A cryptic microdeletion del(12)(p11.21p11.23) within an unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome

In a patient diagnosed with both Kallmann syndrome (KS) and intellectual disability (ID), who carried an apparently balanced translocation t(7;12)(q22;q24)dn, array comparative genomic hybridization (aCGH) disclosed a cryptic heterozygous 4.7 Mb deletion del(12)(p11.21p11.23), unrelated to the translocation breakpoint. This novel discovery prompted us to consider the possibility that the combination of KS and neurological disorder in this patient could be attributed to gene(s) within this specific deletion at 12p11.21-12p11.23, rather than disrupted or dysregulated genes at the translocation breakpoints. To further support this hypothesis, we expanded our study by screening five candidate genes at both breakpoints of the chromosomal translocation in a cohort of 48 KS patients. However, no mutations were found, thus reinforcing our supposition. In order to delve deeper into the characterization of the 12p11.21-12p11.23 region, we enlisted six additional patients with small copy number variations (CNVs) and analyzed eight individuals carrying small CNVs in this region from the DECIPHER database. Our investigation utilized a combination of complementary approaches. Firstly, we conducted a comprehensive phenotypic-genotypic comparison of reported CNV cases. Additionally, we reviewed knockout animal models that exhibit phenotypic similarities to human conditions. Moreover, we analyzed reported variants in candidate genes and explored their association with corresponding phenotypes. Lastly, we examined the interacting genes associated with these phenotypes to gain further insights. As a result, we identified a dozen candidate genes: TSPAN11 as a potential KS candidate gene, TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B, and ETFBKMT as candidate genes for the neurodevelopmental disorder, and INTS13, REP15, PPFIBP1, and FAR2 as candidate genes for KS with ID. Notably, the high-level expression pattern of these genes in relevant human tissues further supported their candidacy. Based on our findings, we propose that dosage alterations of these candidate genes may contribute to sexual and/or cognitive impairments observed in patients with KS and/or ID. However, the confirmation of their causal roles necessitates further identification of point mutations in these candidate genes through next-generation sequencing.

approximately 37 kb upstream of the predicted gene ZNF804B (Zinc Finger Protein 804B) with an unknown function.Consequently, the karyotype was revised to t(7;12)(q21.13;q23.1)dn(Fig. 1A) 11 .Although we initially considered RMST as a potential candidate gene due to other cases involving chromosome 12q24 with IHH during  3B).The breakpoint was refined to 492 bp between the centromeric end of HaeIII and the telomeric end of DraI, which was then isolated with suppression PCR and sequenced.The breakpoint at 12q23.1 is located in intron 2 of RMST (NR_152618.1).our initial positional cloning efforts between 2002 and 2008 11 , screening of five genes, including RMST, at and around both breakpoints in 48 KS patients did not reveal any pathogenic variants.Hence, the translocation breakpoint at 12q23 did not appear to harbor a positional candidate gene for KS.
Using aCGH with increased resolution, it is possible to detect CNVs (copy number variations) that may not be identified through karyotyping alone 13 .As cryptic deletions are frequently observed in seemingly balanced translocations 14,15 , we performed aCGH analysis, which uncovered a heterozygous 4.7 Mb deletion spanning 29 known and predicted genes at 12p11.21-12p11.23(Fig. 2A).This cryptic heterozygous deletion in the patient with an apparently balanced translocation would not have been detected through previous chromosome analysis due to the limited resolution of karyotyping (approximately 5 Mb or higher) 16 .In order to identify positional www.nature.com/scientificreports/candidate gene(s) for KS and/or ID, we recently recruited an additional six patients (Patients 2-7) with microdeletions and microduplications at 12p11.21-12p11.23(Table 1 and Fig. 2A).Notably, these seven patients share common phenotypic features, including developmental delay (DD), ID, learning disability, and language/speech delay.Some patients also exhibit autism, craniofacial anomalies (CFA), and epilepsy (Table 1), suggesting the presence of neurodevelopmental gene loci within this region.Through in-silico comparative mapping of these seven patients, along with eight informative CNVs (https:// decip her.sanger.ac.uk/, version 11.14) 17 encompassing five microdeletions and ten microduplications within the microdeletion observed in Patient 1, we have implicated one KS candidate gene, seven neurodevelopmental candidate genes, and four candidate genes for KS combined with NDDs.
