313Tyr), were re-analyzed through the review of patients’ records.

Results: Experts’ input was found to significantly contribute to an accurate interpretation of variants. The proper use of surrogate biomarkers can optimize the interpretation of variants. When a (likely) benign GLA variant is disclosed, other genes (e.g., sarcomeric genes in case of HCM) should be investigated. Comparing allele frequencies (AF) of GLA VUS is useful in excluding VUS which AF is higher than the disease prevalence or the frequency of the most common pathogenic allele. While databases and in silico prediction softwares are useful tools, they may yield conflicting results.

Conclusion: In genetics, the traditional approach to interpreting the pathogenicity of variants was developed by the American College of Medical Genetics. Our data suggest that through their in-depth knowledge of disease phenotypes, biomarkers, alleles frequencies, and literature data, highly specialized experts bring an important additional value. These lessons from FD give insights for better interpreting the pathogenicity of allelic variants in other genetic diseases.

References:.

Grants:.

Conflict of Interest: Dominique P. Germain speaker’s honoraria from Takeda, Amicus, Sanofi-Genzyme., consultant for Sanofi-Genzyme, Idorsia, Takeda., Thierry Levade hotel/travel grants from Sanofi-Genzyme, Takeda, BioMarin, Enzyvant, Orphan., Eric Hachulla speaker’s honoraria and/or travel grants from Sanofi- Genzyme, GSK, Actelion, Sobi., Bertrand Knebelmann speaker’s honoraria from Travere, Sanofi, Alnylam, Reata; hotel/travel grants from Sanofi, Travere, Reata., Didier LACOMBE speaker’s honoraria and hotel/travel expenses from Amicus, Sanofi-Genzyme., Vanessa Leguy-Seguin speaker’s honoraria from Sanofi-Genzyme; hotel/travel grants from Amicus, Orphan, Takeda, Sanofi-Genzyme., Karine NGUYEN PHONG speaker’s honoraria, travel grants from Sanofi Genzyme; grants from Amicus therapeutics., Esther Noel speaker’s honoraria and/or travel grants from Amicus Therapeutics, Sanofi-Genzyme., Jean-Pierre Rabes hotel/travel expenses from Amicus Therapeutics; speaker’s honoraria from Amgen., consultant for Sanofi-Genzyme.

P07.016.B Detection of single nucleotide and copy number variants in the Fabry Disease-associated GLA gene using nanopore sequencing

Omer Murik ¹, Tzvia Mann¹, David Zeevi¹, Albina Nowak^2;3, gheona Altarescu¹

¹Shaare Zedek Medical Center, Medical Genetics Institute, Jerusalem, Israel; ²University Hospital of Zürich, Department of Endocrinology and Clinical Nutrition, Zürich, Switzerland; ³Psychiatric University Hospital Zurich (PUK), Zürich, Switzerland

Background/Objectives: More than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology.

Methods: 57 male and 42 female Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants.

Results: All the variants (100%) detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology.

Conclusion: Our long-read sequencing-based method was able to detect multiple missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease. Furthermore, our approach can be easily implemented for other monogenic diseases and improve diagnostic precision and efficiency.

References:.

Grants:.

Conflict of Interest: None declared.

P07.018.D An interactive website visualizing newborn screening programs worldwide

Eyyüb Selim Ünlü ¹, Sümeyye Karakaya¹, Gülbin Gökçay², Gülden Gökçay³

¹Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; ²Institute of Child Health, Istanbul University, Istanbul, Turkey; ³Istanbul Faculty of Medicine, Istanbul University, Department of Pediatric Nutrition and Metabolism, Istanbul, Turkey

Background/Objectives: Newborn screening (NBS) strategies have been proven to be successful diagnostics for several life-threatening inherited metabolic disorders and other genetic conditions. Governments are expanding nationwide NBS programs to improve public health. However, due to various challenges, worldwide NBS programs are not yet optimal. Monitoring worldwide NBS programs could accelerate the ongoing efforts for optimization.

Methods: We reviewed the last 10 years of literature to reveal country-level NBS programs. A user-friendly website (www.nbsww.org), consisting of an interactive world map, a filter table, and individual pages for each country, has been developed. Data sources are available with a link to corresponding publications. A data collection form is placed to receive user contributions to keep the content of the website up-to-date.

Results: We identified 115 countries’ nationwide NBS programs. The current status of NBS programs is heterogeneous, covering conditions ranging from a minimum of 0 to a maximum of 41 conditions. Expanded NBS is implemented in 35 (30%) of these countries while 57 (50%) screens less than 5 conditions. Most screened conditions are primary congenital hypothyroidism, phenylketonuria, congenital adrenal hyperplasia, screened by 82 (71%), 75 (65%), 50 (43%) programs, respectively.

Conclusion: The nationwide NBS programs were reported by presenting a website that allows monitoring and facilitates evaluation of the current situation.

References: Martínez-Morillo, E., Prieto García, B., & Álvarez Menéndez, F. V. (2016). Challenges for Worldwide Harmonization of Newborn Screening Programs. Clinical chemistry, 62(5), 689–698.

Grants: The Scientific And Technological Research Council Of Turkey grant 2209-A.

Conflict of Interest: None declared.

P07.019.A Psychosine in dried blood spots of newborns at risk of Krabbe disease due to GALC p.Y319C homozygosity

Amy White¹, Joseph Orsini², Maria Escolar³, Dawn Peck¹, Gisele Bentz Pino¹, April Studinski¹, Dimitar Gavrilov¹, Devin Oglesbee¹, Matthew Schultz¹, Silvia Tortorelli¹, Dietrich Matern ¹

¹Mayo Clinic, Biochemical Genetics Laboratory, Rochester, MN, United States; ²Wadsworth Center, NY State Department of Health, Albany, NY, United States; ³University of Pittsburgh Medical Center, Department of Pediatrics, Pittsburgh, PA, United States

Background/Objectives: GALC p.Y319C (c.956A>G) is considered a variant of uncertain significance. However, several patients homozygous for p.Y319C presented with symptoms consistent with Krabbe disease (KD) at 3 years of age and older (1). We report on psychosine (PSY) in dried blood spots (DBS) from GALC p.Y319C homozygous neonates.

Methods: PSY concentrations were measured in DBS (2) collected in the neonatal period from individuals homozygous for p.Y319C and identified through newborn screening for KD (n = 11).

Results: Neonatal DBS PSY was >2.0 nM (2.1-2.8 nM) in four of eleven p.Y319C homozygotes. Ten of eleven subjects are asymptomatic to date (age range: 4 months – 6.3 years, average age: 2.5 years). One patient identified through newborn screening (PSY: 2.8 nM) displayed clinical signs by 4 years of age and received a hematopoietic stem cell transplant.

Conclusion: A third of p.Y319C homozygotes had PSY concentrations above the reference threshold of 2 nM. The infant with the highest neonatal PSY DBS concentration was the only case developing signs of KD. Ongoing clinical monitoring of these individuals is required to determine whether they are at risk for KD. When KD is considered for newborn screening, programs should define whether all or specific KD forms (infantile vs. later onset KD) are the primary target of screening because this decision will determine if molecular genetic testing of GALC is required as part of the screening process.

References: 1. Bascou et al. al. Front Neurol. 2020;11:563724.

2. Herbst et al. Int J Neonatal Screen. 2020;6(2):29.

Grants:.

Conflict of Interest: Amy White: None declared, Joseph Orsini: None declared, Maria Escolar Forge Biologics, NIH R01NS061965-01(PI). The Legacy of Angels Foundation (PI), Forge Biologics, Dawn Peck: None declared, Gisele Bentz Pino: None declared, April Studinski: None declared, Dimitar Gavrilov: None declared, Devin Oglesbee: None declared, Matthew Schultz: None declared, Silvia Tortorelli: None declared, Dietrich Matern Neurogene advisory board (4 hrs; compensation passed on to Mayo Clinic).

P07.020.B Mitochondrial encephalomyopathy stroke-like episodes and lactate acidosis (MELAS), disease spectrum, lessons from large cohort

Ralitza Gavrilova¹, Benjamin Cox²

¹Mayo Clinic, Neurology, Clinical Genomics, Rochester, United States; ²Mayo Clinic, Neurology, Rochester, United States

Background/Objectives: Evaluate MELAS mutation cohort clinical spectrum, clinical/genetic factors associated with severe or later presentation.

Methods: Retrospective review 81 Mayo Clinic patients with/without meeting MELAS criteria. Clinical, MRI characteristics, mutations/heteroplasmy reviewed. MELAS, standard onset SLE <40 or late >40.

Results: 42 MELAS, 30 symptomatic non-MELAS, and 9 asymptomatic. MELAS, significantly lower BMI (mean 18.6 vs 25.1, 22.0), trend higher heteroplasmy (means 39.3%, 29.3%, 21.8%). MELAS and non-MELAS had similar age of first symptom (mean 20.3 vs 21.5); non-MELAS higher SNHL presentation (51.6% v24.4%, p = 0.014). Neurologic (seizures/SLE/ataxia/dementia) common MELAS first symptom (39%), versus non-MELAS (19.4%). MELAS, significant seizure prevalence (88.1% vs 16.7%), and higher mortality (43% vs 13%). MELAS cohort, 13 late and 29 standard-onset. Mean age of first symptom 14.2y in standard (range 0-36), 34.1 in late (range 16-53). Trend (p = 0.18) towards higher heteroplasmy in standard (mean 44.8% vs 25.3%). Late-MELAS, significantly longer time from first symptom to SLE (mean 16.6 vs 9.3y), no different duration to death. Longer life expectancy in late-MELAS (mean death 62 vs 30y). Standard-MELAS higher incidence neurologic symptoms at onset (51.7% vs 15.4%); late-onset higher prevalence diabetes (69.2% vs 13.8%), nephropathy (53.8% vs 10.3%). Late-MELAS tended more systems involved (mean 4.1 vs 2.7).

Conclusion: Standard-MELAS more likely to present with neurologic symptoms compared to later onset, who are more likely to suffer diabetes, nephropathy, greater organ-involvement. Many MELAS present late >40y. Non-MELAS suffer substantial neurologic symptoms. Lower BMI in MELAS and higher rates SNHL in non-MELAS may be useful differentiator at onset.

References: N/A.

Grants: N/A.

Conflict of Interest: Ralitza Gavrilova Mayo Clinic, Benjamin Cox Mayo Clinic, Neurology fellow.

P07.021.C Development and validation of a new neonatal screening test

Michaella Georgiadou ¹, Charalambos Loizides¹, Skevi Kyriakou¹, Achilleas Achilleos¹, Christos Lemesios¹, Michalis Nicolaou¹, Chrisovalando Soteriou¹, Haris Kkoufou¹, Louiza Constantinou¹, Krystallo Christou¹, Antonia Matsentidou¹, Michalis Spyrou¹, Stelia Pissaridou¹, Demetra Panayiotou¹, Kyriakos Tsangaras¹, Elena Kypri¹, Marios Ioannides¹, George Koumbaris¹, Philippos Patsalis¹

¹NIPD Genetics, Nicosia, Cyprus

Background/Objectives: Many genetic conditions affect normal development in newborns, infants or young children. These disorders can cause death, serious life-long disability or chronic disease if not treated early. Also, they have a vast array of symptoms, making diagnosis complicated. Therefore, prompt identification and timely treatment is essential to prevent or minimize the impact of the conditions. Our aim was to develop an NGS-based product, which tests for 106 neonatal conditions including: metabolic, endocrine, haemoglobin, hearing loss, pulmonary and musculoskeletal disorders.

Methods: Custom target capture sequences (TACS) were designed to capture all gene exons, and intron-exon boundaries. The TACS were immobilized on streptavidin-coated magnetic beads. Sequencing libraries were constructed and subjected to in-solution hybridization with the immobilized TACs. The captured sequences were amplified and sequenced using NGS. A blind validation study was performed to assess the sensitivity and specificity of single nucleotide variant (SNV), indel and copy number variant (CNV) detection.

Results: SNVs and indels were detected at sensitivity of 100% (CI: 87-100%) and specificity of 100% (CI: 99.8-100%). The algorithm was designed to detect CNVs at high-resolution with estimated high sensitivity and specificity when applied to single or few exon CNV. Each positive call was confirmed with an orthogonal method.

Conclusion: We have developed and validated a neonatal screening test for 106 disorders that when detected early, can prevent or reduce serious health consequences such as developmental delay, cognitive impairment, neurological and physical problems and premature death.

References:.

Grants:.

Conflict of Interest: Michaella Georgiadou NIPD Genetics, Charalambos Loizides NIPD Genetics, Skevi Kyriakou NIPD Genetics, Achilleas Achilleos NIPD Genetics, Christos Lemesios NIPD Genetics, Michalis Nicolaou NIPD Genetics, Chrisovalando Soteriou NIPD Genetics, Haris Kkoufou NIPD Genetics, Louiza Constantinou NIPD Genetics, Krystallo Christou NIPD Genetics, Antonia Matsentidou NIPD Genetics, Michalis Spyrou NIPD Genetics, Stelia Pissaridou NIPD Genetics, Demetra Panayiotou NIPD Genetics, Kyriakos Tsangaras NIPD Genetics, Elena Kypri NIPD Genetics, Marios Ioannides NIPD Genetics, George Koumbaris NIPD Genetics, Philippos Patsalis NIPD Genetics.

P07.022.D Beyond the exome: identification and characterization of non-coding variants in inborn errors of metabolism

Alejandro Soriano-Sexto ^1;2;3;4;5, Fátima Leal^1;2;3;4;5, Patricia Alcaide^1;2;3;4;5, Magdalena Ugarte^1;2;3;4;5, Pilar Rodríguez^1;2;3;4;5, Belén Pérez^1;2;3;4;5

¹Centro de Diagnóstico de Enfermedades Moleculares, Madrid, Spain; ²Centro de Biología Molecular Severo Ochoa, Madrid, Spain; ³CIBERER, Madrid, Spain; ⁴IdiPAZ, Madrid, Spain; ⁵Universidad Autónoma de Madrid, Madrid, Spain

Background/Objectives: Inborn errors of metabolism (IEM) are a large group of rare diseases that include more than 1450 defects. Historically, the search for mutations contributing to IEM has been limited to exons, whereas regulatory elements have remained poorly investigated. Our aim here consisted in applying a combination of multiple omics to identify non-coding variants responsible for disease-state which scape exome sequencing, thus reducing the diagnostic gap.

Methods: Genomic analyses by whole exome or whole genome sequencing and functional studies (minigenes and luciferase reporters) were carried out in DNA and RNA extracted from patients’ fibroblasts. The enzymatic activity was also evaluated in these cells when possible.

Results: We have selected a cohort of unsolved patients with clinical or biochemical diagnosis of different IEM (hyperphenylalaninemia, galactosemia, mucopolysaccharidosis or citrullinemia among others). Human Phenotype Ontologies (HPO) and biochemical signatures were used to filter the obtained variants. We prioritized either no candidate variants or only one. Therefore, all remaining undiagnosed. We have attained a complete genetic diagnosis for several patients by combination of transcriptomic and genomic analyses and functional genomics. We have identified pseudoexons insertions caused by deep intronic variants such as c.[83+658C>G;83+758T>A] in PTS or c.598-757G>A in ASS1 and novel variants located in promoter regions like c.-82_-71delins-103_-86 in PTS or c.-87T>C in IDUA. The pathogenic role of all of these changes was assessed by minigenes studies or luciferase reporter studies confirming their clinical significance.

Conclusion: The non-coding portion of the genome should be interrogated when searching for disease-causing mutations.

References:.

Grants: PI19/01155, ERT18TRL746, B2017/BMD3721.

Conflict of Interest: None declared.

P07.023.A Three novel SLC2A1 mutations in GLUT1DS pediatric patients

Alessia Mauri ^1;2, Alessandra Duse¹, Sara Olivotto³, Stefania Bova³, Pierangelo Veggiotti^1;3, Cristina Cereda²

¹University of Milan, Department of Biomedical and Clinical Sciences, L. Sacco, Milan, Italy; ²Neonatal Screening and Metabolic Disorders Unit, V. Buzzi Children’s Hospital, Milan, Italy; ³Pediatric Neurology Unit, V. Buzzi Children’s Hospital, Milan, Italy

Background/Objectives: GLUT1 deficiency syndrome (GLUT1DS; #606777) is a rare genetic metabolic disease, characterized by infantile seizures, neurodevelopmental delay, and movement disorders. GLUT1DS is caused by heterozygous (or rarely, homozygous) mutations in the SLC2A1 gene, which encodes GLUT1, a glucose transporter across the blood-brain barrier. Most commonly these variants arise de novo resulting in sporadic cases, although several familial cases with AD pattern have been described.

Methods: We performed SLC2A1 sequencing analysis on 26 pediatric patients with clinical suspicion of GLUT1DS, and relatives.

Results: Herein, we reported three novel SLC2A1 mutations causing GLUT1DS: two sporadic cases (a, b), and one familial case (c).

In the proband of the first sporadic case (a), characterized by 3 months-onset epileptic encephalopathy, we found a novel de novo c.114+1G>A splicing variant in SLC2A1 gene. In silico analysis showed that the identified variant affects the donor splice-site of intron 2-3, leading probably to exon 2 skipping and exon 1-3 junction.

In the other sporadic case (b), which the proband presents epileptic discharges and movement disorders, the analysis revealed in SLC2A1 a novel de novo frameshift variant c.370delC (p.(Leu124TrpfsTer12)), predicted to result in NMD.

The proband of the familial case (c) shows febrile seizures, while the father and grandfather present paroxysmal exercise-induced dyskinesia without epilepsy. Despite the clinical familial heterogeneity, we identified a novel SLC2A1 heterozygous missense variant c.1363A>G (p.Thr455Ala), reported as disease causing in all three patients.

Conclusion: Our report described three novel variants in both sporadic and familial cases, thus expanding the genotypic spectrum of SLC2A1 mutations causing GLUT1DS.

References:.

Grants:.

Conflict of Interest: None declared.

P07.024.B Establishing the mutational spectrum of Hungarian patients with familial hypercholesterolemia

László Madar ^1;2, Lilla Juhász³, Zsuzsanna Szűcs¹, Lóránt Kerkovits⁴, Mariann Harangi³, Istvan Balogh^5;6

¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Genetics, Debrecen, Hungary; ²University of Debrecen, Doctoral School of Molecular Cell and Immune Biology, Debrecen, Hungary; ³University of Debrecen, Department of Internal Medicine, Division of Metabolic Diseases, Debrecen, Hungary; ⁴South-Budapest Center Hospital St. Imre Teaching Hospital, Department of Nephrology, Budapest, Hungary; ¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Genetics, Debrecen, Hungary; ⁶University of Debrecen, Department of Human Genetics, Debrecen, Hungary

Background/Objectives: Familial hypercholesterolemia (FH) is one of the most common autosomal dominantly inherited diseases affecting the cholesterol metabolism which, in the absence of treatment, leads to the development of cardiovascular complications. The disease is still underdiagnosed, even though an early diagnosis would be of great importance for the patient to receive proper treatment and to prevent further complications. No studies are available describing the genetic background of Hungarian FH patients.

Methods: In this work, we present the clinical and molecular data of 44 unrelated individuals with suspected FH. Sequencing of five FH-causing genes (LDLR, APOB, PCSK9, LDLRAP1 and STAP1) has been performed by next-generation sequencing (NGS). In cases where a copy number variation (CNV) has been detected by NGS, confirmation by multiplex ligation-dependent probe amplification (MLPA) has also been performed.

Results: We identified 47 causal or potentially causal (including variants of uncertain significance) LDLR and APOB variants in 44 index patients. The most common variant in the APOB gene was the c.10580G>A p.(Arg3527Gln) missense mutation, this being in accordance with literature data. We detected 40 mutations in the LDLR gene in a total of 37 patients of which 3 patients carried mutations in compound heterozygous form.

Conclusion: Our study revealed one novel mutation in the LDLR gene, while most of the mutations that are present in the Hungarian population, have already been described. Indicating the importance of establishing a genetic diagnosis, the timing of initiation of lipid-lowering therapies may greatly influence the onset and severity of expected complications.

References:.

Grants:.

Conflict of Interest: None declared.

P07.025.C Biochemical and clinical effects of vitamin E supplementation in Hungarian Smith-Lemli-Opitz syndrome patients

Katalin Koczok¹, László Horváth², Zeljka Korade³, Zoltán András Mezei⁴, Gabriella P. Szabó⁵, Ned A. Porter⁶, Eszter Kovács ¹, Károly Mirnics⁷, Istvan Balogh^1;8

¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Genetics, Debrecen, Hungary; ²University of Debrecen, Department of Pharmaceutical Surveillance and Economics, Debrecen, Hungary; ³University of Nebraska Medical Center, Department of Pediatrics, Omaha, United States; ⁴University of Debrecen, Department of Laboratory Medicine, Debrecen, Hungary; ⁵University of Debrecen, Department of Pediatrics, Debrecen, Hungary; ⁶Vanderbilt University, Department of Chemistry, Nashville, United States; ⁷University of Nebraska Medical Center, Munroe-Meyer Institute, Omaha, United States; ⁸University of Debrecen, Department of Human Genetics, Debrecen, Hungary

Background/Objectives: Smith-Lemli-Opitz syndrome (SLOS) is a severe monogenic inborn error of cholesterol biosynthesis resulting in low cholesterol and high 7-dehydrocholesterol (7-DHC) levels. 7-DHC-derived oxysterols likely contribute to disease pathophysiology, thus antioxidant treatment might be beneficial because of high oxidative stress.

Methods: In a 3-year prospective study we investigated the effects of vitamin E supplementation in six SLOS patients already receiving dietary cholesterol treatment. Plasma vitamin A and E concentrations were determined by a HPLC method. At baseline plasma 7-DHC, 8-DHC and cholesterol levels were determined by a LC-MS/MS method. The clinical effect of the supplementation was assessed by performing structured parental interviews.

Results: At baseline, patients were characterized by low or low-normal plasma vitamin E concentrations (7.19-15.68 μmol/L), while vitamin A concentrations were found to be normal or high (1.26-2.68 μmol/L). Vitamin E supplementation resulted in correction or significant elevation of plasma vitamin E concentration in all patients. We observed reduced aggression, self-injury, irritability, hyperactivity, attention deficit, repetitive behavior, sleep disturbance, skin photosensitivity and/or eczema in 3/6 patients, with notable individual variability. Clinical response to therapy was associated with a low baseline 7-DHC+8-DHC/cholesterol ratio (0.2-0.4).

Conclusion: We suggest that determination of vitamin E status is important in SLOS patients. According to our results, the lower level of vitamin E is most likely attributable to increased consumption due to oxidative stress, rather than malabsorption. Supplementation of vitamin E should be considered and might be beneficial.

References:.

Grants:.

Conflict of Interest: None declared.

P07.026.D Missense variant in PDK1 associated with severe developmental delay and epilepsy

Raquel Vaz ¹, Josephine Wincent^1;2, Najla Elfissi³, Kristina Forsblad⁴, Maria Pettersson^1;2, Karin Naess³, Anna Wedell^5;6;7, Anna Wredenberg^3;5, Anna Lindstrand^1;2, Sofia Ygberg^5;8

¹Karolinska Institutet, Department of Molecular Medicine and Surgery, Stockholm, Sweden; ²Karolinska University Hospital, Department of Clinical Genetics, Stockholm, Sweden; ³Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Stockholm, Sweden; ⁴Akademiska Sjukhuset, Uppsala, Sweden; ⁵Karolinska University Hospital, Centre for Inherited Metabolic Diseases, Stockholm, Sweden; ⁶Karolinska Institutet, Science for Life Laboratory, Department of Molecular Medicine and Surgery, Stockholm, Sweden; ⁷Karolinska Institutet, Molecular Medicine and Surgery, Stockholm, Sweden; ⁸Karolinska Institutet, Department of Women’s and Children’s Health, Stockholm, Sweden

Background/Objectives: The pyruvate dehydrogenase complex (PDC), located in the mitochondrial matrix, is responsible for the conversion of pyruvate into acetyl-CoA, used for energy production in the cells. PDC activity is tightly regulated by phosphorylation, via kinases and phosphatases (PDK/PDP), that respond to, for example, the amount of substrate in the cell (e.g. pyruvate or NADH) and hypoxia. Mutations in all subunits of the PDC and in PDK3 have been reported and the clinical presentation often includes lactic acidosis, neurodevelopmental delay, and seizures. Here we report a missense mutation in PDK1, a gene not previously associated with disease.

Methods: Genetic analysis was done using trio genome sequencing. Functional studies in zebrafish were done by overexpressing human wild type and mutant PDK1 in early-stage embryos.

Results: A de novo missense variant in PDK1 (c.1139G>A, p.Gly380Asp) was identified in a patient with developmental delay and seizures. In zebrafish, mutant PDK1 fails to phosphorylate PDHE1 (pyruvate dehydrogenase) resulting in abnormal PDC activity which, consequently, affects mitochondria activity. This, in turn, affects muscle activity, seen by reduced bouts of movement when compared to control embryos, and neuronal development delay.

Conclusion: Here, we report genetic and functional evidence suggesting that loss of function of PDK1 causes a similar clinical presentation as other PDC genes.

References:.

Grants:.

Conflict of Interest: None declared.

P07.027.A History of a diagnostic errancy: how new technologies may allow for a diagnosis after 40 years

Sabrina Bertoli ¹, Jean-Hubert Caberg¹, François Boemer¹, Vincent Bours¹, Philippe Lousberg¹, Saskia Bulk¹

¹University Hospital of Liège, Human Genetics, Liège, Belgium

Background/Objectives: This clinical case demonstrates that subtle metabolic impairments may not be detected by basic screening performed in routine diagnostics. In addition to metabolic diagnostics, it’s imperative to use more precise molecular biology techniques.

Methods: 46 year old woman, born at full term after a pregnancy and normal delivery, of non-consanguineous parents presented with mild developmental delay since infancy. A waddling walk was noted at the age of 5.Legge-Calvé-Perthes disease diagnosed at the age of 6, necessitating a bilateral hip prosthesis. She underwent surgery for severe scoliosis in childhood.

In her twenties, she was diagnosed with a demyelinating sensory-motor polyneuropathy leading to amyotrophy of the 4 limbs with predominance in the lower limbs.

At age 45, she presented cardiac arrhythmias diagnosed as probable arythmogenic right ventricular dysplasia.

Clinical and genetics investigations over a period of over 40 years had not revealed an explanation for the developmental delay, neuropathy and cardiac condition (Normal EEG and brain MRI).

Results: Normal caryotype and CHG-array, normal metabolic analysis including mucopolysaccharides and oligosaccharides, GJP1 gene analysis.

Genetic analysis of a cardiac arrhythmia panel was normal.

Analysis of a neurodevelopmental panel (859 genes) revealed a compound heterozygous GNPTAB mutation, confirming a diagnosis of mucolipidosis type III. Re-evaluation of skeletal X-rays confirmed associated skeletal changes.

Conclusion: Due to her mild phenotype, a clinical diagnosis of classic mucolipidosis was never evoked.Routine metabolic diagnostic testing may not be sensitive enough to diagnose mild forms of metabolic conditions, gene panels must absolutely cover the genes for storage diseases.

References:.

Grants:.

Conflict of Interest: None declared.

P07.028.B Discovery of Type 2 Diabetes genes using an accessible tissue

David Davtian ¹, Jochen Schwenk², Mark McCarthy³, Anubha Mahajan⁴, Mun-Gwan Hong², Emmanouil Dermitzakis⁵, Hae Kyung Im⁶, Ewan Pearson¹, Ana Viñuela⁷, Andrew Brown¹

¹University of Dundee, Dundee, United Kingdom; ²KTH Royal Institute of Technology, Stockholm, Sweden; ³University of Oxford, Oxford, United Kingdom; ⁴Wellcome Centre for Human Genetics, Oxford, United Kingdom; ⁵University of Geneva, Geneva, Switzerland; ⁶University of Chicago, Chicago, United States; ⁷University of Newcastle, Newcastle, United Kingdom

Background/Objectives: Transcriptome Wide Association Study (TWAS) methods use reference expression datasets to link genes to disease. Ideally, these should use samples from a disease relevant tissue, which may be difficult to collect, limiting sample size. We evaluate the power to discover Type 2 Diabetes (T2D) causal genes using a well powered whole blood expression reference against smaller reference datasets from disease relevant tissues.

Methods: Predictive models were built using whole blood expression and proteomic data (DIRECT consortium, n = 3029), pancreatic islets eQTL data (INSPIRE consortium, n = 420) and eQTL data from 49 tissues (GTEx consortium). These models were combined with GWAS summary statistics (DIAGRAM consortium, n = 898,130) to calculate gene-T2D association scores using S-PrediXcan.

Results: We found 97 significant associations between T2D and predicted gene expression levels using whole blood from DIRECT. We recapitulate known T2D genes such as CAMK1D, PAM, and CKDN1C and find more associations using the larger reference dataset (43 using islets and from 2 to 84 in GTEx). There is a correlation between the sample size and the discovery power (R² = 0.9, p = <2e^-16). We also find DIRECT implicated genes to be enriched around GWAS loci compared to GTEx tissue derived genes, meaning a higher proportion of DIRECT genes have supporting genetic evidence. However, the DIRECT reference also misses important T2D genes, such as TCF7L2 or IGF2BP2, which were only captured when using islets models.

Conclusion: Our findings indicate that large studies in non-relevant tissues identify disease causal genes but miss relevant tissue specific signals.

References:.

Grants:.

Conflict of Interest: None declared.

P07.029.C Mitochondrial ATP-synthase deficiency causes a clinical, biochemical and genetical disease spectrum

Andrea Pietra ^1;2, Marco seri^1;2, Leonardo Caporali³, Valerio Carelli^3;4, Duccio Maria Cordelli⁵, Valentina Del Dotto³, Veronica Di Pisa⁵, Claudio Fiorini³, Emanuela Iovino¹, Chiara La Morgia³, Francesca Montanari^1;2, Giulia Olivucci^1;2, Flavia Palombo³, Tommaso Pippucci², Emanuela Scarano⁶, Giulia Severi², Caterina Garone¹

¹Alma Mater Studiorum University of Bologna, Department of Medical and Surgical Sciences, Bologna, Italy; ²IRCCS Azienda Ospedaliera Universitaria di Bologna, UO Genetica Medica, Bologna, Italy; ³IRCCS Institute of Neurological Sciences, Neurogenetics Unit, Bologna, Italy; ⁴Alma Mater Studiorum University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy; ⁵IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC di Neuropsichiatria dell’Età Pediatrica, Bologna, Italy; ⁶IRCCS Sant’Orsola Hospital, Rare Diseases Unit, Department of Pediatrics, Bologna, Italy

Background/Objectives: Mitochondrial Complex V is composed by 18 protein subunits of which 16 are nuclear DNA encoded and 2 mitochondrial DNA (mtDNA) encoded (MT-ATP8 and MT-ATP6). Damaging genetics variants in protein subunits causes enzymatic activity deficiency and, consequently, severe mitochondrial encephalomyopathy.

Methods: Biochemical, histochemical and molecular genetics analyses including next generation sequencing of the whole mitochondrial genome were performed in biological samples (skeletal muscle, urine epithelium and blood) of probands and relatives from two unrelated families.

Results: Proband of family 1, is a 9 years-old male presenting with cerebellar ataxia, psychomotor delay and hypertrophic cardiomyopathy; brain MRI imaging showed cerebellar vermis hypoplasia and corpus callosum dysmorphism. The latter were also presented in an asymptomatic sister. Proband of family 2, is a 48 years-old male presenting with progressive retinitis pigmentosa, cataract, sensorineural deafness and ataxia. Our analyses revealed in both patients a biochemical deficiency of mt-ATP-synthase confirmed by the inheritance of novel pathogenetic variants in MT-ATP8 gene in family 1 (m.8535A>G (p.Lys57Ter) and MT-ATP6 gene (m.8858G>A (p.Gly111Asp) in family 2 with high level of hereroplasmy (95-98%) in patients’ tissues while absent or very low level of heteroplasmy in unaffected family members.

Conclusion: Our study identifies novel variants in a very rare mitochondrial disorder due mitochondrial ATP-synthase deficiency and describes a clinical spectrum with a common and predominant feature represented by cerebellar ataxia and a variable multisystemic involvement.

References:.

Grants: Italian Minister of University and Research - Rita Levi Montalcini Program - Rientro cervelli RLM2017; CARISBO ricerca medica traslazionale e clinica 2021.

Conflict of Interest: None declared.

P07.031.A Biallelic SLC25A36 mutation causes hyperinsulinism/hyperammonemia syndrome

Amit Safran ¹, Regina Proskorovski-Ohayon¹, Neta Loewenthal², orna staretz-chacham³, Ohad Shmuel Birk⁴

¹Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel; ²Pediatric Endocrinology Unit, Division of Pediatrics, Soroka Medical Center, Beer Sheva, Israel; ³Soroka Medical Center, Metabolic Clinic, Beer Sheva, Israel; ⁴Soroka University Medical Center, Genetics Institute, Beer Sheva, Israel

Background/Objectives: Hyperinsulinism/Hyperammonemia (HIHA) syndrome, the second most common form of congenital hyperinsulinism, is characterized by recurrent symptomatic hypoglycemia combined with persistently elevated serum ammonia levels. HIHA syndrome has been shown to be caused by dominant activating mutations in GLUD1, encoding the intra-mitochondrial enzyme glutamate dehydrogenase (GDH). In this study, we present four children from consanguineous Bedouin kindred that were diagnosed with an autosomal recessive consistent HIHA syndrome phenotype with no mutations in GLUD1.

Methods: Genome-wide linkage analysis of ten family members was performed using 750K SNP arrays, HomozigosityMapper and SuperLink softwares. Whole exome sequencing was performed for one affected individual and analyzed using the Qiagen Clinical Insight software and our in-house database of ~700 samples.

Results: A single ~16 Mbp homozygous disease-associated locus was identified on chromosome 3, between rs6439033 and rs55940906 (maximal LOD score of 2.65). Using our filtering analysis pipeline only one homozygous variant was found within the locus: a novel highly conserved SLC25A36 c.284+3A>T splice site mutation. The variant was validated by Sanger sequencing and segregated as expected within the family. The mutation changes the 5’ splice site consensus sequence and leads to exon 3 skipping.

Conclusion: In parallel to two recent independent studies, we report a homozygous mutation in SLC25A36 as causing HIHA syndrome phenotype. The SLC25A36/PNC2 encodes a mitochondrial transporter that imports/exports pyrimidine and guanine nucleotides. Our study reinforces the involvement of SLC25A36 in the development of the HIHA syndrome through a recessive mode of inheritance.

References:.

Grants: The Morris Kahn Family Foundation.

Conflict of Interest: None declared.

P07.032.B Reverse phenotyping enables diagnosis of Smith-Lemli-Opitz syndrome in an oligosymptomatic female

Angela Abad Perez ¹, Natalie Weinhold², Dorothea Haas³, Denise Horn¹

¹Charité – Universitätsmedizin Berlin, Institute of Medical Genetics and Human Genetics, Berlin, Germany; ²Charité – Universitätsmedizin Berlin, Department of Pediatrics Endocrinology, Gastroenterology and Metabolic medicine, Berlin, Germany; ³University Hospital Heidelberg, Division of Child Neurology and Metabolic Medicine, Center for Child and Adolescent Medicine, Heidelberg, Germany

Background/Objectives: Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital malformation and intellectual disability syndrome. It is caused by biallelic sequence variants in DHCR7, which encodes the enzyme 7-dehydrocholesterol (7-DHC) reductase. Biochemically, cholesterol concentration is low, whereas concentration of 7- and 8-DHC is elevated. The spectrum of clinical severity of SLOS is wide and some patients with mild manifestations have been reported.

Methods: Next generation sequencing and analysis of the DHCR7 gene (NM_001360.2) by Sanger sequencing were performed on two siblings with clinically diagnosed SLOS and their parents. Analysis of 7- and 8-DHC in the affected individuals was performed using gas chromatography mass spectrometry.

Results: We describe a 33-year-old female with normal psychomotor development and apart from mild microcephaly no major or minor anomalies. Her two daughters were clinically diagnosed with SLOS. Analysis of DHCR7 revealed the same paternally inherited pathological variant c.452G>A;p.(Trp151*) in both affected daughters. Each daughter carried a different, maternally inherited pathogenic variant: c.89G>C;p.(Gly30Ala) and c.278C>T;p.(Thr93Met), for which the mother was compound heterozygous. Plasma concentrations of 7- and 8-DHC in this female were elevated, confirming the diagnosis of SLOS.

Conclusion: Biallelic pathogenic DHCR7 variants c.89G>C;p.(Gly30Ala) and c.278C>T;p.(Thr93Met), accompanied by the typical biochemical features are associated with mild microcephaly as sole manifestation for SLOS. Both variants have been previously described in combination with other pathogenic variants, respectively. The genotype of this female leading to only mild microcephaly has not been reported in the literature.

References: Opitz et al., 1987; Fitzky et al., 1998; Blahakova et al., 2007.

Grants:.

Conflict of Interest: None declared.

P07.033.C Trio-exome sequencing reveals Infantile Liver Failure Syndrome Type 1 (ILFS1) in an infant with recurrent hepatorenal failure and status epilepticus

Stefanie Beck-Wödl ^1;2, Tobias Haack^2;3, Olaf Riess^2;3, Ute Grasshoff^2;3, Mona Grimmel^2;3, Marc Sturm³, Marisa Mendes⁴, Ralf A. Husain⁵

¹Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany; ²ZSE, Rare Disease Center, Tuebingen, Germany; ³Institute of Medical Genetics and Applied Genomics, Tuebingen, Germany; ⁴Metabolic Unit, Vrije Universiteit Amsterdam, Department of Clinical Chemistry, Amsterdam UMC, Netherlands; ⁵Jena University Hospital, Department of Neuropediatrics, Jena, Germany

Background/Objectives: Biallelic variants in LARS1, coding for the cytosolic leucyl-tRNA synthetase, cause infantile liver failure syndrome 1 (ILFS1), a rare disorder of aminoacylation. Especially in infancy-onset of inborn errors of metabolism an early diagnosis is crucial to guide downstream clinical management and treatment decisions.

Methods: We report on a female child born in the 32^nd week of gestation with intrauterine growth restriction, metabolic acidosis, hypoglycemia showing in the further course failure to thrive, developmental delay, hepatomegaly, low albumin and microcytic anemia. Many metabolic and endocrine causes were ruled out. At the age of 12 and 16 months episodes of acute liver and renal failure occurred most probably after viral infections with extreme hyperglycemia under moderate diazoxide dosing (6 mg/kg/d) during the first episode and status epilepticus and prolonged encephalopathy during the second episode. A third episode with infection-triggered status epilepticus but without liver failure occurred at the age of 35 month. Trio exome sequencing was performed during the first episode and revealed compound-heterozygous variants of unknown significance in the LARS1 gene.

Results: With the exclusion of other probable causes and LARS1 activity in fibroblasts of 8 % of control fibroblasts we concluded the pathogenicity of the LARS1 variants. A high-protein diet as recommended was probably protective.

Conclusion: In children with acute liver failure rare genetic causes should be considered early. Next generation sequencing shows advantages over a step-wise approach because even a distinct phenotype can be recognized only in retrospect in extremely rare disorders.

References: PMID: 30349989.

Grants: No affiliations.

Conflict of Interest: None declared.

P07.034.D Next-generation diagnosis in patients with suspected mitochondrial neuromuscular diseases through a deep phenotyping and a in silico panel approach

Flavia Palombo ¹, Chiara La Morgia¹, Claudio Fiorini¹, Mariantonietta Capristo¹, Maria Lucia Valentina², Giulia Severi³, Leonardo Caporali¹, Gaetano Cantalupo⁴, Caterina Garone⁵, Marco seri³, Valerio Carelli^1;2

¹IRCCS ISNB, Bologna, Italy; ²DIBINEM UNIBO, Bologna, Italy; ³U.O. Genetica Medica, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; ⁴U.O.C. di Neuropsichiatria Infantile, Dipartimento ad Attività Integrata Materno Infantile - AOUI di Verona, Verona, Italy; ⁵Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Bologna, Italy

Background/Objectives: Mitochondrial neuromuscular diseases are highly heterogeneous disorders occurring at any age and with several clinical features including neurologic, muscular, cardiac, visual and auditory symptoms. These characteristics make the traditional diagnostic approach long, expensive and often inconclusive. Whole Exome Sequencing (WES) has revolutionized the study of rare genetic diseases and has found a wide application in clinical setting allowing to end the diagnostic odyssey typical of many heterogeneous genetic disorders, such as mitochondrial diseases.

Methods: WES strategy analysis based on standardized phenotyping by the Human Phenotype Ontology (HPO) terms and the use of in silico gene panels, in a heterogeneous cohort of 153 patients with suspected mitochondrial neuromuscular diseases. The cohort can be subdivided in patients with isolated optic atrophy (AO, 54), syndromic optic atrophy (AOplus, 16), progressive external ophthalmoplegia isolated or syndromic (CPEO, 34), adult-onset (EM, 16) and neonatal-onset encephalomyopathy (EMped, 22) and myopathy (MIO, 11).

Results: This strategy allowed to reach a diagnostic yield of 46.4%. In patients who had already undergone pre-sequencing panel (79%), very rare genes were identified. In addition, candidate genes could be identified in 4.5% of patients. The diagnosis of mitochondrial disease was confirmed in 50% of patients. The phenotypic traits most associated with mitochondrial genes are optic atrophy and ptosis, while the presence of phenocopies was high in the EMped and in the MIO patients.

Conclusion: The application of WES and a diagnostic algorithm that relies on phenotyping and filtering by in silico panels represents an effective diagnostic strategy in patients with suspected mitochondrial diseases.

References:.

Grants: GR-2016-02361449.

Conflict of Interest: Flavia Palombo: None declared, Chiara La Morgia PI/SI for clinical trials sponsored by GenSight. Biologics and Santhera, Santhera Pharmaceuticals, Chiesi Farmaceutici, Regulatory Pharma Net, Thenewway srl, First Class srl and Biologix, Chiesi Farmaceutici, Regulatory Pharma Net and Thenewway srl, Claudio Fiorini: None declared, Mariantonietta Capristo: None declared, Maria Lucia Valentina: None declared, Giulia Severi: None declared, Leonardo Caporali: None declared, Gaetano Cantalupo: None declared, Caterina Garone: None declared, Marco seri: None declared, Valerio Carelli Stealth BioTherapeutics, Chiesi, GenSight Biologics, Stealth.

BioTherapeutics, Santhera Pharmaceuticals, and Chiesi.

P07.035.A Investigation of the role of the DNM2 gene in mitochondrial dynamics by siRNA gene silencing

Fruzsina Szabo ¹, Domonkos Trager², András Gézsi¹, Anna Süveges¹, Emese Bato³, Krisztina Takács-Vellai⁴, Csilla Nemes⁵, Aniko Gal¹

¹Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary; ²Budapest University of Technology and Economics, Budapest, Hungary; ³Xenovea Ltd, Szeged, Hungary; ⁴Biological Anthroplogy, Eötvös Loránd University, Budapest, Hungary; ⁵Dr. Kenessey Albert Hospital, Balassagyarmat, Hungary

Background/Objectives: The Dynamin2 protein (DNM2) has diverse roles in cell functions, including in clathrin mediated endocytosis at the plasma membrane and together with DRP1, in mitochondrial division. DNM2 depletion blocks mitochondrial division and results in an elongated, hyper-fused mitochondrial network.

Methods: The silencing was performed for 72 hours. The efficiency of siRNA gene silencing was analysed by real-time PCR and Western blotting. RNA sequencing was performed on the properly transfected samples on Illumina NextSeq platform. The bioinformatics analysis focusing on mRNA expression changes.

Results: Gene silencing of siRNA was performed in 3 parallel measurements with 3 different siRNAs. Scrambled siRNA and non-transfected HeLa cells were used as controls for the experiments. After a bioinformatical analysis very strong significance for 12 genes were found. These genes are involved in regulation of cytoskeletal function and trafficking, muscle function, and steroid biogenesis.

Conclusion: In DNM2 depletion samples, the downregulation of the FBLIM1, KRT13, KRT19, TMEM139, TMEM45A genes are involved in cytoskeletal function and trafficking, so they might be indirectly modify mitochondrial dynamics. TNNC1 plays a major role in the regulation of muscle function, so the decrease in expression found may be related to the formation of centronuclear nuclei. CYP4F3 gene expression was also significantly decreased. It is a monooxidase involved in cholesterol and steroid biosynthesis. Its relationship with DNM2 is currently in question. A validaton of RNASeq results currently in process.

References: -.

Grants: The study was supported by the Semmelweis University StartUp, NKFIH_ 132812 and UNKP-21-5 grants.

Conflict of Interest: None declared.

P07.036.B Correlation between GLA rare variants and phenotype in Hungarian patients with Fabry disease

Tamás Szlepák ¹, Robert Sepp², Eva Rakoczi³, Krisztina Nemeth⁴, Tamas Gyimesi⁵, Sandor Molnar⁶, Gyorgy Fekete⁴, Maria Judit Molnár¹

¹Semmelweis University, Institute of Genomic Medicine and Rare Disorders, Budapest, Hungary; ²University of Szeged, Second Department of Internal Medicine and Cardiology Center, Szeged, Hungary; ³University of Debrecen, Department of Rheumatology, Debrecen, Hungary; ⁴Semmelweis University, 2nd Department of Paediatrics, Budapest, Hungary; ⁵University of Pécs, 2nd Department of Internal Medicine and Nephrological Center, Pécs, Hungary; ⁶Soproni Erzsébet Teaching Hospital and Rehabilitation Institute, Neurology-stroke, Sopron, Hungary

Background/Objectives: Fabry disease (FD) is the second most common metabolic disorder with high morbidity and mortality. Hundreds of mutations and non-coding haplotypes in the GLA gene have been described; however, many are variants of unknown significance, prompting doubts about the diagnosis and treatment.