Breakpoint region was refined to 3.5 kb at 12q23.1, and 87 kb at 7q21.13 by FISH mapping.FISH mapping was initiated using clones RP11-11O3 and RP11-77E2 on 7q21 and clones RP11-74K11 and RP11-1K22 on 12q23 to identify the breakpoints.These clones served as flanking markers for the breakpoints, and further experiments were conducted until the clones containing the breakpoints were identified, following the previously described methodology 7 .For the chromosome 12 breakpoint, final experiments utilized clones RP11-492N15 and CTD-2235H23, which hybridized to chromosome 12, der(12) chromosome, and der(7) chromosome, indicating that the translocation breakpoint of chromosome 12 resided within the sequence of these two BAC clones (Fig. 1B).The hybridization of SpectrumGreen labeled RP11-492N15 to chromosome 12 and both derivative chromosomes is illustrated in Fig. 2B.CTD-2542D2 was found to hybridize to chromosome 12 and der(7) chromosome, while CTD-2268E11 hybridized to chromosome 12 and der(12) chromosome, delineating the breakpoint region at 12q23 (Fig. 1B).
These findings indicate that the chromosome 12 breakpoint was located within or adjacent to a 3.5 kb interval (chr12: 97,460,688-97,464,146 / hg38) between CTD-2542D2 and CTD-2268E11, based on the placement of end-sequenced BAC clones on the current genomic sequence map (Fig. 1B and Fig. 3A).This interval was positioned 874 bp upstream from the 5' end of the RMST locus.
For the chromosome 7 breakpoint, mapping revealed its location within clone CTD-2325L19 (Fig. 2B), with clone RP11-46O13 being telomeric to the breakpoint.Based on the positions of these end-sequenced clones, the chromosome 7 breakpoint was determined to be within an 87 kb interval (data not shown).This interval did not contain any genes, and the nearest gene, ZNF804B, was approximately 11 kb beyond the telomeric boundary of the chromosome 7 breakpoint region defined by FISH mapping.
Breakpoint region at 12q23.1 was refined to 492 bp by Southern blot analysis.To map the breakpoints where the disease gene in Patient 1 might be disrupted or dysregulated, Southern blot analysis was performed on the refined breakpoint region of chromosome 12 identified through FISH.Probes KS-1, KS-2, and KS-3 were sequentially hybridized to genomic DNAs from translocation patients and normal controls digested by BglII, DraI, EcoRI, EcoRV, HaeIII, HindIII, RsaI, and ScaI, using a filter (Blot 1).All three probes detected the same rearrangement observed in a 7.3 kb genomic HindIII fragment in the patient, which was absent in controls (data not shown).Thus, the breakpoint was located within a 7.3 kb HindIII restriction fragment (chr12: 97,460,797-97,468,119/hg38), which encompassed 3350 bp of the 3.5 kb putative breakpoint region narrowed down by FISH (Fig. 3A).To confirm that the aberrant band detected by HindIII was not a result of a polymorphism, probe KS-4 was hybridized to nylon membrane Blot 2 containing genomic DNAs digested by BamHI, StuI, HincII, NaeI, Sau3AI, SfoI, SnaBI, and SspI.An aberrant band was observed in the patient's genomic DNA lane with SnaBI, located below the 12.3 kb control SnaBI restriction fragment, which included the putative 7.3 kb breakpoint region narrowed by the HindIII restriction fragment.This confirmed that the aberrant band detected by HindIII was indeed caused by the chromosome rearrangement (data not shown).
As the putative breakpoint region defined by the HindIII rearranged fragment included a new 3.8 kb region, probes KS-5 and KS-6 were separately hybridized to genomic DNAs from the patient and normal control, digested by BamHI, StuI, HincII, DraI, EcoRI, HindIII, RsaI, and SspI, on two nylon membranes of Blot 3. Probe KS-5 detected rearranged fragments in the patient's genomic DNAs digested with BamHI, StuI, Hin-cII, EcoRI, and HindIII, while probe KS-6 detected rearranged fragments in the patient lanes with EcoRI and HindIII (data not shown).The breakpoint region was found within a 3.9 kb StuI restriction fragment (chr12: 97,466,625-97,470,522/hg38), overlapping with the breakpoint region of the 7.3 kb HindIII restriction fragment, further narrowing down the breakpoint to 1.5 kb (chr12: 97,466,625-97,468,119 / hg38).