Methods: We identified GLA mutations in patients with suspicion of FD in Hungary for the last couple of decades. Identification of patients’ genotype was done with Sanger sequencing. Patients tested were participating in FD screening projects or showed typical signs of FD or had low enzyme activity. The detected variants were classified using the current ACMG guideline, Fabry databases and literature.

Results: We found 24 different rare variants in 51 patients overall, of which 16 were classified as pathogenic, 4 likely pathogenic and 4 likely benign. Of the identified variants, 13 cause classic and 7 later onset phenotype. We also identified 2 variants that have conflicting interpretations of pathogenicity. Four of the damaging rare variants were only found in Hungarian patients so far.

Conclusion: We present a descriptive clinical study including 51 patients with 24 different GLA variants. We identified 4 novel rare damaging variants of the GLA gene. In order to better characterise VUS, not only probands but also all asymptomatic variant carriers from Fabry families should be followed prospectively. Data sharing has great importance. These data, in the future, will help to distinguish symptoms attributable to FD from nonspecific comorbidities in benign GLA variants carriers.

References:.

Grants: Nothing to disclose.

Conflict of Interest: None declared.

P07.037.C Molecular diagnosis of Fabry disease in patients with chronic renal failure of unknown etiology

Marina Parezanovic ¹, Maja Stojiljkovic¹, Marina Andjelkovic¹, Nina Stevanovic¹, Vesna Spasovski¹, Milena Ugrin¹, Jovana Komazec¹, Natasa Tosic¹, Sonja Pavlovic¹, Dejan Celic², Jelica Vucenovic³, Anita Skakic¹

¹Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; ²Clinic for Nephrology and Clinical Immunology, Clinical Center of Vojvodina, University of Novi Sad, Novi Sad, Serbia; ³General Hospital Sremska Mitrovica, Hemodialysis Center, Sremska Mitrovica, Serbia

Background/Objectives: Fabry disease (FD) is a rare X-linked disorder caused by variants in the GLA gene leading to the deficiency of lysosomal α-galactosidase-A and progressive accumulation of globotriaosylceramide affecting the heart, nervous system, and kidneys. FD has overlapping phenotypes and often remains undiagnosed. Therefore, the precise molecular-genetic diagnosis and the earliest possible treatment are essential to avoid significant disease progression.

Methods: We analyzed 95 (34 female and 61 male) hemodialysis patients with clinical suspicion of FD using Sanger sequencing of all coding exons (7) and flanking intron regions of the GLA gene, and measured the relative expression of the GLA gene in available samples.

Results: The genetic analysis revealed 3 patients with a missense variant (p.Asp313Tyr), and 10 patients with combinations of non-coding variants, described as complex intronic haplotypes (CIHs). CIH1 (c.-10C>T, c.370-81_370-77delCAGCC, c.640-16A>G, c.1000-22C>T), the most frequent haplotype, was detected in 7 (7.4%) patients. Lyso-Gb3 biomarker levels were within the normal range in each tested patient. However, RT-qPCR analysis revealed decreased relative expression of GLA gene in PBMC of 2 female patients with CIH1 and one female patient carrying only c.-10C>T variant by 9,1%, 7,4%, 46,3%, respectively, pointing out that further analyses are needed to confirm/exclude FD in these patients.

Conclusion: Because the effects of CIHs are not yet fully understood, our work highlights the importance of analyzing intronic regions of the GLA gene as genetic modifiers and the need to include expression analysis in the diagnostic algorithm.

References:.

Grants: Genetic and biomarker analyses are sponsored by Takeda GmbH, Serbia.

Conflict of Interest: None declared.

P07.038.D An unusual case of combined hypolipidaemia and premature peripheral vascular disease

Zuzana Pös^1;2;3, Milad Khedr⁴, Jan Radvanszky^1;2;5, Rastislav Hekel^2;3;5;6, Tomas Szemes^2;3;5, Adela Penesova⁷, Lakshminarayan Ranganath⁴, Andrea Zatkova ¹

¹Biomedical Research Centre of the Slovak Academy of Sciences, Institute of Clinical and Translational Research, Bratislava, Slovakia; ²Comenius University Science Park, Bratislava, Slovakia; ³Geneton Ltd., Bratislava, Slovakia; ⁴Royal Liverpool University Hospital, Department of Clinical Biochemistry and Metabolic Medicine, Liverpool, United Kingdom; ⁵Faculty of Natural Sciences, Comenius University, Department of Molecular Biology, Bratislava, Slovakia; ⁶Slovak Centre of Scientific and Technical Information, Bratislava, Slovakia; ¹Biomedical Research Centre of the Slovak Academy of Sciences, Institute of Clinical and Translational Research, Bratislava, Slovakia

Background/Objectives: Monogenic hypobetalipoproteinemias include a heterogeneous group of disorders characterized by very low plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL), and apolipoprotein B (apoB) that are relatively uncommon in general population. It is thought that low LDL can protect from CVD, but this is not what we found in a case we present. We report on a 57 years old male patient (with BMI 21 kg/m²) with combined hypolipidaemia who presented with premature peripheral vascular disease. We also presented his two sons aged 32 and 27 years, who also manifested tendency to low lipid levels.

Methods: We used Illumina exome analysis in all three individuals. Variant filtering by QCI was performed using two different approaches: one based on candidate genes and the other one based on clinical symptoms.

Results: No pathogenic or likely pathogenic variants within main hypocholesterolaemia/dyslipidaemia candidate genes (ANGPTL3, SAR1B, APOB, PCSK9 or MTTP) were found in any of them. However, all three individuals share a novel ABCA1 variant, possibly responsible for decreased HDL levels. The proband and one of his sons share also the splicing variant rs138326449 within the APOC3 gene, shown to be associated with decreased TG levels.

Conclusion: We can hypothesize that in our patient despite his low TGs and LDL levels obtained thanks to the presence of the protective APOC3 variant, atherosclerosis developed due to the impaired cholesterol efflux caused by ABCA1 variant.

References:.

Grants: APVV-18-0319; VEGA_2/0167/20; VEGA_02/0040/20; ITMS: 313011V446 co-financed by the European Regional Development Fund (LISPER).

Conflict of Interest: None declared.

P07.039.A Untreated PKU patients without intellectual disability: SHANK gene family as a candidate modifier

Kristel Klaassen¹, Maja Djordjevic^2;3, Anita Skakic ¹, Bozica Kecman², Marina Parezanovic¹, Marina Andjelkovic¹, Nina Stevanovic¹, Vesna Spasovski¹, Milena Ugrin¹, Radoje Drmanac^4;5;6, Sonja Pavlovic¹, Maja Stojiljkovic¹

¹Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; ²Mother and Child Health Care Institute of Serbia „Dr Vukan Cupic“, Belgrade, Serbia; ³School of Medicine, University of Belgrade, Belgrade, Serbia; ⁴Complete Genomics Incorporated, San Hose, United States; ⁵MGI, BGI-Shenzhen, Shenzhen, China; ⁶BGI-Shenzhen, Shenzhen, China

Background/Objectives: Phenylketonuria (PKU) is an inborn error of metabolism caused by variants in the phenylalanine hydroxylase (PAH) gene. Although PKU is a monogenic disease, decades of research and clinical practice have shown that the correlation between the genotype and corresponding phenotype is not simple at all. Attempts have been made to discover modifier genes for PKU cognitive phenotype but without any success so far.

Methods: We conducted whole genome sequencing of 4 subjects from unrelated non-consanguineous families who presented with pathogenic mutations in the PAH gene, high blood phenylalanine concentrations and near-normal cognitive development despite no treatment.

Results: We used cross sample analysis to select genes common for more than one patient. Thus, the SHANK gene family emerged as the only relevant gene family with variants detected in 3 of 4 analyzed patients. We detected two novel variants, p.Pro1591Ala in SHANK1 and p.Asp18Asn in SHANK2, as well as SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro and SHANK3:p.Pro1716Thr variants that were previously described. Computational analysis indicated that the identified variants do not abolish the function of SHANK proteins. However, changes in posttranslational modifications of SHANK proteins could influence functioning of the glutamatergic synapses, cytoskeleton regulation and contribute to maintaining optimal synaptic density and number of dendritic spines.

Conclusion: Our findings are linking SHANK gene family and brain plasticity in PKU for the first time. We hypothesize that variant SHANK proteins maintain optimal synaptic density and number of dendritic spines under high concentrations of phenylalanine and could have protective modifying effect on cognitive development of PKU patients.

References:.

Grants: MESTD-RS 451-03-68/2022-14/200042.

Conflict of Interest: None declared.

P07.040.B Clinical, genetic and therapeutic aspects in Menkes disease: study of a French cohort and systematic literature review

Paul Rollier ^1;2, Marie-Pierre Moizard^3;4, Annick Toutain⁵, Sophie Blesson⁵, Eric Bieth⁶, Chrystèle Bonnemains⁷, Aline Cano⁸, Brigitte Chabrol⁸, Annabelle Chaussenot⁹, Léna Damaj¹⁰, François Feillet⁷, Sylvie Joriot¹¹, Manoelle Kossorotoff¹², Christian Richelme¹³, Dominique Bonneau¹⁴, Sylvie Odent¹, Magalie Barth¹⁴

¹Rennes University Hospital, Clinical Genetics, RENNES, France; ²Rennes University Hospital, Molecular Genetics and Genomics, RENNES, France; ³Tours University Hospital, Molecular Genetics, TOURS, France; ⁴INSERM, U1253, TOURS, France; ⁵Tours University Hospital, Clinical Genetics, TOURS, France; ⁶Toulouse University Hospital, Medical Genetics, TOULOUSE, France; ⁷Nancy University Hospital, Pediatrics, NANCY, France; ⁸Marseille Public University Hospital, Pediatric Neurology, MARSEILLE, France; ⁹Nice University Hospital, Medical Genetics, NICE, France; ¹⁰Rennes University Hospital, Pediatric Neurology, RENNES, France; ¹¹Lille University Hospital, Pediatric Neurology, LILLE, France; ¹²Necker-Enfants Malades Hospital, Pediatric Neurology, PARIS, France; ¹³Nice University Hospital, Pediatric Neurology, NICE, France; ¹⁴Angers University Hospital, Medical Genetics, ANGERS, France

Background/Objectives: Menkes disease has multi-organ involvement with neurological, cutaneous, urological, vascular and bone complications and historically death before 3 years. Copper histidinate (CuHis) is the only specific treatment but with variable results. The objective was to describe clinical and genetic characteristics of a French cohort and discuss therapeutic management.

Methods: A cohort was constructed from genetic diagnoses (ATP7A) of Menkes in France and compared with a literature systematic review.

Results: Diagnostic yield was 81% (71/88 cases) with 50% undescribed variants. 24.6% are intragenic deletions/duplications, 50.8% are loss-of-function and 24.6% are missense. Missenses are distributed exclusively from exon 7 onwards and can result on splicing defect. Of the 24 individuals (clinical sub-cohort), average age at diagnosis was 4.68±2.16 months. Symptoms at diagnosis are hypotonia (96%), epilepsy (88%), pili torti (38%), skin pallor (44%). Neonatal hypothermia (53%) and cephalhaematomas (27%), although non-specific, are over-represented. CuHis was started on 55% of individuals (mean age 5.1±2.8 months), without significant improvement on survival or development, or of genotype-phenotype correlation distinguishing best responders. Comparing with the literature, CuHis is only effective (survival and neurodevelopment) when initiated in infants who are not yet neurologically impaired. Epilepsy occurred in all individuals in our cohort even when CuHis was started before first seizures. CuHis may also provide better control of urological complications but not on vascular and bone phenotypes.

Conclusion: Menkes disease remains a non-curable disease with limited therapeutic range and poor prognosis. CuHis should only be offered in first intention in presymptomatic individuals.

References:.

Grants: No funding source.

Conflict of Interest: None declared.

P07.041.C Novel MECR mutation in a Czech patient with childhood-onset dystonia, optic atrophy, and basal ganglia abnormality

Lukáš Ryba ¹, Zuzana Korandová², Eliška Koňaříková², Tomáš Mráček², Markéta Havlovicová¹, Markéta Vlčková¹, Emílie Vyhnálková¹

¹Second Faculty of Medicine, Charles University and University Hospital Motol, Department of Biology and Medical Genetics, Prague, Czech Republic; ²Institute of Physiology, Czech Academy of Science, Prague, Czech Republic

Background/Objectives: Gene MECR encodes a highly conserved mitochondrial trans-2-enoyl-coenzyme-A-reductase which has a crucial role in mitochondrial fatty acid synthesis (mFAS). Here we report a 12-year-old patient with recessive mutations in MECR who presented with childhood-onset dystonia, hypoplasia and atrophy of the optical nerves, and abnormal MRI signaling of basal ganglia and dorsal mesencephalon.

Methods: Family-based whole-exome sequencing of DNA extracted from peripheral blood lymphocytes was followed by bioinformatics analysis of the TRIO using the software VarAFT. Functional measurements of mitochondrial oxidative phosphorylation (OXPHOS) were performed on skin fibrocytes via high-resolution respirometry (OroborosOxygraph). Western blot was used to detect individual mitochondrial proteins and to study the assembly of OXPHOS complexes.

Results: Within gene MECR, we found a novel variant c.610G>C p.(Ala204Pro) and previously described variant c.772C>T p.(Arg258Trp) in a compound heterozygous state. Functional measurements of OXPHOS on fibrocyte cell line found a statistically significant decrease of the mitochondrial electron transport chain (ETC) capacity, complex I and IV capacity, and overall mild decrease of respiration. Western blot showed a decreased quantity of complexes I and IV.

Conclusion: We described a novel variant c.610G>C in MECR in an individual with a phenotype consistent with previously described cases of the MECR-related neurologic disorder (1). The pathogenicity of the variant was confirmed by functional analysis.

References: 1. Heimer G, Kerätär JM, Riley LG, et al. MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder. Am J Hum Genet. 2016;99(6):1229-1244. https://doi.org/10.1016/j.ajhg.2016.09.021.

Grants: IGA MZČR (NT/13770-4/2012 to MMJr, NT/14200 to MH), Norway Grants (PDP3-NorwayGrants) and COST–LD14073.

Conflict of Interest: None declared.

P07.042.D False positive newborn screening for maple syrup urine disease in patients with hydroxyprolinemia

Dimitar Gavrilov ¹, Amy White¹, Faisal Asumda¹, Noemi Vidal Folch¹, Laura Davis-Keppen², Isum Ward², Kari Casas³, Matthew Schultz¹, Devin Oglesbee¹, Silvia Tortorelli¹, Dietrich Matern¹

¹Mayo Clinic, Rochester, United States; ²USD Sanford School of Medicine, Sioux Falls, United States; ³University of North Dakota School of Medicine and Health Sciences, Fargo, United States

Background/Objectives: Newborn screening (NBS) for maple syrup urine disease (MSUD) by tandem mass spectrometry utilizes leucine as primary marker. The isobaric amino acids leucine, isoleucine, alloisoleucine and hydroxyproline (OH-Pro) are not distinguished by this method. Hydroxyprolinemia is an autosomal recessive benign condition (1) and therefore should not be identified by NBS.

Methods: Of six healthy newborns with NBS results presumptive positive for MSUD two were hospitalized until MSUD was excluded. The other four patients had plasma and urine collected for amino acid (PAA) and organic acid (UOA) analyses at an outpatient visit. Original NBS specimens of two cases were available for retrospective measurement of leucine, isoleucine, valine, alloisoleucine and OH-Pro (2).

Results: PAA and UOA analyses excluded MSUD and disclosed elevations of OH-Pro (237-424 mmol/L; controls <61) in all six cases. OH-Pro but not leucine, isoleucine, valine or allo-isoleucine, was elevated in the NBS specimens of two cases. Molecular genetic analysis of PRODH2 is underway.

Conclusion: MSUD is a critical condition that requires rapid initiation of treatment. However, hydroxyprolinemia is more common and therefore a cause of false positive NBS results (1). Total parenteral nutrition can also cause false positive results (2). Employing a second-tier NBS test in the original dried blood spot specimen to differentiate between branch-chain amino acids and OH-Pro can avoid the cost and anxiety associated with false positive results of NBS for MSUD.

References: 1. Staufner et al. J Inherit Metab Dis. 2016;39:625-32.

2. Oglesbee et al. Clin Chem 2008;54:542-9.

Grants:.

Conflict of Interest: Dimitar Gavrilov: None declared, Amy White: None declared, Faisal Asumda: None declared, Noemi Vidal Folch: None declared, Laura Davis-Keppen: None declared, Isum Ward: None declared, Kari Casas: None declared, Matthew Schultz: None declared, Devin Oglesbee: None declared, Silvia Tortorelli: None declared, Dietrich Matern Advisory board, Novogene, 4 hrs (honorarium passed on to my employer, Mayo Clinic).

P07.043.A Clinical and metabolomic peculiarities in monogenic disorders affecting urea cycle or mitochondrial ATP synthase

Botezatu Alina ¹, Adina Chis^2;3, Calin Deleanu^4;5, Cecilia Lazea⁶, Gabriella Horvath⁷, Melinda Baizat⁸, Ligia Blaga⁹, Simona Bucerzan⁶, Alina Nicolescu^4;5, Romana Vulturar^2;3

¹Iuliu Hatieganu University of Medicine and Pharmacy, Medicine, Cluj-Napoca, Romania; ²“Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, Department of Molecular Sciences, Cluj-Napoca, Romania; ³Babeș-Bolyai University Cluj-Napoca, Romania, 3-Cognitive Neuroscience Laboratory, Cluj-Napoca, Romania; ⁴“P. Poni” Institute of Macromolecular Chemistry, Romanian Academy, Cluj-Napoca, Romania; ⁵“C. D. Nenitescu” Centre of Organic Chemistry, Romanian Academy, Cluj-Napoca, Romania; ⁶“I. Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, Ist Paediatric Clinic - Cluj Children Hospital, Cluj-Napoca, Romania; ⁷Faculty of Medicine, University of British Columbia, Vancouver, Canada, Division of Biochemical Diseases, Department of Pediatrics, Cluj-Napoca, Romania; ⁸Neonatology Department, Clinical Hospital Zalau, Cluj-Napoca, Romania; ⁹“Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, Neonatology Department, Cluj-Napoca, Romania

Background/Objectives: The clinical-biochemical findings in born errors of metabolism (IEMs) are sometimes nonspecific. A metabolomic approach involves a quantitative analysis of a set of metabolites present in urine or/and plasma linking them to a selected metabolic pathway and/or particular metabolic condition.

Methods: Results from plasma and/or urine amino acids analyzed by a two-dimensional thin-layer-chromatography (2D-TLC) as an urgent selective screening in patients from pediatric clinical departments were compared with urinary NMR spectroscopic results in several IEMs. ¹H-NMR urine spectroscopy constitutes a complementary technique in the diagnosis of several IEMs, providing an overall view of metabolism, giving a “fingerprint” of almost all hydrogen nuclei in a metabolite.

Results: We summarize clinical data and biochemical comparative markers obtained using these two methods in several urea cycle disorders (ornithine transcarbamylase - OTC and argininosuccinic aciduria - AAS), and a mitochondrial disorder - the TMEM70 defect evolving with mild or severe hyperlactataemia. In TMEM70 defect, several plasma amino acids were outside the normal ranges as nonspecific changes, but the diagnostic was established by molecular DNA investigation. Contrary, the orotic acid and argininosuccinic acid were rapidly detected using urinary NMR spectroscopy in ornithine transcarbamylase defect. In AAS, the urinary 2D-TLC method was useful for rapid identification of high concentration of arginino-succinic acid.

Conclusion: The 2D-TLC and ¹H-NMR spectroscopy methods available for a rapid evaluation may be a versatile association for several specific molecules. Identification of such bio-markers demonstrated important potential for including more rare diseases into current national screening programme and research.

References:.

Grants:.

Conflict of Interest: None declared.

P07.045.C Shifting the border of pathogenicity with a DEL9 mutation in the CEL gene

Maria Grossmann ¹, Janek Wilk², Andrea Bier¹, Silke Reif¹, Manuela Timmer¹, Christian Jänecke¹, Vivien Klaschka¹, Beate Küchler², Albrecht Kobelt³, Stefan Krüger¹, Jens Plaschke^1;2

¹Gemeinschaftspraxis für Humangenetik, member of Praxisnetz Humangenetik Deutschland, Dresden, Germany; ²MEWIGEN, Dresden, Germany; ³Praxis für Medizinische Genetik, Zentrum für Diagnostik am Klinikum Chemnitz, Chemnitz, Germany

Background/Objectives: Maturity-onset diabetes of the young (MODY) is a group of autosomal dominantly inherited disorders of non-autoimmune diabetes mellitus with usual onset in adolescence or young adulthood. Most MODY mutations are located in genes GCK, HNF1A, HNF4A and HNF1B. Rarely, mutations in other contributing genes (APPL1, ABCC8, BLK, CEL, INS, KCNJ11, KLF11, NEUROD1, PAX4, PDX1) are causative for MODY.

In the last exon of the CEL gene, single-base deletions within the 33bp VNTR (Variable Number of Tandem Repeat) region causing different C-terminal aberrant proteins have been described. The pathogenic potential is higher for mutations located in more 5’ VNTR parts. For DEL1 (named after the affected repeat number) and DEL4 a negative gain-of-function effect has been established by functional studies. In contrast, DEL13 was considered as benign variant due to a behavior like wildtype, whereas DEL9 showed intermediate effects and was concluded to be likely benign because of observation in healthy controls.

Here we report two sisters (34 and 37 years) and their mother (61 years) affected by MODY.

Methods: NGS analysis for all above-mentioned 14 genes was performed.

Results: For all three patients we identified the CEL mutation NM_001807.6:c.1941delC, p.(Val648Cysfs*45) in a heterozygous state affecting VNTR repeat 9 (DEL9 regarding individual VNTR haplotype).

Conclusion: Due to co-segregation in this family we consider the CEL DEL9 as pathogenic. This shifts the previously assumed border of pathogenicity to a more 3’end of the gene.

References: Gravdal et al. J Biol Chem. Jan-Jun 2021;296:100661.

Grants:.

Conflict of Interest: None declared.

P07.046.D MELAS: clinical and laboratory-based diagnostic algorithm

Sebastian Romeo Pintilie ¹, Ilinca Maria Farcas², Alina Botezatu¹, Laura Damian³, Adina Chis^4;5, Romana Vulturar^4;5

¹”Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; ²University of Oxford, Oxford, United Kingdom; ³Emergency Clinical County Hospital, Rheumatology, Center for Rare Autoimmune and Autoinflammatory Diseases, Cluj-Napoca, Romania; ⁴Babes-Bolyai University, Cognitive Neurosciences Laboratory, Cluj-Napoca, Romania; ⁵”Iuliu Hatieganu” University of Medicine and Pharmacy, Molecular Sciences, Cluj-Napoca, Romania

Background/Objectives: Mitochondrial Encephalopathy with Lactic Acidosis Syndrome (MELAS) is a mitochondrial disease caused by point mutations or deletions in the mitochondrial or even nuclear genome. The pathogenesis of MELAS is not fully understood, so several hypotheses have been proposed. Some of the most obvious processes involved are related to the accumulation of RNA19 in active metabolic tissues, mitochondrial proliferation due to energy and nitric oxide deficiency, impaired glucose metabolism with lactic acidosis.

Methods: Following a review of the current literature on the diagnosis methods and criteria of MELAS, we have developed a quick algorithm for helping clinicians in establishing the diagnosis of MELAS.

Results: The Hirano et al. (1992) and Yatsuga et al. (2012) criteria remain the benchmark for selecting patients suspected of MELAS, based on their clinical picture such as: seizures, headache with vomiting, and myopathy. However, to secure the diagnosis, performing PCR-RFLP in a common set of affected-genes, including the prevalent m.3243A>G substitution, should be done first on a MELAS-suspected, only then followed by full mitochondrial and even nuclear genomic testing or, if sequencing is not possible, by allele-specific oligonucleotide (ASO) dot blot hybridization, which is more sensitive than PCR.

Conclusion: Beside the proposed clinical algorithm, muscle biopsy/blood lactate cannot provide certain evidence for MELAS diagnosis, which is why molecular testing should be prioritized over other clinical tests when establishing a diagnosis; yet a genetic test positive for a mutation in the mitochondrial DNA should still be correlated with any of the first two tests.

References:.

Grants:.

Conflict of Interest: None declared.

P08 Immunology and Hematopoietic System

P08.001.A The value of genetic data from 665,460 individuals in predicting anemia and ability to donate blood

Jarkko Toivonen ¹, Johanna Castrén¹, FinnGen Consortium², Mikko Arvas¹

¹Finnish Red Cross Blood Service, Helsinki, Finland; ²FinnGen Consortium, Helsinki, Finland

Background/Objectives: We aim to find out whether genetic information has significant effect in predicting iron deficiency anemia or blood donation ability.

Methods: Genetic data from FinnGen release 6 (230,000 participants), Blood Service Biobank (30,000 participants) and UK Biobank (400,000 participants) was analyzed. In addition, age, sex, weight, height, anemia status and blood donation histories were used when available.

We performed GWAS for anemia and blood donation ability and computed polygenic risk score weights for anemia, ferritin¹ and hemoglobin². A Bayesian logistic regression for anemia was fitted on all FinnGen participants and for donation ability on blood donors, stratified into three demographic groups.

Results: A single significant SNP rs199598395 in gene RNF43 was revealed by our anemia and deferral GWAS. The meta-analysis from FinnGen and UKBB for anemia provided three more significant lead SNPs. The largest effect of the genetic data in anemia model was by the RNF43 SNP for pre-menopausal females (OR 4.3, CI 2.6 – 6.7) and in the deferral model again the RNF43 SNP for pre-menopausal females (OR 3.2, CI 1.9 – 5.4). PRSs didn’t have significant effects in either model.

Conclusion: A single SNP can have a strong effect on prediction of both anemia and blood donation ability. PRSs are not yet informative enough to be usable predictors.

References: ¹ Bell et al. A genome-wide meta-analysis yields 46 new loci associating with biomarkers of iron homeostasis. Commun Biol 4, 156 (2021).

² Vuckovic et al. The Polygenic and Monogenic Basis of Blood Traits and Diseases. Cell. 2020 Sep 3;182(5):1214-1231.e11.

Grants:.

Conflict of Interest: Jarkko Toivonen Full-time employee at Finnish Red Cross Blood Service. Significant contribution., Johanna Castrén Full-time employee at Finnish Red Cross Blood Service. Significant contribution., FinnGen Consortium: None declared, Mikko Arvas Full-time employee at Finnish Red Cross Blood Service. Significant contribution.

P08.002.B Added value of reanalysing whole exome- and whole genome sequencing data with an extended gene panel and structural variant calling in patients suspected of having primary immune deficiency

Sara Mørup ¹, Lusine Nazaryan-Petersen², Migle Gabrielaite², Joanne Reekie³, Hanne V. Marquart⁴, Hans Jakob Hartling⁴, Rasmus Marvig², Terese L. Katzenstein⁵, Tania N. Masmas⁶, Jens D. Lundgren^1;5, Daniel D. Murray¹, Marie Helleberg^3;5, Line Borgwardt²

¹Copenhagen university hospital, Rigshospitalet, Persimune, Centre of excellence for health, immunity, and infections, Copenhagen, Denmark; ²Copenhagen university hospital, Rigshospitalet, Center for genomic medicine, København Ø, Denmark; ³Copenhagen university hospital, Rigshospitalet, Persimune, Centre of excellence for health, immunity, and infections, København Ø, Denmark; ⁴Copenhagen university hospital, Rigshospitalet, Department of clinical immunology, København Ø, Denmark; ⁵Copenhagen university hospital, Rigshospitalet, Department of infectious diseases, København Ø, Denmark; ⁶Copenhagen university hospital, Rigshospitalet, The child and adolescent department, København Ø, Denmark

Background/Objectives: Knowledge of genetic variation underlying Primary Immune Deficiency (PID) is steadily increasing(1). Reanalysis of genome-wide sequencing data from patients suspected of PID may improve the diagnostic rate.

Methods: We included patients monitored at the Department of Infectious Diseases, Rigshospitalet, Denmark, for a suspected PID, who had been analysed during 2015-2020 using a targeted PID gene panel (457 PID-related genes) on whole exome- (WES) or whole genome sequencing (WGS) data. A literature review was performed to extend the PID gene panel used for reanalysis of single nucleotide variation (SNV) and small indels. Structural variant (SV) calling was added on WGS data.

Results: In total, genetic data from 94 patients (86 adults) was reanalysed a median of 23 months after the initial analysis (38 WES and 56 WGS). The extended gene panel included 208 additional PID-related genes. The total proportion of patients with ACMG class 3-5 variants increased from 43 (46 %) to 50 (53 %). The proportion of patients with a causal genetic diagnosis was constant, but after reanalysis 13 patients (14 %) had a new potentially disease causing/contributing variant of unknown significance (VUS) identified. Among these, 8 of 94 (9 %) had a SNV and 5 of 56 (9 %) patients had a SV.

Conclusion: These data indicate a possible diagnostic gain of reassessing WES/WGS data from patients with suspected PID. Reasons for the possible gain included improved knowledge of genotype-phenotype correlation, expanding the gene panel, and adding SV analyses.

References: (1) PMID: 31953710.

Grants: This work is funded by Danish National Research Foundation (DNRF126).

Conflict of Interest: Sara Mørup: None declared, Lusine Nazaryan-Petersen: None declared, Migle Gabrielaite: None declared, Joanne Reekie: None declared, Hanne V. Marquart: None declared, Hans Jakob Hartling: None declared, Rasmus Marvig: None declared, Terese L. Katzenstein Gilead Sciences., Advisory boards for Gilead Sciences, GlaxoSmithKline/ViiV and MSD., Teaching for Takeda and CSLBehring., Tania N. Masmas: None declared, Jens D. Lundgren: None declared, Daniel D. Murray: None declared, Marie Helleberg Advisory boards for AstraZeneca, Gilead, GSK, MSD, Roche and Sobi., Teaching for Gilead and GSK., Line Borgwardt: None declared.

P08.003.C Copy number variants in pediatric patients with suspected inborn errors of immunity

Breanna Beers ¹, Morgan Similuk¹, Magdalena Walkiewicz¹, Michael Kamen¹, Michael Setzer¹, Colleen Jodarski¹, Rachel Gore¹, Bryce Seifert¹, Rajarshi Ghosh¹, Rylee Duncan¹, Devin Hunt¹, Madison Mixer¹, Kathleen Jevtich¹, Yunting Yu¹, Luis M. Franco¹, Steven Holland¹, Jia Yan¹

¹National Institutes of Health, Bethesda, United States

Background/Objectives: Exome sequencing (ES) results do not rule out genetic contribution to disease, including contribution from structural variation. We evaluated 332 pediatric patients with suspected inborn errors of immunity (IEI) to determine the relative contribution of ES and chromosomal microarray (CMA).

Methods: ES and CMA customized for exonic coverage of immune-related genes were performed to study 332 unrelated pediatric probands referred to the National Institute of Allergy and Infectious Diseases Centralized Sequencing Program for suspected IEI.

Results: Nearly one-third (107/332) of patients’ phenotypes involved 10 or more top-level Human Phenotype Ontology categories, most commonly the immune system, integument, respiratory system, digestive system, and blood/blood-forming tissues, in decreasing frequency.

Of the 332 probands, 131 (39.5%) received at least one molecular diagnosis. In total, 113/131 (86.2%) probands were diagnosed by ES alone; 15/131 (11.5%) were diagnosed by CMA alone, including two de novo changes. ES and CMA both contributed to diagnoses for 3/131 (2.3%) probands, including two compound heterozygotes.

Nine of the 18 patients with CMA contribution to diagnosis had copy number variants in at least one gene not listed by the International Union of Immunological Sciences as causative of IEI. Six were primarily neurological, one was cardiovascular, one was metabolic, and one was respiratory.

Conclusion: CMA findings contributed to over one-in-ten (18/131) molecular diagnoses. Although most diagnoses were made by ES, CMA can increase the likelihood of molecular diagnosis when ES is inconclusive. Half of CMA diagnoses at least partially involved non-immune phenotypes, and so would not typically appear on commercial panels for immune disorders.

References:.

Grants:.

Conflict of Interest: None declared.

P08.004.D Trio-based sequencing in patients with sporadic inborn errors of immunity: a retrospective cohort study

Caspar van der Made ^1;2, Anne Hebert¹, Annet Simons¹, Janneke Schuurs-Hoeijmakers¹, Esther van Rijssen³, Simon van Reijmersdal¹, Evelien Zonneveld-Huijssoon⁴, Stefanie Henriet⁵, Wendy Van Zelst-Stams¹, David Koolen¹, Erika Leenders¹, Etienne Janssen⁶, Gijs Santen⁷, Sonja de Munnik¹, Simone Kersten¹, Hans Koenen³, Mihai Gheorghe Netea², Ruben Smeets³, Frank van de Veerdonk², Alexander Hoischen^1;2

¹Radboud University Medical Center, Department of Human Genetics, Nijmegen, Netherlands; ²Radboud University Medical Center, Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Nijmegen, Netherlands; ³Radboud University Medical Center, Laboratory Medicine, Laboratory for Medical Immunology, Nijmegen, Netherlands; ⁴University Medical Center Groningen, Department of Genetics, Groningen, Netherlands; ⁵Radboud University Medical Center, Department of Pediatric immunology, Pediatrics, Nijmegen, Netherlands; ⁶Maastricht University Medical Center+, Department of Clinical Genetics, Maastricht, Netherlands; ⁷Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, Netherlands

Background/Objectives: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) in patients with inborn errors of immunity (IEI). This study explored the additional value of systematic DNV assessment in a retrospective cohort of 123 patients with sporadic PIDs.

Methods: Patient-parent trios sequenced at the Radboud University Medical Center were eligible for inclusion when 1) the IEI in-silico gene panel was analysed and 2) the phenotype of the index patient suggested sporadic disease. Exome-wide analysis was performed to retain rare, coding, non-synonymous de novo SNVs. Variants were further prioritized based on gene and variant level metrics. In immune cells from one selected patient, functional validation experiments were performed at the level of RNA splicing, NF-κB signalling and cytokine production.

Results: Candidate DNVs were identified in 15 (12.2%) trios, in addition to 12 (9.8%) with inherited (likely) pathogenic mutations. These potentially disease-causing DNVs were identified in the known IEI genes NLRP3 and RELA, and novel candidate genes including PSMB10, DDX1, KMT2C and FBXW11. Furthermore, the FBXW11 canonical splice site DNV, carried by a patient with autoinflammatory disease, was shown to cause defective RNA splicing, increased NF-κB signalling and elevated IL-1β production.

Conclusion: This retrospective cohort study advocates the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI to improve the solve rate. Furthermore, we have provided functional evidence in support of a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease.

References:.

Grants: This research was part of a Radboud Institute for Molecular Life Sciences PhD grant.

Conflict of Interest: None declared.

P08.005.A Genetic characterization of non-familial hemophagocytic lymphohistiocytosis patients: monogenic defects in HAVCR2, TNFRSF9 and MADD genes

Laura Batlle-Masó ^1;2, Clara Franco-Jarava³, Laura Viñas-Gimenez³, Marina Garcia-Prat¹, Alba Parra-Martinez¹, Cristina Díaz-de-Heredia⁴, Anna Maria Cueto-González⁵, Marta Codina-Sola⁵, Berta Campos⁵, Eduardo Tizzano⁵, Jacques G Rivière¹, Pere Soler-Palacin¹, Laia Alsina⁶, Monica Martínez-Gallo³, Ferran Casals^2;7, Roger Colobran^3;5

¹VHIR - Vall d’Hebron Institut de Recerca / HUVH - Hospital Universitari Vall d’Hebron, Pediatric Infectious Diseases and Immunodeficiencies Unit, Barcelona, Spain; ²Genomics Core Facility, Department of Experimental and Health Science - Universitat Pompeu Fabra (UPF), Barcelona, Spain; ³VHIR - Vall d’Hebron Institut de Recerca / HUVH - Hospital Universitari Vall d’Hebron, Immunology Division - Translational Immunology Research Group, Barcelona, Spain; ⁴HUVH - Hospital Universitari Vall d’Hebron, Department of Pediatric Hematology and Oncology, Barcelona, Spain; ⁵HUVH - Hospital Universitari Vall d’Hebron, Department of Clinical and Molecular Genetics, Barcelona, Spain; ⁶Institut de Recerca Sant Joan de Déu - Hospital Sant Joan de Déu, Clinical Immunology and Primary Immunodeficiencies Unit, Barcelona, Spain; ⁷Universitat de Barcelona, Department of Genetics, Microbiology and Statistics, Barcelona, Spain

Background/Objectives: Hemophagocytic lymphohistiocytosis (HLH) is a severe and life-threatening syndrome characterized by a strong hyperactivation of the immune system. Familial HLH (FHL) is caused by biallelic variants in the genes PRF1, UNC13D, STX11 and STXBP2. Non-familial HLH (nFHL) may be caused by other genetic entities or may be sporadic or secondary to other conditions.

Methods: To elucidate other genetic mechanisms driving nFHL we studied a cohort of 31 nFHL patients (25 European, 5 North-African and 2 Asian). All cases had an immunological diagnosis of HLH but remained unsolved at a genetic level after excluding pathogenic variants in FHL genes.

Results: Using WES data, we first evaluated the possibility of the disease being casued by a monogenic defect. We found the genetic diagnosis for three of the patients i) one patient who carried a compound heterozygous variant at HAVCR2, ii) one with a homozygous nonsense variant at TNFRSF9, and iii) one with a homozygous deletion at MADD. Next, we looked for pathogenic variants over-represented in our cohort in comparison to healthy populations, identified monoallelic variants that could be acting as risk factors, and explored the digenic model in genes of the cytotoxic pathway.

Conclusion: Altogether, we showed that WES is a valuable diagnostic tool in nFHL patients, provided new insights into the physiopathology of the disease, and insisted on the importance of a genetic diagnosis on the clinical management and genetic counseling of the patients and their families.

References:.

Grants:.

2018_FI_B00072 by Generalitat de Catalunya, RTI2018-096824-B-C22 by Spanish Government-ERDF, PI17/00660-PI20/00761 by Instituto de Salud Carlos III-ERDF.

Conflict of Interest: None declared.

P08.006.B First description of a bone marrow failure syndrome in Spain caused by compound heterozygous truncating mutations in ERCC6L2

Perla Bandini ^1;2, Nina Borràs^1;2, Laura Martin-Fernandez^1;2, Eugenia Fernández-Mellid³, Paula Melero-Valentín³, Natàlia Comes^1;2, Lorena Ramírez^1;2, Patricia Cadahia Fernández³, Matilde Rodriguez Ruíz³, Manuel Mateo Perez Encinas³, Francisco Vidal^1;2;4, Irene Corrales^1;2

¹Banc de Sang i Teixits (BST), Coagulopaties Congènites, Barcelona, Spain; ²Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona (VHIR-UAB)., Medicina Transfusional, Barcelona, Spain; ³Hospital Clínico Universitario de Santiago de Compostela (CHUS – SERGAS)., Servicio de Hematología, Santiago de Compostela, Spain; ⁴Instituto Carlos III (ISCIII)., Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV)., Madrid, Spain

Background/Objectives: Biallelic variants in ERCC6L2 have been recently described to cause inherited bone marrow failure syndrome type 2 (BMFS-2) which is characterized by hypocellular bone marrow and, frequently, microcephaly. We describe the case of two siblings diagnosed at 8 and 14 years with medullary aplasia and thrombocytopenia (60-80e9platelets/L) without extra-hematopoietic manifestations. The aim was to identify the molecular defect associated with the patients’ phenotype using a whole exome sequencing (WES) strategy.

Methods: WES was performed using the DNA Prep with Enrichment protocol (Illumina) and sequenced in NextSeq500 system (Illumina). Variants were identified with BWA Enrichment and annotated and filtered with Variant Interpreter (Illumina). Family based trio analysis including the affected siblings and their parents (unaffected) was carried out using recessive inheritance pattern.

Results: Two truncating ERCC6L2 pathogenic variants, according to ACMG guidelines, were identified in both patients: 1) NM_020207:c.1930C>T (p.Arg644Ter) in exon 13, the second most recurrent variant, only found in homozygous state; 2) NM_020207:c.2156del (p.Gly719AspfsTer50), located in exon 15, previously described in a single patient.

Conclusion: This is the first description in Spain of medullar aplasia caused by ERCC6L2 mutations. Our findings contribute to the small worldwide cohort of 20 families with BMFS-2, with only four caused by compound heterozygous mutations in ERCC6L2. Early detection of the genetic defect will help in clinical management, which is relevant due to the susceptibility of BMFS-2 patients to myelodysplastic syndrome and acute myeloid leukaemia.

References: PMID: 29633571; 29987015; 29146883; 30936069.

Grants: ISCIII (PI18/01492 and CIBERCV), co-funded by ERDF, “A way to make Europe”. Fundació Privada Catalana de l’Hemofília.

Conflict of Interest: None declared.

P08.007.C A homozygous STING1 gene variant causes STING-associated vasculopathy with onset in infancy (SAVI) in two patients

Rensheng Wan ¹, Sandra Von Hardenberg¹, lisa isabel olfe¹, Bernd Auber¹, Martin Wetzke², Doris Steinemann¹

¹Hannover Medical School, Department of Human Genetics, Hannover, Germany; ²Hannover Medical School, Department of Pediatric Pneumology, Allergology and Neonatology, Hannover, Germany

Background/Objectives: Stimulator of interferon response cGAMP interactor 1 (STING1) gain of function (GoF) variants cause STING-associated vasculopathy with onset in infancy (SAVI; AD inheritance). While most causative heterozygous variants arise de novo, Lin et al. (2020) showed that STING1 variant p.(R281W) causes SAVI only in homozygous state. Here we report two further patients carrying STING1 homozygous variant p.(R281W).

Methods: Whole exome sequencing (WES) and whole genome sequencing (WGS) were applied respectively to identify causative variant in patient 1 and 2.

Results: STING1 (NM_198282.3): c.841C>T p.(R281W) was identified in two patients and classified as “pathogenic”. Patient 1 presented with recurrent severe hypoxaemia, hypoventilation, diffuse alveolar hemorrhage, interstitial lung disease, without skin vasculitis. Segregation analysis within the consanguineous family showed that healthy parents and one brother carried variant heterozygously. The interferon signature was highly elevated in patient 1 but not in healthy heterozygous family members. This correlates to published cases displaying that the heterozygous p.(R281W) variant did not affect the interferon signature. No significant improvement was observed after therapy with Janus kinase inhibitor Baricitinib. Patient 2 showed hypoventilation with scarring fibrotic changes, bronchiectasis, tachypnea, failure to thrive with marked dystrophy, hypocalcemia, and also no skin vasculitis.

Conclusion: Our report supports the possibility that p.(R281W) causes an atypical SAVI phenotype without demonstrating vasculopathy. It is important to consider an autosomal recessive inheritance pattern when evidence of STING-associated vasculopathy is given.

References: No references.

Grants: German Research Foundation (DFG) under Germany’s Excellence Strategy - EXC 2155 - RESIST project number 390874280.

R. Wan holds DAAD scholarship.

Conflict of Interest: None declared.

P08.009.A Influence of HBG2, BCL11A and HMIP polymorphisms on the clinical phenotype in Iraqis with beta-thalassemia

Nasir AS Al-Allawi¹, Sulav D Atroshi², Christian Oberkanins ³, Regir K Sadullah⁴, Adil A Eissa¹, Gernot Kriegshäuser⁵, Shaima SM Al-Zebari⁶, Shatha MA Qadir²

¹Department of Pathology, College of Medicine, University of Duhok, Duhok, Iraq; ²Department of Hematology, Azadi Teaching Hospital, Duhok, Iraq; ³ViennaLab Diagnostics, Vienna, Austria; ⁴Medical Laboratory Technology Department, College of Health and Medical Techniques, Duhok Polytechnic University, Shekhan, Iraq; ⁵IHR LABOR Medical Diagnostic Laboratories, Vienna, Austria; ⁶Research Center, College of Science, University of Duhok, Duhok, Iraq

Background/Objectives: The variability in β-thalassemia phenotype has been attributed to several genetic modifiers. The significance of the latter varies in different populations. The objective of the current study was to determine the significance of six genetic modifiers as they relate to phenotype in Iraqi β-thalassemia patients and to create a genetic scoring system that could predict phenotype in patients from this part of the world.

Methods: A total of 224 Iraqi patients homozygous or compound heterozygous for β-thalassemia were assessed for five polymorphisms at HbF QTLs, namely: rs7482144 C>T at HBG2, rs1427407 G>T and rs10189857 A>G at BCL11A, and rs28384513 A>C and rs9399137 T>C at HMIP.

Results: The enrolled patients had a median age of 14 years, with 96 males and 128 females. They included 144 thalassemia major (TM) and 80 thalassemia intermedia (TI) patients. Multivariate logistic regression revealed that three genetic modifiers, namely β⁺ alleles, HBG2 rs7482144 and BCL11A rs1427407, could significantly predict phenotype (TM versus TI) with an overall prediction rate of 82.1%. A cumulative favorable allele score based on these significant predictors had an area under curve of 0.862 (95% CI 0.816-0.909), which was highly significant (P = 3.2656E-19).

Conclusion: The current study identified three genetic predictors of phenotype in Iraqi patients with β-thalassemia, with an overall prediction rate of 82.1%. Furthermore, the population-specific cumulative favorable allele scoring system created had a good ability to discriminate between TM and TI. The application of the latter may help in providing more informed management and therapeutic options in this region.

References:.

Grants:.

Conflict of Interest: None declared.