Breakpoint cloning of t(7;12)(q21.13;q23.1)identified RMST truncated at 12q23.1.Cloning of the breakpoint from derivative chromosome 12.The 3.5 kb junction fragment from derivative chromosome 12, which was detected on Blot 3 using probes KS-5 and KS-6 from EcoRI digestion, was amplified through two independent suppression PCRs 18 .The primers sets mentioned in Materials & Methods section were employed under the specific condition.The size of the resulting PCR products was 0.6 kb and 0.8 kb, respectively.Sequence analysis confirmed that these fragments corresponded to the junction fragments from the der (12).The genomic breakpoint was identified between positions 97,466,873 and 97,466,877 (hg38) at 12q23.1 (Fig. 4A), specifically within the second intron of RMST (Fig. 1B).
Since the junction fragment contained chromosome 7 sequences adjacent to the breakpoint, a BAC clone named CTD-2325L19 was identified through BLAT analysis on the Human Genome Browser (hg38).This clone mapped to 7q21 and was located within the 3.5 kb region narrowed down by FISH.BAC clone CTD-2325L19 also contained the chromosome 7 sequence of the junction fragment.FISH analysis (Fig. 2B, right picture) revealed hybridization signals of SpectrumGreen on chromosomes 7, der (7), and der (12), confirming the presence of the 7q21 breakpoint in the patient.Therefore, the results indicate that BAC clone CTD-2325L19 harbors the 7q21 breakpoint observed in the patient's genome.
Cloning of the breakpoint from derivative chromosome 7.Since the sequence of the chromosome 7 region adjacent to the breakpoint of der (12) had been determined, a 2.3 kb junction fragment from der(7) was generated using nested PCR.This was achieved by employing primers located proximal to the 7q21.13breakpoint and distal to the 12q23.1 breakpoint, as specified in the Materials & Methods section.Sequence analysis confirmed that this fragment corresponded to the junction fragment from der (7).The genomic breakpoint was identified between positions 88,722,752 and 88,722,753 (hg38) at 7q21.13 (Fig. 4A).
To investigate if there were any chromosomal alterations at the translocation site, two junction fragment sequences were examined.A comparison between the normal chromosome sequence at 7q21.13, the normal chromosome sequence at 12q23.1, and the sequences from the two junction fragments revealed an unknown 17 bp insertion (GCA ATT GCA ATG AATAT) in the der(12) junction fragment, as well as a 3 bp CTC deletion from chromosome 12 in the der(7) junction fragment 11 (Fig. 4A).
Identification of three candidate genes at 12q23.1 from t(7;12)(q21.13;q23.1).To identify the gene associated with KS, we conducted mapping and sequencing of both translocation breakpoints.Previous studies reported two balanced translocations and one deletion 12q24 related to hypogonadism [2][3][4] .Therefore, we analyzed sequences upstream and downstream of the chromosome 12 breakpoint to search for a potential candidate gene.This analysis led to the identification of RMST.By comparing the genomic sequence, we determined that the translocation directly disrupted RMST, with the 12q23 breakpoint located within intron 2, downstream of the second exon of this gene (Fig. 1B) 11 .RMST is a long non-coding RNA specifically expressed in the developing brain.
Identification of two candidate genes at 7q21.13 from t(7;12)(q21.13;q23.1).In de novo balanced translocation with an associated phenotype, the disease gene is often found near one of the two breakpoints 6,7,9,19 .We also examined the genes located at the chromosome 7 breakpoint.The nearest gene to the 7q21.13breakpoint is ZNF804B, which is mapped 37 kb distal to the breakpoint.Another gene, STEAP4 (Six-Transmembrane Epithelial Antigen of Prostate 4, also known as STEAP4 Metalloreductase, MIM 611098), was mapped 416 kb proximal to the breakpoint 11 .7) and der(12) at the breakpoints junctions.Three bp sequence CTC from chr12 is deleted at the junction of der (7), and a 17 bp insertion was found at the junction of the der (12).(B) Transcript levels of INTS13, TM7SF3, STK38L, ARNTL2, PPFIBP1, REP15, FAR2, ERGIC2, TMTC1, TSPAN11, and DENND5B in five different human tissues (i.e.brain, fetal brain, muscle, ovary and testis) were determined by RT-qPCR.Varying levels of expression of these candidate genes were detected in different tissue samples.

Discussion
To date, there have been reports of three balanced translocations and one microdeletion involving 12q24 associated with hypogonadism or KS 1-4 .In a case reported in 1983, all three brothers of Vietnamese Chinese origin were found to have severe primary hypogonadism with a karyotype of 46,XY,t(1;12)(p32;q24) 3 .Another case, published in 1990, described an apparently balanced translocation t(7;12)(q22;q24) in a male with KS and ID 1 .In 1994, a Turkish male patient exhibited isolated hypogonadotropic hypogonadism (IHH) and a lack of secondary sexual characteristics, but with normal olfaction, due to a balanced chromosomal translocation between the distal q arms of chromosomes 4 and 12, t(4;12)(q25;q24.2) 2 .Following the publication of these three cases involving hypogonadism, a de novo interstitial deletion del(12)(q24.3q24.33)was described in a male individual with ambiguous genitalia and DD in 1999 4 .
We postulated that the four previously reported chromosomal rearrangements affecting the 12q24 region could be attributed to the haploinsufficiency of a specific gene responsible for KS or isolated hypogonadotropic hypogonadism (IHH).Successful positional cloning of KS-related genes at the translocation breakpoints has been demonstrated in previous studies 9,19 .To explore the potential contribution of the translocation to the phenotypes observed in male Patient 1 with t(7;12)(q22;q24) 1 , we conducted mapping and sequencing of both translocation breakpoints with the aim of identifying genes that might be involved in KS, ID, or both 11 .
The translocation breakpoint was precisely mapped through physical mapping using FISH and Southern blot hybridization, leading to the identification of five genes at both breakpoints.The gene ZNF804B, located 37 kb distal to the 7q21.13breakpoint, was found to be the closest gene to this breakpoint, while STEAP4 mapped 416 kb proximal to it.At the 12q23.1 breakpoint, the non-coding RNA RMST was directly disrupted, with NEDD1 and PAFAH1B2P2 mapping closest to the proximal and distal sides of the breakpoint, respectively.Based on the molecular analysis results, the cytogenetic band locations on both chromosomes 7 and 12 of the apparently balanced chromosome translocation were accurately revised as t(7;12)(q21.13;q23.1) 8,11,12.Considering the overlapping phenotypes of KS observed in previously reported chromosomal rearrangements, we hypothesized that the causative gene for KS is located on chromosome 12.We screened three selected candidate genes (RMST, NEDD1, PAFAH1B2P2) for mutations based on their proximity to the 12q23.1 breakpoint.In intron 2, RMST was found to be disrupted, and mutation screening of this gene was performed in 48 KS patients who tested negative for ANOS1 and FGFR1.In a KS male patient, we identified a heterozygous nucleotide change (C/C to C/T at the 214th nucleotide in exon 10 of RMST NR_152618.1),which was later determined to be a polymorphism as the patient's two healthy sisters shared the same nucleotide change.Interestingly, the patient and his mother with anosmia were found to have an FGFR1 mutation (c.821G>A, p.E274G) 25 , suggesting FGFR1 as the cause of the phenotype in this family.We also screened PAFAH1B2P2 and NEDD1, which map 248 kb downstream and 513 kb upstream from the breakpoint, respectively, in the same cohort of 48 recruited KS patients, but no evidence of mutations was found.