P08.012.D Genetics in inborn errors of immunity: pediatric autoinflammatory phenotypes and the underlying genetic causes in 125 families

Yvonne Poker¹, Sandra Von Hardenberg ¹, Winfried Hofmann¹, Michelle Tang¹, Ulrich Baumann², Nicolaus Schwerk², Martin Wetzke², Viola Lindenthal³, Bernd Auber¹, Brigitte Schlegelberger¹, Hagen Ott⁴, Philipp von Bismarck⁵, Dorothee Viemann⁶, Frank Dressler², Christian Klemann², Anke Katharina Bergmann¹

¹Department of Human Genetics, Hanover, Germany; ²Department of Pediatric Pneumology, Allergology and Neonatology, Hanover, Germany; ³Department of Pediatrics and Pediatric Hematology/Oncology, Oldenburg, Germany; ⁴Division of Pediatric Dermatology, Children’s Hospital Auf der Bult, Hanover, Germany; ⁵Department of Pediatrics, University Medical Center Schleswig‐Holstein, Hanover, Germany; ⁶Translational Pediatrics, Department of Pediatrics, Würzburg, Germany

Background/Objectives: Diagnosis of monogenic autoinflammatory diseases (AID) requires an accurate description of the patients´ phenotype and the identification of highly penetrant genetic variants in single genes is pivotal.

Methods: In a routine genetic diagnostic setting, we performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy: First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis could be established, followed by the analysis of a virtual panel including 420 genes published by the International Union of Immunological Societies associated with all known inborn errors of immunity (IEI). Subsequently WES data were analyzed without pre-filtering for known AID/IEI genes.

Results: Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 420 genes to 21.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis nor in trio-WES analysis.

However variants of unknown significance were detected in 43.2%, (n = 54) which could become clinically relevant in the future.

Conclusion: The study highlights that analyzing virtual gene panels based on WES that include the majority of known genes causing AID or differential diagnosis can rapidly confirm the diagnosis for a large number of pediatric patients. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of minor use.

References: /.

Grants: /.

Conflict of Interest: None declared.

P08.013.A Transcriptomes of MPO-deficient patients with generalized pustular psoriasis reveals expansion of CD4+ cytotoxic T cells and an involvement of the complement system

Stefan Haskamp¹, Benjamin Frey², Ina Becker², Imke Atreya², Carola Berking², Rotraut Mößner³, Dagmar Wilsmann-Theis⁴, Steffen Uebe¹, Philipp Kirchner¹, Ulrike Hüffmeier ⁵

¹FAU Erlangen-Nürnberg, Erlangen, Germany; ¹FAU Erlangen-Nürnberg, Erlangen, Germany; ³University of Göttingen, Göttingen, Germany; ⁴University of Bonn, Bonn, Germany; ⁵FAU Erlangen-Nürnberg, Human Genetics, Erlangen, Germany

Background/Objectives: Generalized pustular psoriasis (GPP) is a severe psoriatic subtype characterized by epidermal neutrophil infiltration. Although variants in IL36RN and MPO have been shown to affect immune cells contributing to GPP’s pathogenesis, a systematic analysis of neutrophils and of a wide variety of PBMC subsets and their differential gene expression dependent on MPO genotypes was not performed yet.

Methods: We assessed transcriptomes of MPO-deficient patients using single cell RNA-sequencing (scRNAseq) of peripheral blood mononuclear cells (PBMCs) and RNA-sequencing of neutrophils in relatively stable disease state.

Results: Cell type annotation by multimodal reference mapping of scRNAseq data was verified by flow cytometry of surface and intracellular markers; proportions of CD4⁺ cytotoxic T-lymphocytes (CTLs) and other CD4⁺ effector cells were increased in GPP, while frequencies of naïve CD4⁺ T cells were significantly lower. The expression of the marker gene for CD4⁺ CTLs and CD8⁺ effector memory T-cells (TEMs) FGFBP2 was elevated in GPP patients with disease-contributing variants compared to healthy and diseased non-carriers (p = 0.0015) based on quantitative RT-PCR. Differentially expressed genes (DEGs) involved in the complement system were enriched in patients’ neutrophils.

Conclusion: Future studies assessing affected cell types and pathways will show their contribution to GPP’s pathogenesis, and indicate whether findings can be transferred to the situation in affected skin and whether depletion or inactivation of CD4⁺ CTLs may be a reasonable therapeutic approach.

References:.

Grants: CRC1181, project A05.

Conflict of Interest: None declared.

P08.014.B Filaggrin loss-of-function mutations are associated with the persistence of egg and milk allergy

Ingo Marenholz ^1;2, Birgit Kalb^2;3, Alexander CSN Jeanrenaud^1;2, Lara Meixner³, Ahla Ghauri^1;2, Aleix Arnau-Soler^1;2, Katharina Blümchen⁴, Rupert Schlags⁵, Gesine Hansen⁶, Jürgen Seidenberg⁷, Susanne Lau³, Bodo Niggemann³, Kirsten Beyer³, Young-Ae Lee^1;2

¹Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany; ²Clinic for Pediatric Allergy, Experimental and Clinical Research Center, Charité-Universitätsmedizin Berlin, Berlin, Germany; ³Department of Pediatric Respiratory Medicine, Immunology, and Intensive Care Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany; ⁴Department of Allergy, Pulmonology and Cystic Fibrosis, Children’s Hospital, Goethe University, Frankfurt, Germany; ⁵Department of Pediatric Pneumology and Allergology, Wangen Hospital, Wangen, Germany; ⁶Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; ⁷Department of Pediatric Pneumology and Allergology, Neonatology and Intensive Care, Medical Campus of University Oldenburg, Oldenburg, Germany

Background/Objectives: A genetic defect in the epidermal barrier protein filaggrin plays a major role in the etiology of eczema and associated allergic airways diseases. However, it is still controversial to what extend loss-of-function (LOF) mutations in the filaggrin gene (FLG) contribute to the development of food allergies. We tested association of FLG LOF mutations with allergic reactions to diverse foods and investigated their effect on the persistence of early food allergies.

Methods: We recruited 890 children with challenge-proven food allergy for the German Genetics of Food Allergy Study (GOFA). All children were genotyped for the four most common LOF mutations in FLG; R501X, 2282del4, R2447X, and S3247X. Associations between FLG mutations and food allergies were analyzed by logistic regression using the German Multicenter Allergy Study cohort as control population.

Results: FLG mutations were associated with allergies to diverse foods including hen’s egg (HE), cow’s milk (CM), peanut, hazelnut, fish, soy, cashew, walnut, and sesame with similar risk estimates. Effects remained significant after adjusting for the eczema status. Interestingly, FLG mutations increased the risk of a persistent course of HE and CM allergy.

Conclusion: Using the gold standard for food allergy diagnosis, we demonstrate that FLG LOF mutations confer risk of any food allergy independent of eczema. They predispose to the persistence of HE and CM allergy and should be considered in the assessment of tolerance development.

References:.

Grants: The study was funded in part by the Federal Ministry of Education and Research (CHAMP; 01GL1742C), and the German Research Foundation (Clinical Research Group 339 “Food@”; Project B2).

Conflict of Interest: None declared.

P08.015.C Ethnicity-specific GWAS to explore the genomic architecture of platelet count in the Northeast Indian subcontinent

Kate Burley ¹, Dragana Vuckovic², David van Heel³, Andrew Mumford⁴

¹University of Bristol, School of Physiology, Pharmacology and Neuroscience, Bristol, United Kingdom; ²Imperial College London, School of Public Health, London, United Kingdom; ³Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London, United Kingdom; ⁴University of Bristol, School of Cellular and Molecular Medicine, Bristol, United Kingdom

Background/Objectives: Blood cell traits are highly heritable, yet most known genomic associations have been identified in broad population sub-groups. We compared the genomic determinants of platelet count (PLT) in Pakistanis and Bangladeshis, a genetically distinct population from the Northeast Indian subcontinent where thrombocytopenia (PLT<150 x10⁹/L) is reported in 35%.¹.

Methods: Data were analysed from British Bangladeshi (BAN, n = 20,292) and British Pakistani (PAK, n = 9,237) individuals (https://www.genesandhealth.org/). Associations between TOPMed-imputed genotypes and mean PLT were calculated using BOLT-LMM and LD score regression software. Variants were annotated using the Ensembl Variant Effect Predictor.

Results: PLT was slightly lower in BAN (mean 266.4 x10⁹/L) than PAK (mean 271.5 x10⁹/L, p = 1.16e^-10) but respective rates of thrombocytopenia (1.5% vs 1.8%) and trait heritability (0.214 vs 0.216) were comparable. PLT was lower (mean 255.2 x10⁹/L) and had higher heritability (0.230) in a previously reported European (EUR) population (n = 166,066).² Genetic correlation for PLT was higher between BAN and PAK (r_g 1.11, SE 0.2) than for BAN and EUR (r_g 0.82, SE 0.07). GWAS-significant variants in BAN were in regions associated with PLT in EUR, except for a MAST2 intronic loci previously associated with PLT in East Asian populations.

Conclusion: Loci associated with PLT were concordant between BAN and PAK, and largely reproduced previous findings in EUR. Thrombocytopenia observed in the Northeast Indian subcontinent was not reproduced in the British BAN population in which there was also no discernible genomic explanation for lower PLT.

References: 1. Nania et al., J Thromb Haemost. 2005, 2, 2581-1582.

2. Astle et al., Cell 2016, 167, 1415-1429.

Grants:.

Conflict of Interest: None declared.

P08.016.D Italian ancestral HLA haplotype predisposing to severe COVID-19 by low spike and high IFNα binding affinity

Susanna Croci^1;2, Silvia Brugiapaglia^3;4, Ticiana D.J. Farias⁵, Paola Magistroni⁶, Kristina Zguro², Claudia Curcio^3;4, Chiara Fallerini^1;2, Francesca Fava^1;2;7, Francesco Pettini², Margherita Baldassarri^1;2, Mirella Bruttini ^1;2;7, Gen-Covid Multicenter Study⁷, Paul Norman⁵, Alessandra Renieri^1;2;7, Ilaria Meloni^1;2, Simone Furini², Antonio Amoroso^6;8, Franco Novelli^3;4;9

¹Medical Genetics, University of Siena, Siena, Italy; ²Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy; ³Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; ⁴Laboratory of Tumor Immunology Center for Experimental Research and Medical Studies, Città della Salute e della Scienza di Torino, Turin, Italy; ⁵Division of Biomedical Informatics and Personalized Medicine, Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, Aurora, Colorado, US, Aurora, United States; ⁶Immunogenetics and Transplant Biology, Azienda Ospedaliera Universitaria, Città della Salute e della Scienza di Torino, Turin, Italy; ⁷Genetica Medica, Azienda Ospedaliero-Universitaria Senese, Siena, Italy; ⁸Department of Medical Sciences, University of Turin, Italy, Turin, Italy; ⁹Molecular Biotechnology Center, University of Turin, Turin, Italy

Background/Objectives: Validated association between COVID-19 and the most obvious candidate genes, e.g. HLA, is still missing. A weak association with class I HLA- C*04:01 was found for infection in Sardinians and for severity in another mixed population. Auto-antibodies to interferon type I have been implicated in the severity of COVID-19 in two studies.

Methods: The binding affinity between HLA molecules and SARS-CoV-2 spike protein and IFNα subunits was evaluated in silico. The presence of antibodies against one or more of the 12 IFNα subunits was evaluated in 160 hospitalized COVID-19 patients. The 10 most frequent haplotypes in the Italian population were tested in 1.997 SARS-CoV-2 infected patients (hospitalized versus not hospitalized).

Results: The presence of auto-antibodies against at least one IFNα subunit was detected in 26% of patients. The haplotype A*24:02-B*35:02-C*04:01-DRB1*11:04-DQB1*03:01 was found to predispose to severity (p = 0.0018; p = 0.07 after Bonferroni correction) in patients <50 years. The haplotype includes alleles able to bind spike with low affinity (i.e. C*04:01 and DRB1*11:04) and IFNα with high affinity (i.e. DRB1*11:04).

Conclusion: One of the 10 most frequent ancestral haplotype of the Italian population predisposes to severity likely reducing both innate immunity through IFNα auto-antibodies induction and adaptive immunity through weaker spike protein presentation.

References: Daga S et al. Employing a systematic approach to biobanking and analyzing clinical and genetic data for advancing COVID-19 research. Eur J Hum Genet. 2021 Jan 17:1-15.

Grants: FISR 2020 / Tuscany Region COVID-19 / INTERVENE - GA No. 101016775 / Soka Gakkai PAT-COVID.

Conflict of Interest: None declared.

P08.017.A High prevalence of Netherton syndrome in Latvian population caused by founder SPINK5 variant

INGA NARTISA ^1;2, Lota Ozola³, Ineta Grantina³, Gita Taurina², Rasa Insberga², Linda Gailite¹, Natalja Kurjane^1;2

¹Riga Stradins University, Riga, Latvia; ²Children’s Clinical University Hospital Clinic of Medical Genetics and Prenatal Diagnostics, Riga, Latvia; ³Children’s Clinical University Hospital, Riga, Latvia

Background/Objectives: Netherton syndrome (NTS) is a rare autosomal recessive inborn error of immunity caused by biallelic SPINK5 loss-of-function variants. NTS is characterized by skin and immune system abnormalities, but with variable expressivity. Therefore, we aimed to clinically and molecularly characterize NTS in individuals with homogenous genotype and ethnicity.

Methods: We selected all Latvian NTS patients diagnosed in Children’s Clinical University Hospital by using exome sequencing (ES). Unrelatedness, as well as haplotype lengh for variant’s age estimation in individuals with homozygous pathogenic variant was analysed using high-quality variants from ES.

Results: We identified pathogenic biallelic variants in 9 individuals (age 1 month to 17 years) from 7 families, confirming NTS diagnosis. Clinically, all patients have ichthyosis, hair abnormalities, developmental delay, high total IgE levels (371-6977 kU/L), and recurrent skin and respiratory infections with variable severity. Surprisingly, 5/7 families had the same homozygous SPINK5 variant (NM_006846.4:c.1048C>T,p.(Arg350*)) and two siblings carried the same variant in compound heterozygous state (c.1048C>T,p.(Arg350*); c.1430+4A>G,r.spl.). Therefore, we suspected that the variant is founder in Latvian population. NTS prevalence in Latvian population is higher: 1:256,000 (vs. ~1:1,000,000 in France by Dreifus I. et. Al. 2014) and carrier frequency is estimated 1:300. Assuming a ‘correlated’ genealogy, the mutation arose 47.4(95%CI 14.9-167.2) generations and 1175 years (assuming one generation 25y) ago.

Conclusion: We report high prevalence of NTS in Latvia due to a common founder variant that arose ~50 generations ago. Interestingly, despite similar ethnical and identical genotype, NTS expressivity was variable among the individuals.

References:.

Grants: Latvian Council of Science project:lzp-2020/1-0269.

Conflict of Interest: None declared.

P08.018.B Microenvironmental determinants of clonal hematopoiesis expansion rate

Taralynn Mack ^1;2, Michael Raddatz³, Joshua Weinstock⁴, Jaiswal Siddhartha⁵, Alexander Bick²

¹Vanderbilt University Medical Center, Vanderbilt Genetics Institute, Nashville, United States; ²Vanderbilt University Medical Center, Division of Genetic Medicine, Nashville, United States; ³University of California, Department of Medicine, Los Angeles, United States; ⁴University of Michigan School of Public Health, Department of Biostatistics, Ann Arbor, United States; ⁵Stanford University School of Medicine, Department of Pathology, Stanford, United States

Background/Objectives: Clonal hematopoiesis of indeterminate potential (CHIP) is a clonal expansion of blood cells caused by somatic mutations. CHIP confers risk of multiple aging diseases including blood cancer and cardiovascular disease. It is presently unknown what causes CHIP clones with identical driver mutations to expand at different rates in humans. Here, we leverage genetically predicted traits to identify factors that modify CHIP clonal growth rate.

Methods: We used the passenger-approximated clonal expansion rate (PACER) method to quantify clonal growth rate for 4,370 individuals with CHIP mutations in the NHLBI TOPMed cohort. [1] We calculated polygenic risk scores (PRS) for DNA methylation clocks, inflammation related lab values, disease traits, and circulating protein levels. We tested for associations between PRS and PACER score with linear regression, controlling for covariates and correcting for multiple-hypothesis testing.

Results: CHIP clonal growth rate was significantly associated with both genetically predicted and measured methylation clocks (p < 0.01). No associations were identified with any of the inflammation-related lab values, proteins or diseases. An unbiased proteome wide search identified circulating levels of myeloid zinc finger 1, anti-müllerian hormone, TIMP metallopeptidase inhibitor 1 and glycine N-methyltransferase as altering CHIP clonal expansion rate (p < 4.1 x 10^-6).

Conclusion: An aging microenvironment was associated with increased CHIP expansion rate as well as four specific circulating micro-environment proteins. Contrary to prior murine models, we do not identify inflammation as a root cause of CHIP clonal expansion rate in humans.

References: [1] Weinstock (2021) bioRxiv https://doi.org/10.1101/2021.12.10.471810.

Grants: HBPRP T32 HL144446.

Conflict of Interest: Taralynn Mack: None declared, Michael Raddatz: None declared, Joshua Weinstock: None declared, Jaiswal Siddhartha TenSixteen Bio, TenSixteen Bio, Alexander Bick TenSixteen Bio, TenSixteen Bio.

P08.019.C Assessing new RNA-based biomarkers in multiple sclerosis

Giulia Cardamone^1;2, Federica Airi³, Giuseppe Liberatore⁴, Giulia Solda’^1;3, Valeria Rimoldi¹, Eduardo Nobile-Orazio^4;5, Rosanna Asselta ^1;3, Elvezia Maria Paraboschi^1;3

¹Humanitas University, Department of Biomedical Sciences, Pieve Emanuele, Italy; ²Institute of Biochemistry I, Goethe-University, Frankfurt, Germany; ³IRCCS Humanitas Research Hospital, Rozzano, Italy; ⁴Neuromuscular and Neuroimmunology Service, IRCCS Humanitas Clinical and Research Center, Rozzano, Italy; ⁵Milan University, Department of Medical Biotechnology and Translational Medicine, Milano, Italy

Background/Objectives: Multiple sclerosis (MS) is an autoimmune neurodegenerative disease characterized by chronic inflammation, and demyelination. Accumulating evidence suggests a pathogenic link between MS and abnormalities in alternative splicing (AS) [1], a process that increases the information content of the transcriptome through the expression of different mRNAs from single genes [2]. Our objective is to define an AS-based signature able to discriminate patients from controls, starting from a blood sample.

Methods: RNA was extracted from peripheral blood mononuclear cells of 29 relapsing-remitting MS patients and 20 controls. Fluorescent RT-PCR assays, focusing on three specific AS events were performed, and PCR products were separated using capillary electrophoresis. The relative exon inclusion level (percent-spliced in, psi) was calculated for each sample.

Results: For each AS event, we divided the psi range in quantiles and we assigned each sample to its relevant class. We calculated a risk score for each sample, resulting from the sum of the quantiles they fit in in each AS assay. We used this score to perform a ROC analysis. Using this 3-marker-AS signature, we obtained a good predictive model (AUC = 0.72, specificity = 0.9, sensitivity = 0.48). We are testing the predictive value of our model on an independent cohort (32 MS cases and 50 matched controls).

Conclusion: Considering that the current diagnostic methods for MS are based on complicated procedures, the implementation of a diagnostic test, centered on RNA biomarkers use, would be useful in a clinical perspective.

References: 1. https://doi.org/10.1016/j.autrev.2019.05.010.

2. https://doi.org/10.1146/annurev.biochem.72.121801.161720.

Grants: This work is supported by Fondazione Regionale per la Ricerca Biomedica (FRRB), Early Career Award.

Conflict of Interest: None declared.

P08.020.D Immunodeficiency 68 - a life-threatning primary immunodeficiency

Joana Azevedo Silva¹, Rosário Leite ¹, Osvaldo Moutinho¹, Márcia Martins¹

¹Centro Hospitalar de Trás-os-Montes e Alto Douro, Vila Real, Portugal

Background/Objectives: MYD88 deficiency is an inherited disease of the immune system that leads to abnormally frequent severe infections by pyogenic bacteria such as Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa that can develop into sepsis and meningitis. Infections can be fatal in infancy and childhood and become less frequent around the age of 10.

Diagnostic and genetic counselling.

Methods: Case Report: First male child of a healthy nonconsanguineous couple, with congenital oculocutaneous hypopigmentation, nystagmus and ophthalmoscopy of the left eye compatible with partial oculocutaneous albinism. WES revealed a heterozygous mutation in TYR gene. At ten months, he had an inguinal abscess due to Escherichia Coli, with blood culture positive to Campylobacter. Normal Immunodeficiency investigation. At fourteen months, the child presented a periorbital cellulitis and, at 21 months, a right thumb recurrent cellulitis to Methicillin-susceptible Staphylococcus aureus. He died at 29 months from fulminant meningitis due to Pseudomonas aeruginosa.

Results: An WES trio analysis was performed and revealed ac.625C>T(Arg209Cys) in the MYD88 gene in homozygosity.

Conclusion: The MYD88 gene encodes a cytosolic adapter protein that plays a central role in the innate and adaptive immune response that interferes in the activation of numerous proinflammatory genes. The phenotypic characteristics observed in the present case are consistent with those described in the literature, with a severe meningitis responsible for the child’s death. This condition has an autosomal recessive pattern and both parents are heterozygous, with no signs or symptoms of this disease. To our knowledge, there are less than 30 affected individuals described in the literature.

References:.

Grants:.

Conflict of Interest: None declared.

P08.021.A Differential diagnostic algorithm for Inherited Macrothrombocytopenia enables the genetic characterization of 52 families in a single tertiary referral centre

Catarina Monteiro ^1;2;3;4, ana gonçalves^1;3;4, mónica pereira^2;3;4, catarina lau^3;4;5, marta gonçalves^3;4;5, eugenia cruz², sara morais^2;3;4, Rosário Santos^1;3;4

¹Centro de Genética Médica Jacinto Magalhães (CGMJM), Centro Hospitalar Universitário do Porto (CHUPorto), Unidade de Genética Molecular, Porto, Portugal; ²Serviço de Hematologia Clínica, Centro Hospitalar Universitário do Porto (CHUPorto), Unidade de Trombose e Hemostase, Porto, Portugal; ³UMIB - Unidade Multidisciplinar de Investigação Biomédica, ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; ⁴ITR - Laboratory for Integrative and Translational Research in Population Health, Porto, Portugal; ⁵Serviço de Hematologia Clínica, Centro Hospitalar Universitário do Porto (CHUPorto), Unidade de Diagnóstico Hematológico Margarida Lima, porto, Portugal

Background/Objectives: Inherited Macrothrombocytopenias (IMTP) are platelet (PLT) disorders characterized by reduced PLT counts, increased PLT volume and often disproportionate bleeding, with up to 30 genes currently implicated. We aimed to develop an efficient diagnostic algorithm for IMTP, contributing towards the understanding of these disorders and their underlying defects in our population.

Methods: The proposed algorithm comprises three assessment steps: (i) clinical data (personal/family history, physical examination, bleeding tendency); (ii) preliminary laboratory tests including PLT counts and indices [mean PLT volume (MPV) and immature PLT fraction (IPF)], PLT morphology and function assays (occlusion time by PFA100/200 and lumi-aggregometry); (iii) flow cytometry to assess surface glycoproteins and PLT size (forward scatter indices). PLT phenotypic patterns orientate towards candidate gene(s); in the absence of a suspected underlying cause, targeted next-generation sequencing (NGS) is used.

Results: The algorithm distinguished patient subgroups, guiding appropriate molecular approaches. The genetic cause was identified in 37 families by direct Sanger sequencing and in 15 families by NGS. Differential diagnosis achieved was: 4 biallelic Bernard-Soulier syndrome (BSS), 5 monoallelic BSS, 1 Platelet-type vWD, 13 ITGB3/ITGA2B-related thrombocytopenia (-RT), 9 ACTN1-RT, 9 TUBB1-RT, 3 GFI1B-RT, 7 MYH9-related disease (-RD) and 1 DIAPH1-RD.

Conclusion: Knowledge emerging from new genotype-phenotype correlations, particularly by reassessment of PLT function and phenotype in NGS-diagnosed cases, will be incorporated into the algorithm for further refining. Genetic diagnosis is essential for prognosis and preventive treatments, to flag syndromic entities with probable multi-organ involvement, as well as for determining carrier status.

References:.

Grants: FCT grants to CM (UI/BD/150741/2020), UMIB and ITR (UIDB/00215/2020; UIDP/00215/2020; LA/P/0064/2020).

Conflict of Interest: None declared.

P08.022.B Making the invisible visible: Understanding what’s helpful in learning to live with a suspected Inborn Error of Immunity

Madison Mixer ¹, Rylee Duncan¹, Michael Setzer¹, Morgan Similuk¹

¹National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, United States

Background/Objectives: Prior research has established the potential for negative quality-of-life consequences among individuals with suspected Inborn Errors of Immunity (IEI). Relatively underrepresented in this literature, however, are qualitative methods that explore patient perceptions of their challenges and opportunities. This study aims to interrogate psychosocial impacts of having an IEI to better understand how patients adapt to their condition.

Methods: A survey including open-ended questions was sent to patients enrolled on the Centralized Sequencing Protocol and were suspected of having an IEI. Here, we present responses to the question: What has been most helpful in learning to live with your illness? And why?.

Results: Out of 1038 invited participants, 252 (24% response rate) responses were coded and analyzed thematically (59% female, mean age = 48 years). Many patients reported that they benefit from competent providers with a nuanced understanding of their disease and who provide effective treatments. Patients also reported seeking support in coping from family, friends, and the disease community. Many also reported the benefit of lifestyle changes and reconceptualizing their illness to gain a sense of control. An important minority of individuals indicated they had not found anything helpful in living with their illness.

Conclusion: Beyond encouraging further biomedical research into new disease associations and therapies, patient responses suggest healthcare providers can promote adaptation by encouraging patients to play an active role in their treatment and connecting them to support systems and psychological interventions. Patients who claim no adaptation can benefit from practices to better identify their challenges and corresponding interventions.

References: None.

Grants: None.

Conflict of Interest: None declared.

P08.023.C Mutation spectrum of Fanconi anemia associated genes in five patients from Azerbaijan

AGHARZA AGHAYEV ^1;2, Valeh Hüseynov¹, Güven Toksoy², Samira Hasanova¹, Güler Mammadova¹, Zehra Oya Uyguner²

¹National Hematology and Transfusiology Center, Department of Medical Genetics, Baku, Azerbaijan; ²Istanbul Medical Faculty, Istanbul University, Department of Medical Genetics, Istanbul, Turkey

Background/Objectives: Fanconi anemia (FA) is a rare genetic disorder caused mutations in genes which protein products are involved in replication, cell cycle control and DNA repair. FA proteins are required for the proper repair of DNA interstrand crosslinks (ICL), a deleterious type of DNA damage that covalently binds DNA strands. FA is characterized by congenital malformations, bone marrow failure, and predisposition to malignancies. Presently, 22 autosomal and one X linked genes are held responsible from >85% of the disease and the FANCA mutations accounts for almost 60%-70%.

Methods: 5 patients, with FA clinic are included into this investigation. We performed exome sequencing and copy number variant analyses and detected variants considered to be pathogenic are verified by Sanger. Mutation un-identified patients and patients carrying heterozygous pathogenic variants are further tested by MLPA.

Results: Three known variants in four alleles (c.[2679G>A];[2679G>A], c.[1343A>G];[2495_2497delTCT], and two novel variants in four alleles (c.[495delC];[495delC], c.[2941T>C];[2941T>C] in FANCA, and a novel variant in two alleles (c.[283_284delCT];[283_284delCT]) in FANCF are identified.

Conclusion: This is the first study looking at clinical and genetic features of FA in Azerbaijan. We aimed to identify the mutation frequencies of FA genes, outcome, overall condition, and genetic features of patients in Azerbaijan to optimize management, identify the most common genes, describe new mutations, and offer prenatal diagnosis and counseling to the affected families.

References: Toksoy, Güven, et al. “Clinical and Molecular Characterization of Fanconi Anemia Patients in Turkey.” Molecular syndromology 11.4 (2020):183-196.

Grants:.

Conflict of Interest: None declared.

P08.024.D Variant of CXCL8 gene associated with IBD pathogenesis?

Oliwia Zakerska-Banaszak ¹, Marzena Skrzypczak-Zielinska¹, Joanna Zuraszek¹, Aleksandra Zielińska¹, Ryszard Słomski¹

¹Institute of Human Genetics PAS, Poznan, Poland

Background/Objectives: Inflammatory bowel diseases (IBD) is a group of chronic pathologies of the digestive system with not well known etiology. Interleukin 8 encoded by the CXCL8 gene is a cytokine that is a chemotactic factor responsible for the targeted migration of leukocytes to the site of inflammation. Its pro-inflammatory properties suggest that it plays a key role in the development of the inflammatory process. The aim of this analysis was to examine the two variants rs4073 (c.-251A>T) and rs188378669 (c.91G>T, p.Glu31Ter) in CXCL8 gene distribution in a group of IBD patients and populations.

Methods: Using pyrosequencing, Competitive Allele-Specific PCR (CASP) and Sanger sequencing, the two variants, rs4073 (c.-251A>T) and rs188378669 (c.91G>T, p.Glu31Ter) of the CXCL8 gene were analyzed in a group of 353 IBD patients and 200 subjects from Polish population in order to check if there is any correlations of them with a disease.

Results: Polymorphism c.91G>T, located in exon 2 of CXCL8 gene, causing premature termination of the polypeptide chain and consequently forming a non-functional protein was observed significantly more often in a group of IBD patients (MAF of 2.12%) compared to population group (MAF of 0.25%) p = 0,012, OR = 8,661, C.I.=[1,140-65,816].

Conclusion: This is the first report concerning the correlation of IBD presence and p.Glu31Ter variant in CXCL8 gene, what suggest the role of CXCL8 gene in IBD pathogenesis.

References: Dakal TC et al. Predicting the functional consequences of non-synonymous single nucleotide polymorphisms in IL8 gene. Sci Rep 2017.

McGovern DP et al. Genetics of Inflammatory Bowel Diseases. Gastroenterology 2015.

Grants:-.

Conflict of Interest: None declared.

P09 Intellectual Disability

P09.001.A Parental mosaicism is underestimated in rare intellectual disability syndromes

Sofia Frisk ^1;2, Alexandra Wachtmeister¹, Tobias Laurell^1;3, Anna Lindstrand^1;2, Nina Jäntti^1;2, Helena Malmgren^1;2, Kristina Lagerstedt-Robinson^1;2, Bianca Tesi^1;2, Fulya Taylan¹, Ann Nordgren^1;2

¹Karolinska Institutet, Molecular Medicine and Surgery, Stockholm, Sweden; ²Karolinska University Hospital, Clinical Genetics, Solna, Sweden; ³Södersjukhuset, Hand surgery, Stockholm, Sweden

Background/Objectives: De novo variants are a common cause to rare intellectual disability syndromes, associated with low recurrence risk. However, when such variants occur pre-zygotically in parental germ cells, the recurrence risk might be higher. Still, the recurrence risk estimates are mainly based on empirical data and the prevalence of germline mosaicism is often unknown.

Methods: To establish the prevalence of mosaicism in parents of children with intellectual disability syndromes caused by de novo variants, we performed droplet digital PCR on DNA extracted from blood (43 trios), and sperm (31 fathers).

Results: We detected low-level mosaicism in sperm-derived DNA but not in blood in the father of a child with Kleefstra syndrome caused by an EHMT1 variant. Additionally, we found a higher level of paternal mosaicism in sperm compared to blood in the father of a child with Gillespie syndrome caused by an ITPR1 variant.

Conclusion: By employing droplet digital PCR, we detected paternal germline mosaicism in two intellectual disability syndromes. In both cases, the mosaicism level was higher in sperm than blood, indicating that analysis of blood alone may underestimate germline mosaicism. Therefore, sperm analysis can be clinically useful to establish the recurrence risk for parents and improve genetic counselling.

References:.

Grants: This work was supported by grants from the Swedish Research Council, the Region Stockholm (combined residency and PhD training program), Karolinska Institutet, the Swedish Brain Foundation, the Swedish Rare Diseases Research Foundation (Sällsyntafonden) and The Hållsten Research Foundation.

Conflict of Interest: None declared.

P09.003.C A novel splice site mutation in the CNOT3 gene is associated with syndromic intellectual disability with clinical variability

Lucia Bruno ^1;2, Flavia Privitera^3;4, Caterina Lo Rizzo⁵, Michele Carullo^4;5, Kristina Zguro⁴, Simone Furini⁴, Alessandra Renieri^1;4;5, Francesca Ariani^1;4;5

¹Medical Genetics, University of Siena, Siena, Italy, Siena, Italy; ²Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy, Siena, Italy; ³Medical Genetics, University of Siena, Siena, Italy, Siena, Italy; ⁴Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy, Siena, Italy; ⁵Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy, Siena, Italy

Background/Objectives: Variants in CNOT3 have been associated with intellectual developmental disorder with speech delay, autism, and dysmorphic features (IDDSADF). To date, 22 mutated patients are known, and the phenotype is poorly described.

Methods: Via Exome sequencing, we detected the first splice site mutation) in intron 2 in CNOT3 (c.26-2A>G). In silico tools revealed a damaging effect on the splicing due to an alteration on the canonical splice site at 3’end, no longer recognized. Analysis of the transcript confirmed its damaging effect, consisting of intron 2 retention and of an in-frame duplication of exons 1 and 2.

Results: This is the third family with an inherited mutation in CNOT3, associated with a condition displaying intrafamilial variability. In fact, the proband showed intellectual disability (ID), language delay, structural cerebral anomalies, cardiac defects, physical dysmorphisms, whereas his mother manifested a mild ID and shared her son’s dysmorphisms.The mutation was related to anorectal dysplasia, firstly detected in CNOT3-patients.

Conclusion: The rectum is generated from the differentiation of the endoderm in the primordial gastrointestinal tract during the 4th week of the fetal life. CNOT3 takes part in the mesendoderm differentiation, which originates mesoderm and endoderm, suggesting a connection between its alterations and rectal atresia.This study contributes to define the CNOT3 phenotypic spectrum and highlights its clinical variability.

References: R. Meyer, et al., Inherited cases of CNOT3-associated intellectual developmental disorder with speech delay, autism, and dysmorphic facies, Clinical Genetics 2020.

Martin R, et al., De novo variants in CNOT3 cause a variable neurodevelopmental disorder. Eur J Hum Genet. 2019.

Grants: None.

Conflict of Interest: None declared.

P09.004.D Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein

Elke de Boer ^1;2, Charlotte Ockeloen¹, Rosalie A. Kampen³, Juliet Hampstead^1;4, Alexander Dingemans^1;2, Dmitrijs Rots^1;2, Tazeen Ashraf^5;6, Rachel Baker⁷, Mouna Barat-Houari⁸, Brad Angle⁷, Nicolas Chatron^9;10, Anne-Sophie Denommé-Pichon^11;12, Orrin Devinsky¹³, Christele Dubourg^14;15, Frances Elmslie¹⁶, Kristin Monaghan¹⁷, Houda Elloumi¹⁷, Laurence Faivre^11;18;19, Sara Fitzgerald-Butt²⁰, David Geneviève²¹, Jacqueline Goos²², Benjamin Helm^20;23, Usha Kini²⁴, Amaia Lasa-Aranzasti²⁵, Gaetan Lesca^9;10, sally ann Lynch²⁶, Irene Mathijssen²², Ruth McGowan²⁷, Sylvie Odent²⁸, Rolph Pfundt¹, Audrey Putoux^9;29, Jeroen van Reeuwijk^1;2, Gijs Santen³⁰, Erina Sasaki²⁴, Arthur Sorlin^11;18, Peter van der Spek²², Alexander Stegmann³¹, Sigrid Swagemakers²², Irene Valenzuela²⁵, Eléonore Viora-Dupont¹⁸, Antonio Vitobello^11;12, Stephanie Ware^20;32, Mathys Weber¹⁸, Christian Gilissen^1;4, Karen Low³³, Simon Fisher^2;3, Lisenka Vissers*^1;2, Maggie Wong³, Tjitske Kleefstra^1;2;34

¹Radboudumc, Human Genetics, Nijmegen, Netherlands; ²Donders Institute, NIjmegen, Netherlands; ³MPI for Psycholinguistics, Nijmegen, Netherlands; ⁴4. RIMLS, Nijmegen, Netherlands; ⁵Great Ormond Str Hospital for Children NHS, London, United Kingdom; ⁶Guys and St Thomas’ NHS, London, United Kingdom; ⁷Advocate Children’s Hospital, Park Ridge, United States; ⁸CHU Montpellier, Montpellier, France; ⁹Hospices Civils de Lyon, Lyon, France; ¹⁰Institut NeuroMyoGene, Lyon, France; ¹¹Université de Bourgogne Franche-Comté, Dijon, France; ¹²Laboratoire de Génétique chromosomique et moléculaire, Dijon, France; ¹³NYU Grossman School of Medicine, New York, United States; ¹⁴Service de Génétique Moléculaire et Génomique, Rennes, France; ¹⁵University of Rennes, Rennes, France; ¹⁶St George’s, London, United Kingdom; ¹⁷GeneDx, Gaithersburg, United States; ¹⁸Centre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l’Interrégion Est, Dijon, France; ¹⁹Fédération Hospitalo-Universitaire Médecine TRANSLAD, Dijon, France; ²⁰Indiana University, Indianapolis, United States; ²¹Montpellier University, Montpellier, France; ²²Erasmus MC, Rotterdam, Netherlands; ²³Indiana University Fairbanks School of Public Health, Indianapolis, United States; ²⁴Oxford University Hospitals NHS, Oxford, United Kingdom; ²⁵Vall d’Hebron, Barcelona, Spain; ²⁶Children’s Health, Dublin, Ireland; ²⁷Queen Elizabeth University Hospital, Glasgow, United Kingdom; ²⁸CHU Rennes, Rennes, France; ²⁹Centre de Recherche en Neurosciences de Lyon, Lyon, France; ³⁰Leiden UMC, Leiden, Netherlands; ³¹Maastricht UMC+, azM, Maastricht, Netherlands; ³²Indiana University School of Medicine, Indianapolis, United States; ³³University Hospital Bristol, Weston NHS, Bristol, United Kingdom; ³⁴Vincent van Gogh Institute, Venray, Netherlands

Background/Objectives: Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders [1], the role of rare ANKRD11 missense variation remains unclear. We characterized the clinical, molecular and functional spectra of ANKRD11 missense variants.

Methods: We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants by in silico analyses and cell-based experiments.

Results: We identified 29 individuals with (mostly de novo) ANKRD11 missense variants, who presented with syndromic neurodevelopmental disorders and were phenotypically similar to individuals with KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in Repression Domain 2. Cellularly, most variants caused reduced ANKRD11 stability. One variant resulted in decreased proteasome degradation and loss of ANKRD11 transcriptional activity.

Conclusion: Our study indicates that pathogenic heterozygous missense variants in ANKRD11 cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges, as the KBG-associated phenotype may be mild, and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.

References: 1. Ockeloen, C.W., et al., Further delineation of the KBG syndrome caused by ANKRD11 aberrations. Eur J Hum Genet, 2015. 23(9): p. 1270.

Grants: Aspasia grants, Dutch Research Council (015.014.036, 015.014.066), Netherlands Organization for Health Research and Development (91718310), the Max Planck Society, Solve-RD project (Horizon 2020, No 779257).

Conflict of Interest: Elke de Boer: None declared, Charlotte Ockeloen: None declared, Rosalie A. Kampen: None declared, Juliet Hampstead: None declared, Alexander Dingemans: None declared, Dmitrijs Rots: None declared, Tazeen Ashraf: None declared, Rachel Baker: None declared, Mouna Barat-Houari: None declared, Brad Angle: None declared, Nicolas Chatron: None declared, Anne-Sophie Denommé-Pichon: None declared, Orrin Devinsky: None declared, Christele Dubourg: None declared, Frances Elmslie: None declared, Kristin Monaghan This co-author is an employee of GeneDx, Inc, Houda Elloumi This co-author is an employee of GeneDx, Inc, Laurence Faivre: None declared, Sara Fitzgerald-Butt: None declared, David Geneviève: None declared, Jacqueline Goos: None declared, Benjamin Helm: None declared, Usha Kini: None declared, Amaia Lasa-Aranzasti: None declared, Gaetan Lesca: None declared, sally ann Lynch: None declared, Irene Mathijssen: None declared, Ruth McGowan For this centre, the WGS data were generated in the Scottish Genomes Partnership. The Scottish Genomes Partnership was funded by the Chief Scientist Office of the Scottish Government Health Directorates [SGP/1] and The Medical Research Council Whole Genome Sequencing for Health and Wealth Initiative (MC/PC/15080)., Sylvie Odent: None declared, Rolph Pfundt: None declared, Audrey Putoux: None declared, Jeroen van Reeuwijk: None declared, Gijs Santen: None declared, Erina Sasaki: None declared, Arthur Sorlin: None declared, Peter van der Spek: None declared, Alexander Stegmann: None declared, Sigrid Swagemakers: None declared, Irene Valenzuela: None declared, Eléonore Viora-Dupont: None declared, Antonio Vitobello: None declared, Stephanie Ware: None declared, Mathys Weber: None declared, Christian Gilissen: None declared, Karen Low: None declared, Simon Fisher This work was financially supported the Max Planck Society, Lisenka Vissers* This work was financially supported by Aspasia grants of the Dutch Research Council (015.014.066). In addition, the collaborations in this study were facilitated by ERN ITHACA, one of the 24 European Reference Networks (ERNs) approved by the ERN Board of Member States, co-funded by European Commission. The aims of this study contribute to the Solve-RD project which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257., Maggie Wong This work was financially supported by the Max Planck Society, Tjitske Kleefstra This work was financially supported by Aspasia grants of the Dutch Research Council (015.014.036) and the Netherlands Organization for Health Research and Development (91718310) In addition, the collaborations in this study were facilitated by ERN ITHACA, one of the 24 European Reference Networks (ERNs) approved by the ERN Board of Member States, co-funded by European Commission. The aims of this study contribute to the Solve-RD project which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257.

P09.005.A A novel pathogenic FMR1 splice site variant in a man with normal intelligence and Klinefelter syndrome - A case report

Malou Schadeck ¹, Nicole Meier¹, Eva Wohlleber¹, Juergen Kohlhase¹

¹SYNLAB Center for Human Genetics, Freiburg, Germany

Background/Objectives: We describe a 30-year-old man referred to genetic counselling regarding a possible Marfan syndrome. He presented with marfanoid features such as tall stature, an aortic root enlargement, and a systemic score of 7 points.

Methods: We performed whole exome sequencing based on the marfanoid phenotype including HPO-terms for cardiac defects.

Results: No variants associated with Marfan syndrome were detected. The exome based CNV (copy number variation) analysis revealed a duplication of all X-chromosomal genes, strongly suggesting a Klinefelter syndrome (KS) (karyotype 47,XXY), that does not fully explain the patient’s phenotype. Exome analysis detected a novel splice-site variant c.1738-1G>A, p.? in the FMR1 gene. The variant has not been described previously. Splice-site in silico prediction tools (SSF, MaxEnt, NNSplice and GeneSplicer) indicated a disrupted splice acceptor site of intron 16.

KS is a sex-chromosomal disorder with a frequency of ca. 1:500 men. Phenotypic features can include tall stature, gynecomastia and often azoospermia.

In more than 99% of patients diagnosed with a Fragile-X-syndrome the mutational mechanism is a CGG-trinucleotid repeat-expansion in FMR1, whereas other types of FMR1 mutations, affecting the coding region or splicing of FMR1, account for less than 1% of cases. Males previously described to carry FMR1 splice mutations had moderate to severe mental retardation.

Conclusion: We describe a patient with normal intelligence despite a pathogenic splice variant in FMR1, probably due to a skewed X-inactivation because of his Klinefelter syndrome.

References: 1. Intragenic FMR1 disease-causing variants: a significant mutational mechanism leading to Fragile-X syndrome, Quartier et al. (2017).

Grants:.

Conflict of Interest: None declared.

P09.006.B Deep phenotyping of patients with developmental disorders and their relatives to support interpretation of rare inherited copy number variants

Elise Pelgrims ¹, Laurens Hannes^1;2, Marta Atzori¹, Catia Attanasio¹, Jeroen Breckpot^1;2, Ann Swillen^1;2

¹KU Leuven, Department of human genetics, Leuven, Belgium; ²UZ Leuven, Centre for human genetics, Leuven, Belgium

Background/Objectives: In patients with developmental disorders (DD), copy number variants (CNV) inherited from seemingly unaffected parents are typically disregarded in variant interpretation. However, understanding the phenotypic contribution of inherited rare variants is crucial for genetic counselling. Therefore, an extended family-oriented approach is proposed, including deep familial phenotyping and multi-omics in carriers and non-carriers.

Methods: Deep phenotyping (including medical, developmental and behavioral, using standardized instruments) of carriers and non-carriers within the nuclear family, enables familial segregation analysis of a CNV with DD (sub)phenotypes. Trio whole genome sequencing is used to identify additional pathogenic variants causing or contributing to the phenotype. RNA and capture Hi-C sequencing on EBV cell lines is used to examine the regulatory effect of the CNV. This combined approach was applied to interpret a rare paternally inherited deletion ([GRCh37]4:141693186-142147039x1) in a 15-year-old boy with moderate intellectual disability, ASD, DCD, hypotonia and facial dysmorphic features.

Results: CNV carriers (FSIQ; index 40, father 71) scored significantly lower on cognitive abilities compared to non-carriers (FSIQ; mother 100, sibling 92). Behavioral profiles of carriers were more overlapping than profiles of non-carriers. De novo, recessive, X-linked and paternally inherited variant analyses of SNV, indels and additional CNVs were negative. RNA-seq shows four differentially expressed genes in the CNV or flanking regions (INPP4B, SETD7, MAML3, ZNF330). Their contribution to DD is subject to further study.

Conclusion: This multi-omics approach and correlation through deep familial phenotyping is suggestive of a contributory effect of this CNV to DD in this family.

References:.

Grants: This work is supported by a KULeuven grant (C24M/19/075).

Conflict of Interest: None declared.