Within the breakpoint region at 7q21.13, two additional genes were mapped.ZNF804B, located 37 kb downstream from the breakpoint, is the closest gene to the breakpoint 8 .ZNF804B belongs to the zinc finger protein family and consists of four exons.Although it has not been extensively studied, previous research suggests its implication in autism spectrum disorder (ASD) and NDDs 26,27 .Therefore, dysregulation of ZNF804B due to position effect may contribute to ID observed in Patient 1.On the other hand, STEAP4, located at 7q21.12, is situated 416 kb upstream from the breakpoint.Acting as a metalloreductase, STEAP4 plays a role in adipocyte development and metabolism, primarily within the Golgi apparatus.Mutation screening of both ZNF804B and STEAP4 in the cohort of 48 recruited KS patients did not reveal any potential disease-causing mutations; however, polymorphisms were detected.Furthermore, we also screened for mutations in ANOS1 and FGFR1 in the patient with the t(7;12) translocation but did not find any potential disease-causing mutations (data not  136 , two missense [134][135][136] , one splice 136 , and three frameshift 136,137 135 , and one missense 26,27,45,117 variants in NDD patients DENND5B is interacting with RAB11A 60 , and GRB10 84 , two genes involved in NDDs ETFBKMT (615256) NDD 12p11.21Enables heat shock protein binding activity and protein-lysine N-methyltransferase activity One missense 134,135 and one synonymous 27,134,135 variants in NDD patients ETFBKMT is interacting with TUBB2A 90 , TUBB4A 90 , DARS2 55 , and GLS Approximately 6% of carriers of balanced translocations exhibit abnormal phenotypes 28 due to disruption of genes at the breakpoints or dysregulation (position effect), resulting in reduced gene expression caused by separation from their cis regulatory elements 29 .However, it is worth mentioning that 40% of patients with apparently balanced translocations have been reported to carry at least one deletion at one of the breakpoints or in other genomic regions, indicating that deletions may be common in seemingly balanced chromosome rearrangements 14 .
After not finding any mutations in the five candidate KS genes at or near the breakpoints of the apparent balanced translocation, we conducted aCGH analysis, which revealed a significant finding.We identified a cryptic 4.7 Mb submicroscopic microdeletion at 12p11.21-12p11.23.Interestingly, this cryptic microdeletion was not previously detected in molecular-level studies of the breakpoints in Patient 1 (DGAP032) 8,11,12 .
While there have been reports of chromosomal variations, including copy number variations (CNVs), in lymphoblastoid cell lines (LCLs), it is worth noting that these studies did not associate chromosome 12 with significantly different numbers of CNVs 30 .Therefore, it is unlikely that del(12)(p11.21p11.23) is solely a cultureinduced artifact, although we cannot completely rule out this possibility.Unfortunately, due to the unavailability of parental samples, we were unable to determine the de novo status of this microdeletion.
In cases where a patient has concomitant genomic rearrangements, such as an unbalanced translocation and a simultaneous translocation-unrelated microdeletion or microduplication, it has been observed that the disease gene can be located at one of the translocation genomic breakpoints, although this is rare.For instance, in a female patient affected with autism and ID with t(14;21)(q21.1;p11.2)dnand 2.6 Mb of microdeletion 15 genes at 2q31.1, the causative gene LRFN5 (Leucine-Rich Repeat and Fibronectin Type III Domain-Containing Protein 5, MIM 612811) was found dysregulated at the 14q21.1 translocation breakpoint 31 .However, in most cases, the disease gene is within a copy number variant (CNV), as demonstrated by the identification of two positional ID candidate genes, VAMP8 (Vesicle-Associated Membrane Protein 8, MIM 603177) and RNF181 (Ring Finger Protein 181, MIM 612490) at 2p11.2, in a patient with an unbalanced t(8;10)(p23.3;q23.2) involving a cryptic 390 kb duplication region at 2p11.2 20 .
In addition to KS, Patient 1, with an unbalanced chromosome translocation, also presented with ID 1 .While recent reports have identified genes associated with IHH and ID 32,33 , multiple genes contributing to the comprehensive phenotype are plausible explanations, as seen in contiguous gene deletion syndromes like Potocki-Shaffer-Syndrome 6 or a deletion on chromosome X causing KS coupled with ID 34 .
Given the shared neurodevelopmental phenotypes observed in our seven CNV patients, as well as additional eight unpublished CNV cases from the DECIPHER database, this microdeletion, encompassing 29 genes, is likely to harbor the disease genes involved in these common phenotypes (Fig. 2A, Tables 1 and 3).