P09.007.C Establishing the neurodevelopmental phenotype and genotype-phenotype correlations in individuals with a TRIP12 mutation

Mio Aerden ¹, Anne-Sophie Denommé-Pichon², Saskia Koene³, Amelie Piton⁴, Eric Legius¹, Miel Theunis¹, Tzung-Chien Hsieh⁵, Peter Krawitz⁵, Mala Misra-Isrie⁶, Sandra Whalen⁷, Dagmar Wieczorek⁸, Theresia Herget⁹, Guillaume Debray¹⁰, Nuria Brämswig¹¹, Vanesa López González¹², Guillaume Jouret¹³, Berta Almoguera Castillo¹⁴, Maria Isis Atallah Gonzalez¹⁵, Maria Kibaek¹⁶, Lucia Bruno¹⁷, Katrin Ounap¹⁸, Fernando Santos-Simarro¹⁹, Hilde Van Esch¹

¹University Hospitals Leuven, KU Leuven, Department of Human Genetics, Leuven, Belgium; ²Université de Bourgogne, INSERM UMR1231 Equipe GAD, Dijon, France; ³Leiden University Medical Center, Department of Clinical Genetics, Leiden, Netherlands; ⁴Hôpitaux Universitaires de Strasbourg, Laboratoire de Diagnostic Génétique, Strasbourg, France; ⁵University of Bonn, Institute of Genomic Statistics and Bioinformatics, Bonn, Germany; ⁶Amsterdam University Medical Center, Department of Clinical Genetics, Amsterdam, Netherlands; ⁷UF de Génétique Clinique, Centre de Référence des Anomalies du Développement et Syndromes Malformatifs, APHP, Hôpital Armand Trousseau, Paris, France; ⁸Heinrich-Heine-Universität, Institut für Humangenetik, Düsseldorf, Germany; ⁹Universitätsklinikum Hamburg-Eppendorf, Institut für Humangenetik, Hamburg, Germany; ¹⁰Centre Hospitalier Universitaire de Liège, Center of Human Genetics, Liège, Belgium; ¹¹Universität Duisburg-Essen, Institut für Humangenetik, Essen, Germany; ¹²Hospital Clinico Universitario Virgen de la Arrixaca, Sección de Genética Médica, Servicio de Pediatria, Murcia, Spain; ¹³Laboratoire National de Santé, National Center of Genetics, Dudelange, Luxembourg; ¹⁴Fundación Jiménez Díaz Hospital, Department of Genetics and Genomics, Madrid, Spain; ¹⁵Lausanne University Hospital, Division of Genetic Medicine, Lausanne, Switzerland; ¹⁶Odense University Hospital, Department of Clinical Genetics, Odense, Denmark; ¹⁷University of Siena, Medical Genetics Unit, Siena, Italy; ¹⁸Tartu University Hospital, Department of Clinical Genetics, Tartu, Estonia; ¹⁹Hospital Universitario La Paz, Instituto de Genética Médica y Molecular, Madrid, Spain

Background/Objectives: Haploinsufficiency of TRIP12 causes a neurodevelopmental disorder characterized by intellectual disability associated with epilepsy, autism and dysmorphism, also named Clark-Baraitser syndrome. Less than 25 individuals harboring pathogenic TRIP12 variants have been reported. We aim to further delineate the TRIP12-associated phenotype and review genotype-phenotype correlations. In addition, characteristic facial traits are objectified through image analysis based on deep-learning algorithms.

Methods: 37 individuals were recruited through a collaborative call via ERN-ITHACA. Clinical data was collected and the pictures of 21 individuals were uploaded into the GestaltMatcher database for analysis of facial morphology.

Results: One inherited and 35 de novo TRIP12 variants were identified, including frameshift (n = 16), nonsense (n = 6), missense (n = 5) and splice (n = 3) variants as well as intragenic deletions (n = 5) and a multigene deletion disrupting TRIP12.

Though variable in severity, global developmental delay was noted in all individuals, with language deficit most pronounced. Half of the individuals showed autistic features, but there was no clear correlation with the mutation type. Susceptibility to obesity seemed to be a recurrent feature in older individuals. Seizures were reported in a minority and proved to be refractory in individuals with a missense variant.

Facial analysis shows a clear gestalt including deep-set eyes with narrow palpebral fissures, downturned corners of the mouth and large, low-set ears with prominent earlobes.

Conclusion: We report the largest cohort to date of individuals with pathogenic TRIP12 variants, further delineating the associated phenotype including introduction of a facial gestalt and expanding genotype-phenotype correlations. These findings will improve future counseling and patient guidance.

References: /.

Grants: /.

Conflict of Interest: None declared.

P09.008.D The clinical benefit of trio-based whole-exome sequencing for the detection of rare pathogenic sequence variants in paediatric patients with undiagnosed neurodevelopmental disorders

Marketa Wayhelova ^1;2, Vladimira Vallova^1;2, Jan Smetana², Petr Broz², Aneta Mikulasova³, Renata Gaillyova⁴, Petr Kuglik^1;2

¹University Hospital Brno, Centre of Molecular Biology and Genetics, Brno, Czech Republic; ²Masaryk University, Department of Experimental Biology, Faculty of Science, Brno, Czech Republic; ³Newcastle University, Biosciences Institute, Newcastle upon Tyne, United Kingdom; ⁴University Hospital Brno, Department of Medical Genetics and Genomics, Brno, Czech Republic

Background/Objectives: Thanks to more than 50% diagnostic yield the whole-exome sequencing (WES) has become an effective and powerful approach to identify molecular genetic causes of neurodevelopmental disorders (NDDs) and multiple congenital abnormalities (MCA).

Methods: We present our experience with WES as an effective tool for the detection of rare and novel pathogenic sequence variant using the commercial kit Human Core Exome (Twist Biosciences) and Illumina NovaSeq 6000. Our pilot study included 45 families (trios or quatros) of children with severe NDDs and MCA.

Results: Using our in-house bioinformatic pipeline and unique algorithm for variant prioritization we identified recurrent de novo pathogenic sequence variants in clinically relevant SHANK3, GRIN1, NSD1, CDK13 genes, novel de novo pathogenic variants in KDM1A, KMT2E, GNAI1, MEIS2, SMARCA2, RAI1 and CHD8 genes, pathogenic X-linked variants in EDA and OPHN1 genes of maternal origin, pathogenic sequence variants in ZGRF1 of paternal origin and biallelic pathogenic sequence variants in the BLM gene. Moreover, two pathogenic sequence variants in the CTNNB1 and DYNC1H genes were present as low-level mosaicism in healthy fathers. All clinically important variants including secondary findings (in ”ACMG“ genes) were manually verified using Sanger sequencing and interpreted based on relevant information in integrated databases of genomic variants, relevant scientific literature, and individual anamnesis.

Conclusion: With an achieved diagnostic yield of 37.5% (18/48 children with NDDs and MCA), trio-based WES represents as an effective first-tier diagnostic test in the genetic evaluation of children with NDDs.

References:.

Grants: Supported by Ministry of Health of the Czech Republic, grant nr. NU20-07-00145. All rights reserved.

Conflict of Interest: None declared.

P09.009.A RAB11B-associated neurodevelopmental disorder - confirmed and extended to milder manifestations through 13 individuals with 5 novel variants

Natalie Ahmad¹, Walid Fazeli², Sophia Schließke¹, Gaetan Lesca³, Zeynep Gokce-Samar⁴, Kristopher Kahle⁵, Kedous Mekbib⁵, Jennifer Burton⁶, George Hoganson⁶, Andrea Petersen⁷, Sara Gracie⁷, Leslie Granger⁷, Enrika Bartels⁸, Henry Oppermann¹, Sheng Chih Jin⁹, Adam Kundishora⁵, Marianne Till³, Shane Dangerfield¹⁰, Dave Viskochil¹⁰, Katherine Anderson¹¹, George E Tiller¹², Wolfram Kunz¹³, Sebastian Burkart¹⁴, Matias Simons¹⁴, Ingrid M Wentzensen¹⁵, Hui Yang¹⁵, Rami Abou Jamra¹, Sonja Neuser ¹

¹University of Leipzig Medical Center, Institute of Human Genetics, Leipzig, Germany; ²University Hospital Bonn, Department of Pediatric Neurology, Bonn, Germany; ³Lyon University Hospital, Department of Medical Genetics, Lyon, France; ⁴Lyon University Hospital, Department of Epileptology, Lyon, France; ⁵Yale University School of Medicine, Department of Neurosurgery, New Haven, United States; ⁶University of Illinois College of Medicine, Peoria, United States; ⁷Randall Children’s Hospital, Portland, United States; ⁸Institute of Clinical Genetics and Tumor Genetics, Bonn, Germany; ⁹School of Medicine, Washington University, Department of Genetics, St. Louis, United States; ¹⁰University of Utah, Salt Lake City, United States; ¹¹University of Vermont Medical Center, Department of Pediatrics, Burlington, United States; ¹²Kaiser Permanente, Department of Genetics, Los Angeles, United States; ¹³University Hospital Bonn, Department of Epileptology, Bonn, Germany; ¹⁴University Hospital Heidelberg, Institute of Human Genetics, Heidelberg, Germany; ¹⁵GeneDx Inc., Gaithersburg, United States

Background/Objectives: The family of Rab GTPase proteins are molecular switches acting through GDP/GTP-exchange and involved in vesicle trafficking. RAB11B has been associated with a Mendelian neurodevelopmental disorder (NDD) in 2017 as two recurrent de novo missense-variants (amino acids 22,68) were identified in five children with severe intellectual disability (ID), absent speech, ataxic gait, and decreased cortical white matter. No additional cases have since been reported. We aim at confirming the relevance of RAB11B and delineating the genotypical and phenotypical spectrum.

Methods: Through international matchmaking, we gathered eight individuals with RAB11B variants identified through exome sequencing or array CGH. Clinical details of novel and previously reported five individuals were standardized to HPO-terms. We performed a cross-sectional analysis regarding the clinical manifestations. Three-dimensional analysis using a ProteinDataBank structure was done by manual inspection of variant localization in PyMol as well as cluster calculation according to mutation3d-algorithm.

Results: The variant spectrum comprises six missense-variants (four novel; amino acids 21,33,72,75) as well as one 319kb duplication, all except one confirmed de novo. In silico parameters indicate damaging effects. All variants affect or lay close by the GTP-binding sites and five of them showed significant clustering. Data from 13 individuals confirm main clinical findings to be NDD/ID (75%), muscular hypotonia (75%) and small cerebral cortex (70%). However, cognitive function ranges from severe ID (67%) to few cases with normal cognitive development only with epilepsy.

Conclusion: Confirmation of RAB11B as an NDD gene and extension to milder manifestations, generally de novo-variants within the GTP-binding regions are highly suspicious as disease-causing.

References:.

Grants:.

Conflict of Interest: Natalie Ahmad: None declared, Walid Fazeli: None declared, Sophia Schließke: None declared, Gaetan Lesca: None declared, Zeynep Gokce-Samar: None declared, Kristopher Kahle: None declared, Kedous Mekbib: None declared, Jennifer Burton: None declared, George Hoganson: None declared, Andrea Petersen: None declared, Sara Gracie: None declared, Leslie Granger: None declared, Enrika Bartels: None declared, Henry Oppermann: None declared, Sheng Chih Jin: None declared, Adam Kundishora: None declared, Marianne Till: None declared, Shane Dangerfield: None declared, Dave Viskochil: None declared, Katherine Anderson: None declared, George E Tiller: None declared, Wolfram Kunz: None declared, Sebastian Burkart: None declared, Matias Simons: None declared, Ingrid M Wentzensen employee of GeneDx Inc., Hui Yang employee of GeneDx Inc., Rami Abou Jamra: None declared, Sonja Neuser: None declared.

P09.010.B Comparison of methylation epi-signatures in KMT2B and KMT2D-related human disorders

Sunwoo Lee ¹, Eguzkine Ochoa¹, Katy Barwick², Laura Cif³, Fay Rodger^1;4, France Docquier^1;4, Belen Perez², Graeme Clark^1;4, Jose-Ezequiel Martin Rodriguez^1;4, Siddharth Banka⁵, Manju Kurian², Eamonn R Maher¹

¹University of Cambridge, Department of Medical Genetics, Cambridge, United Kingdom; ²UCL Great Ormond Street Institute of Child Health, Zayed Centre for Research into Rare Disease in Children, Developmental Neurosciences, London, United Kingdom; ³Unite des Pathologies Cerebrales Resistantes, Unite de Recherche sur les Comportements et Mouvements Anormaux, Hopital Gui de Chauliac, Centre Hospitalier R´gional Montpellier, Departement de Neurochirurgie, Montpellier, France; ⁴Stratified Medicine Core Laboratory NGS Hub, Cambridge Biomedical Campus, Cambridge, United Kingdom; ⁵University of Manchester and Manchester Centre for Genomic Medicine, St. Mary’s Hospital, Manchester University Foundation NHS Trust, Health Innovation Manchester, Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine, and Health, Manchester, United Kingdom

Background/Objectives: Many neurodevelopmental disorders caused by mutations in genes regulating chromatin function and/or structure display abnormal DNA methylation patterns (episignatures) in peripheral blood. DYT-KMT2B is unique among “chromatin neurodevelopmental disorders” in that the most prominent clinical feature and most frequent presentation is childhood onset dystonia rather than developmental delay or congenital anomalies (as seen in many chromatin neurodevelopmental disorders such as Kabuki syndrome).

Methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), we undertook genome-wide methylation profiling of ~2M CpGs using a next-generation sequencing based assay and compared the findings to those in controls and patients with Kabuki syndrome Type 1 (KS1-KMT2D).

Results: Methylation profiling revealed 1,812 significantly differentially methylated CpG positions (DMPs) (FDR < 0.05) in 10 DYT-KMT2B samples compared to controls, covering 40 CpG. Multi-dimensional scaling analysis showed that the DYT-KMT2B samples clustered together and separately from 29 control individuals and 10 individuals with pathogenic variants in KMT2D. Most DMPs were specific to one disorder and that all (DYT-KMT2B) and most (KS1) methylation alterations in CpG islands were gain of methylation events. Analysis of genes associated with CpG islands suggested potential; candidate genes for the molecular pathogenesis of DYT-KMT2B.

Conclusion: Using higher resolution methodology for methylation profiling, we confirmed a peripheral blood methylation signature for DYT-KMT2B. Methylation episgnatures could be used to aid pathogenicity interpretation of KMT2B variants and can complement mechanistic investigations of the pathogenesis of DYT-KMT2B.

References:.

Grants: This research was co-funded by the NIHR Cambridge Biomedical Research Centre and Rosetrees Trust.

Conflict of Interest: None declared.

P09.011.C Exome sequencing detects two novel variants in SNRPN in two patients with Prader-Willi/ Prader-Willi-like syndrome

Arrate Pereda ¹, Purvi Majethia², Guiomar Perez de Nanclares¹, Anju Shukla²

¹Bioaraba Health Research Institute, Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, Araba University Hospital-Txagorritxu, Vitoria-Gasteiz, Spain; ²Kasturba Medical College, Department of Medical Genetics, Manipal Academy of Higher Education, Manipal, India

Background/Objectives: Imprinting disorders (IDs) are a group of congenital disorders caused by (epi)genetic alterations in imprinted chromosomic regions. Prader-Willi syndrome (PWS) is one of the best known IDs, and is consequence of the loss of paternally expressed genes (MAGEL2, NDN, SNURF-SNRPN and SNORD116) within the 15q11.2-q13, caused either by a CNV (65–75% of cases), a upd(15)mat (20–30%), or an epimutation (1–3%) (1). Recently, point variants in SNRPN have been reported as causative of PWS in two independent families (2, 3) and in this report we described two new cases.

Methods: Two independent probands were referred for genetic testing due to seizures, mild intellectual disability and obesity (P1); and clinical suspicion of PWS (P2). After negative MS-MLPA testing for 15q11 region, exome sequencing (ES) was carried out. Variant confirmation and cosegregation studies were analysed by Sanger sequencing. Parental origin of the allele was tested by allele-specific RT-PCR amplification, and sequencing.

Results: ES studies identified a novel missense variant in SNRPN in each proband. Complementary studies determined that each variant was carried in the proband’s paternal allele.

Conclusion: According to these and previous results (2, 3), genetic testing for SNRPN point variants should be performed in PWS patients with negative results for classical causes.

References: 1. C. K. Cheon, Ann. Pediatr. Endocrinol. Metab. 21, 126 (2016).

2. K. Pellikaan et al., Genes (Basel). 12, 875 (2021).

3. Y. Huang et al., J. Med. Genet. 6, 1–4 (2021).

Grants: ESPE RU Grant 2020 (UE19_ESPE); Institute of Health Carlos III (PI20/00950); Basque Department of Health (GV2021/111056).

Conflict of Interest: Arrate Pereda ESPE RU Grant 2020 (UE19_ESPE); Institute of Health Carlos III (PI20/00950), Purvi Majethia: None declared, Guiomar Perez de Nanclares ESPE RU Grant 2020 (UE19_ESPE); Institute of Health Carlos III (PI20/00950); Basque Department of Health (GV2021/111056), Anju Shukla: None declared.

P09.012.D LoF variants can cause TUBB-associated disorder

Olga Levchenko ¹, Elena Dadali¹, Sabina Nagieva¹, Alexander Lavrov¹

¹Research Centre for Medical Genetics, Moscow, Russian Federation

Background/Objectives: TUBB encodes one of nine beta-tubulin proteins and is widely expressed in all tissues, especially in the developing brain. Pathogenic variants in TUBB are associated with two main phenotypes: cortical dysplasia, complex, with other brain malformations 6 (OMIM: 615771) and symmetric circumferential skin creases, congenital, 1 (OMIM: 156610). Both diseases are autosomal dominant and result in impaired intellectual development. There are only 10 pathogenic variants currently described and none of them demonstrate loss of function (LoF) effect. We report for the first time that LoF variants in TUBB may be responsible for impaired intellectual development.

Methods: Whole exome sequencing (WES) was performed using the IlluminaTruSeq® ExomeKit, IDT xGen® Exome Research Panel, and Illumina NextSeq 500.

Results: A 38-year-old man was born from a full-term pregnancy with a weight of 2600 g and length of 48 cm. Motor development was delayed: he could seat at 10 months, walk at 1 year 8 months. He cannot speak. On examination: normal height and weight, thoracolumbar kyphosis, strabismus, ocular hypertelorism, short filter, protruding lower jaw, no focal neurological symptoms. Karyotype is 47, XYY, normal methylation status of the FMR1 promotor. WES revealed variant of unknown significance NM_178014.4(NP_821133.1):p.(Tyr208*) in TUBB gene, which has low tolerance to LoF (pLI = 0.98). Segregation analysis by Sanger sequencing revealed de novo status of the variant. The proband’s clinical features overlap with both TUBB-associated phenotypes.

Conclusion: We propose that LoF variants in TUBB can cause TUBB-associated disorder.

References:.

Grants:.

Conflict of Interest: None declared.

P09.013.A Delineating the MAPK8IP3-related neurodevelopmental disorder reveals consistent variant specific phenotypes

Tobias Bartolomaeus ¹, Rami Abou Jamra¹, Johannes Lemke¹, International Study Group Mapk8ip3², Konrad Platzer¹

¹Institute of Human Genetics, University of Leipzig Medical Center, Leipzg, Germany; ²International Study Group MAPK8IP3, New York, United States

Background/Objectives: We and others have recently described de novo variants in MAPK8IP3 gene as a cause of neurodevelopmental disorder (NDD) with intellectual disability (ID), seizures, microcephaly, and brain anomalies.

Methods: We present an overview of 18 published and 20 novel individuals with causative MAPK8IP3 variants and a HPO based phenotypic analysis.

Results: Missense variants were identified in 31 individuals, truncating variants in six, and whole gene deletion in one. About 58% (22/38) harbour one of the previously reported recurrent missense variants p.Arg578Cys (10), p.Leu444Pro (3), or p.Arg1146Cys/His (9). ID ranges from mild to profound. Additional symptoms encompass brain anomalies (22/26), obesity (9/16), spasticity or dystonia (16/21), seizures (10/27), ataxia (6/8), microcephaly (5/15), and precocious puberty (4/6). The recurrent variant p.Arg578Cys is consistently associated with severe ID, spasticity, hypoplastic corpus callosum, white matter abnormalities, seizures, small hands and feet and early-onset obesity with insatiable appetite. In contrast, recurrent variants affecting Arg1146 are less frequently linked with obesity or seizures but instead with autism and ataxia. Three individuals do not fall into the MAPK8IP3—related NDD spectrum: two brothers with the de novo variant p.Ser984Leu have recurrent pain during urination with one of them also mildly delayed. Another six year old boy with the de novo variant p.Pro822Ser initially showed infantile spasm, which subsequently resolved with normal development.

Conclusion: MAPK8IP3-related NDD encompasses ID and consistent variant-specific phenotypes including novel observations of obesity, precocious puberty and small hands and feet. Causality of novel de novo variants remain to be elucidated.

References:.

Grants:.

Conflict of Interest: None declared.

P09.014.B BAFfling: Microduplications of ARID1A and ARID1B cause a novel clinical and epigenetic distinct BAFopathy

Eline van der Sluijs ¹, Sebastien Moutton^2;3, Denisa Weis⁴, Kym Boycott^5;6, Claudia Arberas⁷, Margherita Baldassarri^8;9, Claire Beneteau^10;11, Charles Coutton^12;13, Tabib Dabir¹⁴, Koenraad Devriendt¹⁵, Laurence Faivre^2;16, Khadije Jizi^17;18, Jennifer Kerkhof¹⁹, Mira Kharbanda²⁰, Katherine Lachlan²¹, Michael Levy¹⁹, Angelica Maris²², Nathalie Marle²³, Haley McConkey¹⁹, Maria Antonietta Mencarelli⁸, David Mowat^24;25, Claire Nicolas^2;16, olivier pichon¹⁰, Julia Rankin²⁶, Raissa Relator¹⁹, Jill A. Rosenfeld^27;28, Norbert Winer^29;30, Jeremy Woods^31;32, Bekim Sadikovic¹⁹, Marielle Alders³³, Gijs Santen¹

¹Leiden University Medical Center, Clinical Genetics, Leiden, Netherlands; ²FHU TRANSLAD, CHU Dijon, Centre de Référence Anomalies du Développement et Syndromes Malformatifs, Dijon, France; ³Maison de Santé Protestante Bordeaux Bagatelle, CPDPN, Pôle mère enfant, Talence, France; ⁴Johannes Kepler University Linz, Institute of Medical Genetics, Linz, Austria; ⁵University of Ottawa, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada; ⁶Children’s Hospital of Eastern Ontario, Department of Genetics, Ottawa, Canada; ⁷Hospital de Niños Dr. Ricardo Gutiérrez, Sección Genética Médica, Buenos Aires, Argentina; ⁸Azienda Ospedaliera Universitaria Senese, Genetica Medica, Siena, Italy; ⁹University of Siena, Medical Genetics, Siena, Italy; ¹⁰Centre Hospitalier Universitaire de Nantes, Service de Génétique médicale, Nantes, France; ¹¹Centre Hospitalier Universitaire de Nantes, UF de Foetopathologie et Génétique, Nantes, France; ¹²Université Grenoble-Alpes, Grenoble, France; ¹³Hôpital Couple-Enfant, CHU de Grenoble, Département de Génétique et Procréation, Grenoble, France; ¹⁴Belfast City Hospital, Department of Genetic Medicine, Belfast, United Kingdom; ¹⁵University Hospitals Leuven, Center for Human Genetics, Leuven, Belgium; ¹⁶INSERM - Bourgogne Franche-Comté University, Genetics of Developmental Disorders, Dijon, France; ¹⁷Université de Montréal, Department of Pediatrics, Montréal, Canada; ¹⁸Centre de recherche et Centre Hospitalier Universitaire Sainte-Justine, Montréal, Canada; ¹⁹Western University, Verspeeten Clinical Genome Centre, London Health Sciences Centre, and Department of Pathology and Laboratory Medicine, London, Canada; ²⁰University Hospital Southampton, Princess Anne Hospital, Wessex Clinical Genetics Service, Southampton, UK., United Kingdom; ²¹University Hospital Southampton, Princess Anne Hospital, Wessex Clinical Genetics Service, Southampton, United Kingdom; ²²Universidade Federal de Santa Catarina, Florianópolis, Brazil; ²³CHU de Dijon, Dijon, France, Laboratoire de Génétique Chromosomique et Moléculaire, Pôle de Biologie, Dijon, France; ²⁴Sydney Children’s Hospital, Center for Clinical Genetics, Randwick, New South Wales, Australia; ²⁵University of New South Wales, School of Women’s and Children’s Health, Faculty of Medicine and Health, Kensington, New South Wales, Australia; ²⁶Royal Devon and Exeter NHS Foundation Trust, Department of Clinical Genetics, Exeter, United Kingdom; ²⁷Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, United States; ²⁸Baylor Genetics Laboratories, Houston, United States; ²⁹Centre Hospitalier Universitaire de Nantes, Service de Gynécologie-Obstétrique, Nantes, France; ³⁰Université de Nantes, NUN, INRAE, UMR 1280, PhAN, Nantes, France; ³¹Valley Childrens Hospital, Department of Genetics, Madera, CA, United States; ³²Stanford University, Palo Alto, CA, United States; ³³Amsterdam UMC, University of Amsterdam, Department of Human Genetics, Amsterdam, Netherlands

Background/Objectives: The clinical relevance of ARID1B whole gene duplications has been unclear until now. ARID1B and ARID1A have the same role within the BAF complex and are mutually exclusive. ARID1A duplications appear to lead to a distinct clinical syndrome, while ARID1B duplications have not been linked to a clinical phenotype.

Methods: To investigate whether an ARID1B duplication phenotype exists, and how this relates to the ARID1A phenotype, we included patients with an ARID1A or ARID1B duplication, compared their phenotypes and determined DNA methylation.

Results: We included data of 9 ARID1A and 11 ARID1B duplication patients. Duplication size ranges between 0.1-1.2 Mb with 1-44 genes for ARID1A and 0.4-10.3 Mb with 2-101 genes for ARID1B. Main features shared by ARID1A and ARID1B patients were intellectual disability, microcephaly, growth delay and cryptorchidism. Even though there is overlap between the two groups, the phenotype of ARID1A patients appeared to be more severe compared to that of ARID1B patients. DNA methylation Episign analysis showed that ARID1A and ARID1B duplication patients have a similar DNA methylation pattern in blood, which is different from controls and opposite to the pattern of more common ARID1A or ARID1B loss-of-function variants.

Conclusion: We report for the first time that duplications of ARID1B lead to a clinical phenotype with significant overlap with ARID1A duplications, and that these patients have overlapping episignatures providing further evidence for an overlapping phenotype distinct from other BAFopathies. A new type of BAFopathies caused by a duplication, rather than haploinsufficiency, is emerging here.

References:.

Grants:.

Conflict of Interest: None declared.

P09.015.C Expanding Rett syndrome landscape: identification of candidate genes from a WES study

Elisabetta Di Fede ¹, Angela Peron^2;3;4, Elisa Colombo¹, Simone Tamburri^1;5, Diego Pasini^1;5, Cristina Gervasini^1;6, Aglaia Vignoli^1;7

¹Università degli Studi di Milano, Department of Health Sciences, Milan, Italy; ²ASST Santi Paolo e Carlo, Human Pathology and Medical Genetics, Milan, Italy; ³Università degli Studi di Milano, Child Neuropsychiatry Unit, Epilepsy Center, Department of Health Sciences, Milan, Italy; ⁴University of Utah School of Medicine, Department of Pediatrics, Salt Lake City, United States; ⁵IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Milan, Italy; ⁶Università degli Studi di Milano, Aldo Ravelli Center for Neurotechnology and Experimental Brain Therapeutics, Milan, Italy; ⁷ASST Grande Ospedale Metropolitano Niguarda, Child NeuroPsychiatry Unit, Milan, Italy

Background/Objectives: Rett syndrome (RTT) is a neurodevelopmental disorder (incidence of 1:10,000 live female births) most frequently affecting girls during infancy, after an early normal development. The genetic cause is known for about 90% of patients (MECP2 mutations in the classic form, CDKL5 and FOXG1 mutations for the two variants), while the 10% remain without molecular diagnosis.

Methods: We applied whole exome sequencing (WES) analysis to Rett-like probands and their healthy parents. We enrolled in the study patients negative for mutations in RTT genes to identify the genetic cause and expand the knowledge of the pathogenetic mechanisms underlying RTT.

Results: We found one girl (#1) being compound heterozygote for two unreported variants in NBEA gene, encoding for a brain-specific protein involved in vesicle trafficking. In addition, patient #2 was found being carrier of a de novo missense mutation in DYNC1H1, known to be associated to a recently classified neurodevelopmental disorder. Finally, in patient #3 was identified a novel heterozygous missense variant in SLC35F1, mainly expressed in the brain and coding for a putative solute carrier whose role is currently under investigation.

Conclusion: The identification of new RTT genes could expand the genotype-phenotype correlation for RTT syndrome, and the characterization of new candidate RTT genes could give insights into the pathogenesis of this neurodevelopmental disorder.

References: Neul et al., 2010; Mulhern et al., 2018; Poirier et al., 2013; Szafranski et al., 2015; Di Fede et al., 2021.

Grants: Grant Aldo Ravelli Center for Neurotechnology and Experimental Brain Therapeutics and Intramural funding of Università degli Studi di Milano.

Conflict of Interest: None declared.

P09.016.D A novel missense mutation in SLC2A1 gene responsible for Glut1 Deficiency Syndrome

Renata Szalai ^1;2, Agnes Till³, Judit Bene^2;3, Anita Maasz^2;3, Kinga Hadzsiev^2;3

¹University of Pecs, Department of Medical Genetics, Pecs, Hungary; ²University of Pecs, Szentagothai Research Center, Pecs, Hungary; ¹University of Pecs, Department of Medical Genetics, Pecs, Hungary

Background/Objectives: Glut1 Deficiency Syndrome (Glut1DS) is a brain energy failure disease caused by impaired glucose transport across brain tissue barriers. The definite diagnosis of Glut1DS is confirmed by the presence of characteristic clinical features, hypoglycorrhachia, and a pathogenic variant in SLC2A1 gene. The type of genetic mutation correlates with severity of disease; missense variants (mild severity); splice site, nonsense variants, insertions, deletions (moderate and severe severity); complete gene microdeletions (severe severity). Heterozygous de novo pathogenic variants in SLC2A1 responsible for the phenotype of 81-89% of Glut1DS patients. Ketogenic dietary treatment (KDT) can achieve seizure freedom within a couple of days with normalization of EEG changes, frequently allowing for the withdrawal of any antiepileptic medications.

Methods: We present a patient with positive family history (mother with intellectual disability and seizures) and characteristic features for Glut1DS, including mild microcephaly, developmental and speech delay, paroxysmal eye movement abnormality and starvation-induced atypical absence seizures. NGS-based targeted custom epilepsy gene panel analysis was performed using QIAGEN QIAseq library kit and Illumina sequencing technology.

Results: NGS analysis identified a c.1288G>T (p.Gly430Cys) heterozygous, likely pathogenic variant in SLC2A1 gene. This alteration is a missense variant, has never been reported. Application of KDT resulted in marked clinical improvement of the motor and seizure symptoms in our patient.

Conclusion: Accurate molecular diagnosis is critical for the clinical management and prognosis of subjects suspected with Glut1 deficiency, especially in case of mild severity, where early diagnosis and intervention would substantially influence the cognitive and motor functions.

References:.

Grants:.

Conflict of Interest: None declared.

P09.017.A AutoCaSc: Prioritizing candidate genes for neurodevelopmental disorders

Johann Lieberwirth¹, Benjamin Büttner¹, Chiara Klöckner¹, Konrad Platzer¹, Maximilian Radtke¹, Meret Wegler¹, Bernt Popp¹, Rami Abou Jamra ¹

¹Institute of Human Genetics, University of Leipzig Medical Center, Leipzig, Germany

Background/Objectives: Exome sequencing(ES) in individuals with developmental disorders (DD) remains inconclusive in ~50%. Evaluation unsolved cases to identify candidate genes is subjective, slow, and uncomparable between labs. We developed AutoCaSc to prioritize candidate genes.

Methods: webAutoCaSc was developed in Python and vcfAutoCaSc was designed for VCFs scoring from the command line. The tools automate our fine-tuned candidate scoring scheme (CaSc), which is composed of the four categories “Variant attributes”, “Inheritance”, “Gene constraint” and “Gene plausibility”. The first three categories were implemented as decision trees, while “Gene plausibility” is a precomputed score of expression, model organism, protein-protein interaction, literature, and de novo occurrence in DD cohorts.

Results: As a proof of principle, we injected into two public ES trios 79 variants in recently published DD genes, thus simulating the identification of candidate genes and variants. AutoCaSc consistently (94.5%) scored all variants and genes in the top three ranks (mean rank of 1.5 and 2.3 in the two ES trios). Furthermore, in 93 in-house trios, AutoCaSc identified all previously identified candidate variants by a human evaluator. AutoCaSc placed these in the top ranks while evaluating additional highly scoring variants that were missed in the initial manual evaluation. weAutoCaSc is thus in standard use at our institute and is publically available: https://autocasc.uni-leipzig.de/.

Conclusion: AutoCaSc enables anybody to quickly screen a variant of interest for its plausibility for DDs even if the gene is still not described. We provide usage recommendations, based on our experience in projects describing novel DD genes to accelerate deciphering the genetics of DD.

References:.

Grants:.

Conflict of Interest: None declared.

P09.018.B Novel in-frame deletion in RBMX leads to X-linked intellectual disability by disturbed RNA processing, splicing regulation and potentially reduced SH3 binding

Josefin Johansson ¹, Sarah Lidéus¹, Filip Mihalic², Mauno Vihinen³, Carina Frykholm¹, Sanna Gudmundsson^4;5, Adam Ameur¹, Per Jemth², Marie-Louise Bondeson¹, Maria Wilbe¹

¹SciLifeLab, Uppsala University, Immunology, genetics and pathology (IGP), Uppsala, Sweden; ²Uppsala University, Medical Biochemistry and Microbiology, Uppsala, Sweden; ³Lund University, Experimental Medical Science, Lund, Sweden; ⁴Broad Institute of MIT and Harvard, Program in Medical and Population Genetics, Boston, United States; ⁵Boston Children’s Hospital, Harvard Medical School, Division of Genetics and Genomics, Boston, United States

Background/Objectives: RNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) important for regulation of splicing, sister chromatid cohesion and genome stability. Deletion of the RGG/RG motif in hnRNP G have been associated with Shashi syndrome, however other hnRNP G domains’ association to X-linked intellectual disability (XLID) remains unknown.

Methods: Whole exome sequencing, whole genome sequencing, and X-chromosome inactivation studies were used to genetically characterize a Swedish five-generation family with XLID. The variant effect on biological processes and alternative splicing events were investigated by transcriptomics analyses of an SH-SY5Y cell line overexpressing the mutant or wildtype RBMX. The variant effect on hnRNP G was investigated by using prediction tools and fluorescence polarization assays with SH3 domains and peptides spanning the variant.

Results: A novel in-frame RBMX deletion segregated with disease in this family, where affected individuals were hemizygous, and asymptomatic individuals were non-carriers or heterozygous females with skewed X-chromosome inactivation. The affected individuals presented poor phenotypic overlap with Shashi syndrome, suggesting a different disease mechanism. Transcriptomics analyses revealed differentially expressed splicing events and enrichment for genes involved in RNA processing and neurodevelopment. Protein analyses imply a novel SH3-binding motif in hnRNP G and potentially lower affinity binding to SH3 domains caused by the variant.

Conclusion: We present a novel in-frame deletion in RBMX as a cause to XLID, by disrupted RNA processing, splicing regulation and potentially reduced SH3-binding. The results implies that disruption of different motifs impact the severity of disease.

References:.

Grants: Swedish Society for Medical Research and Sävstaholm Foundation.

Conflict of Interest: None declared.

P09.019.C Genome Sequencing is a sensitive first-line test to diagnose individuals with intellectual disability

Anna Lindstrand^1;2, Marlene Ek ^1;2, Malin Kvarnung^1;2, Britt M. Anderlid^1;2, Erik Björck^1;2, Jonas Carlsten^1;2, Jesper Eisfeldt^1;2;3, Giedre Grigelioniene^1;2, Peter Gustavsson^1;2, Anna Hammarsjö^1;2, Hafdís Helgadóttir^1;2, Maritta Hellström Pigg^1;2, Ekaterina Kuchinskaya^1;2, Kristina Lagerstedt-Robinson^1;2, Lars-Åke Levin⁴, Agne Lieden^1;2, Hillevi Lindelöf^1;2, Helena Malmgren^1;2, Daniel Nilsson^1;2;3, Eva Svensson⁵, Martin Paucar Arce^1;2, Ellika Sahlin^1;2, Bianca Tesi^1;2, Emma Tham^1;2, Johanna Winberg^1;2, Max Winerdal^1;2, Josephine Wincent^1;2, Maria Johansson Soller^1;2, Maria Pettersson^1;2, Ann Nordgren^1;2

¹Karolinska Institutet, Department of Molecular Medicine and Surgery, Solna, Sweden; ²Karolinska University Hospital, Department of Clinical Genetics, Solna, Sweden; ³Science for Life Laboratory, Department of Molecular Medicine and Surgery, Solna, Sweden; ⁴Linköping University, Department of Health, Medicine and Caring Sciences, Linköping, Sweden; ⁵Karolinska University Hospital, Department of Pediatric Neurology, Huddinge, Sweden

Background/Objectives: Individuals with intellectual disability (ID) and/or neurodevelopmental disorders (NDD) are currently investigated with several different approaches in clinical genetic diagnostics. A molecular diagnosis can facilitate better individualized health care, improved genetic counselling and quality of life.

Methods: We compare the results from three diagnostic pipelines in patients with ID/NDD; genome-first (n = 100), genome as a secondary test (n = 129) or chromosomal microarray (CMA) with or without FMR1 screening (n = 421).

Results: The diagnostic yield was 37% (genome-first), 26% (genome as a secondary test) and 12% (CMA/FMR1). Notably, the age of diagnosis was delayed by 1 year when genome was done as a secondary test and the cost per diagnosed individual was 36% lower with genome-first compared to CMA/FMR1. Furthermore, 91% of those with a negative result after CMA/FMR1 screening (338 individuals) have not yet been referred for additional genetic testing and remain undiagnosed.

Conclusion: Our findings strongly suggest that genome analysis outperforms other testing strategies and should replace traditional CMA and FMR1 screening as a first-line genetic test in individuals with ID/NDD. Genome is a sensitive, time- and cost-effective method that results in a confirmed molecular diagnosis in 37% of all referred patients.

References: None.

Grants: The Swedish Research Council, the Swedish Brain Foundation, the Swedish Rare Diseases Research Foundation, the Hållsten Research Foundation.

Conflict of Interest: None declared.

P09.020.D Characterization of de novo variants in PPP2R5C expands the spectrum of PP2A-related neurodevelopmental disorders

Iris Verbinnen ¹, Elise Brimble², Lisa Lenaerts¹, David Geneviève³, Laurence Faivre⁴, Julien Thevenon⁵, Jessica X. Chong⁶, Mike Bamshad^6;7, Karynne Patterson⁷, Elysa Marco⁸, Dmitriy Niyazov⁹, Aida Telegrafi¹⁰, Sumit Punj¹⁰, Kimberly Foss¹¹, William Dobyns^6;11;12, Ghaydda Mirzaa^6;11, Chin-To Fong¹³, Sue White¹⁴, Lynn Pais¹⁵, Emily O’Heir^15;16, Celia van der Merwe^17;18, Raphaela Itzikowitz¹⁹, Kirsty Donald¹⁹, Tony Roscioli^20;21;22, Kerith-Rae Dias^22;23, Carey-Anne Evans^22;23, Alessandro Mussa²⁴, Raffaela Cervini²⁵, Elisa Giorgio²⁶, Anna Ruiz²⁷, Juan Pablo Trujillo Quintero²⁸, Rachel Rabin²⁹, John Pappas²⁹, Xiaodong Wang³⁰, j Wang³⁰, Hua Yuan³¹, Maura R.Z. Ruzhnikov³², Veerle Janssens¹

¹Univ. of Leuven, Lab of Protein Phosphorylation & Proteomics, Dept. of Cellular & Molecular Medicine, Leuven, Belgium; ²Invitae, San Francisco, CA, United States; ³Montpellier Univ., Génétique clinique, Dépt. de Génétique Médicale, Maladies Rares et Médecine Personnalisée, Centre de Référence Anomalies du développement SOOR,CHU Montpellier, INSERM U1183, ERN ITHACA, Montpellier, France; ⁴Hôpital d’enfants, CHU Dijon Bourgogne – Hôpital François Mitterrand, Centre de génétique, Dijon, France; ⁵Univ. Grenoble-Alpes, CNRS UMR 5309, Inserm U1209, Institute of Advanced Biosciences, Grenoble Cedex, France; ⁶Univ. of Washington School of Medicine, Div. of Genetic Medicine, Dept. Pediatrics, Seattle, WA, United States; ⁷Univ. of Washington, Dept. of Genome Sciences, Seattle, WA, United States; ⁸Cortica Healthcare, San Rafael, CA, United States; ⁹Univ. of Queensland, Medical Genetics Ochsner Health System, Queensland, Australia; ¹⁰GeneDx, Gaithersburg, MD, United States; ¹¹Seattle Children’s Research Institute, Center for Integrative Brain Research, Seattle, WA, United States; ¹²Univ. of Washington, Dept. Neurology, Seattle, WA, United States; ¹³Univ. of Rochester Medical Center, Dept. Pediatrics, Rochester, NY, United States; ¹⁴Royal Children’s Hospital, VCGS, Victoria, Australia; ¹⁵Broad Institute, Center for Mendelian Genomics, Cambridge, MA, United States; ¹⁶Boston Children’s Hospital, Div. of Genetics & Genomics, Dept. Pediatrics, Boston, MA, United States; ¹⁷Broad Institute, Stanley Center for Psychiatric Research, Cambridge, MA, United States; ¹⁸Massachusetts General Hospital, Analytic & Translational Genetics Unit, Boston, MA, United States; ¹⁹Red Cross War Memorial Children’s Hospital, Dept. Pediatrics & Child Health, Cape Town, South Africa; ²⁰Sydney Children’s Hospital, Centre for Clinical Genetics, Sydney, Australia; ²¹Prince of Wales Hospital, NSW Health Pathology Randwick Genomics, Sydney, NSW, Australia; ²²Univ. of NSW, NeuRA, Prince of Wales Clinical School, Sydney, Australia; ²³Univ. of NSW, Prince of Wales Clinical School, Faculty of Medicine, Sydney, NSW, Australia; ²⁴Univ. of Torino, Regina Margherita Children’s Hospital, Dept. Public Health & Pediatric Sciences, Torino, Italy; ²⁵Maria Vittoria Hospital, Child Neuropsychiatry Dept., Torino, Italy; ²⁶Univ. of Pavia, Dept. Molecular Medicine, Pavia, Italy; ²⁷Universitat Autònoma de Barcelona, Genetics Lab, Parc Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí I3PT, Sabadell, Spain; ²⁸Universitat Autònoma de Barcelona, Unitat de Genètica Clínica, Servei de Medicina Pediàtrica, Parc Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí I3PT, Sabadell, Spain; ²⁹NYU Grossman School of Medicine, Clinical Genetic Services, Dept Peds, New York, NY, United States; ³⁰Cipher Gene, Ltd, Genetic Testing Company, Beijing, China; ³¹Guangxi Medical Univ., Dept. Pediatrics, The First Affiliated Hospital, Nanning, Guangxi, China; ³²Stanford Medicine, Div. of Medical Genetics, Dept. Pediatrics, Stanford, CA, United States

Background/Objectives: Protein Phosphatases of type 2A (PP2A) regulate brain function and development by catalyzing phospho-Ser/Thr dephosphorylations in various substrates. PP2A holoenzymes comprise a catalytic C, scaffolding A and regulatory B-type subunit, which determines substrate specificity and enzyme regulation. De novo mutations in genes encoding Aα-, B56δ- and Cα-subunits were recently identified as new genetic causes of intellectual disability and (neuro)developmental delay (ID/NDD). A single case report describes an overgrowth phenotype associated with a de novo mutation in PPP2R5C, encoding the regulatory B56γ-subunit.

Methods: Matchmaker Exchange and international collaborations enabled us to identify 12 additional individuals with de novo PPP2R5C mutations, and 2 individuals with a PPP2R5C variant of unclear inheritance. Variants were biochemically characterized for interaction with other PP2A subunits and a potential PP2A substrate, and for phosphatase activity.

Results: Besides ID/NDD, clinical features of PPP2R5C-affected cases included hypotonia and commonly, epilepsy, behavioral and brain size abnormalities (macrocephaly). Most PPP2R5C variants affected the same, highly conserved B56 acidic loop or other orthologous amino acids that are also recurrently mutated in PPP2R5D-affected cases. Six variants were new. All de novo variants showed varying defects in A, C and/or substrate binding, while phosphatase activity, measured on phospho-peptide substrates, did not seem majorly affected. Both variants of unsure inheritance behaved normally in these assays, and were thus classified as likely non-pathogenic (VUS).

Conclusion: We report the first cohort of patients with pathogenic de novo PPP2R5C variants that show impaired functionality and are a novel cause of ID/NDD, with high clinical and biochemical similarities to PPP2R5D-affected cases.

References:.

Grants: JGA/FWO.

Conflict of Interest: None declared.

P09.021.A Copy number detection from exome sequencing data for patients with neurodevelopmental disorder: an effective approach

Erika D’haenens ¹, Sarah Delbaere², Toon Rosseel², Marieke De Bruyne¹, Hannes Syryn¹, Bert Callewaert¹, Björn Menten¹, Annelies Dheedene¹

¹Center for Medical Genetics Ghent - Ghent University Hospital, Department of Biomolecular Medicine, Ghent, Belgium; ²Center for Medical Genetics Ghent - Ghent University Hospital, Department of Biomolecular Medicine, Ghent, Belgium

Background/Objectives: Copy number variant (CNV) sequencing and exome-based gene panel analysis became state-of-the art in routine diagnostics. However, small CNVs may not be routinely detected or reported and in recessive disease, the combination of both a CNV and single nucleo