Out of the 15 heterozygous CNVs at 12p11.2, ten cases were duplications, while the remaining five cases were deletions (Fig. 2A, Tables 1 and 3).Each case exhibited at least one neurodevelopmental phenotype.In some instances, the CNVs were inherited from one parent with an unknown phenotype, while the inheritance status of the others remains unknown (Tables 1 and 3).By examining the pHaplo/pTriplo scores, we evaluated the potential haploinsufficiency and triplosensitivity of our 12 candidate genes using a dosage-sensitivity metrics catalog encompassing data for 18,641 genes 35 .Among the analyzed genes, seven demonstrated concurrent  23.This observation is supported by the phenotypic similarities observed in patients with both deletions and duplications in this region.Any copy number variation (CNV) within this region has the potential to disrupt the precise stoichiometric control of gene expression at the protein level, thereby contributing to phenotypic alterations 36 .
Based on in silico comparative genomic analysis at 12p11.21-12p11.23,we propose TSPAN11 (Tetraspanin 11) as a putative candidate gene for KS.A missense variant c.203G>A (NM_001080509.3) in TSPAN11 was identified in a KS patient, resulting in an amino acid substitution from Glycine to Aspartic acid at position 68 37 .This variant shows high deleterious CADD score of 25.8 (HG38).
Furthermore, we have identified seven candidate genes for ID or NDD: TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B and ETFBKMT (Table 2).Candidate genes for ID and NDDs were identified by searching multiple human disease databases, such as HGMD, MGI, BioGrid, and STRING.Selection criteria included reported nucleotide variants associated with NDDs in the candidate genes or their interacting genes.We also assessed their interactions with known NDD genes and considered the behavioral phenotypes observed in knockout mice.
The putative position effect of TM7SF3, along with the inclusion of STK38L and ARNTL2 in CNVs at 12p11.23, in addition to their sporadic variants reported in NDD patients, is likely to provide an explain their candidacy.Specifically, a patient with NDD was found to have a de novo missense variant in TM7SF3 (Transmembrane 7 Superfamily Member 3, MIM 605181) 27,38 .Furthermore, an interacting protein of TM7SF3 39 , HNRNPL (Heterogeneous Nuclear Riboprotein L, MIM 603083), was described to have one missense variant in an ID patient 40 .
In mouse hippocampal neurons 41 , STK38L (Serine/Threonine Kinase 38 Like, aka NDR2, Nuclear Dbf2 Related Kinase 2, MIM 615836) regulates the morphology and division of neuronal cells [42][43][44] , as well as integrindependent dendritic and axonal growth.Consequently, Stk38l KO mice exhibit arbor-specific alterations of dendritic complexity in the hippocampus 41 .De novo nonsense and missense variants in STK38L have been identified in individuals with ASD 45 and schizophrenia 46 , respectively.Genes mutated in schizophrenia are also mutated in autism and ID 46 .
The third candidate gene of ID at 12p11.23 is ARNTL2 (Aryl Hydrocarbon Receptor Nuclear Translocator-Like Protein 2, MIM 614517), and its missense and nonsense variants were reported in patients with autism 47 , as well as in those with developmental and epileptic encephalopathy 48 .Proteins that physically interact with one another frequently participate in the same biological activity, and mutations in these genes may result in similar clinical features.Among the interactors of ARNTL2, CTTNBP2 (Cortactin Binding Protein 2, MIM 609772) 49 stands out, as it has been associated with 26 de novo genetic variants in probands with autism/DD 50 .Furthermore, another noteworthy interactor UBE3A (Ubiquitin-Protein Ligase E3A, MIM 601623) 51 , a well-known Angelman syndrome gene 52 , has two frameshift variants reported in autistic individuals 53,54 .Another interactor 55 PER2 (Period Circadian Regulator 2, MIM 603426), has been described with eight variants in individuals with autism 45,56,57 .
INTS13 (Integrator Complex Subunit 13, MIM 615079), also known as ASUN (Asunder, Spermatogenesis Regulator) is mapped 65 kb distal from the 12p11.23 telomeric breakpoint of the 4.7 Mb microdeletion.Although it is not directly encompassed in the 4.7 Mb deletion at 12p11.21-12p11.23,it might be dysregulated by a positional effect 29 contributing to the KS phenotype seen in this Patient 1 with an unbalanced chromosome translocation 1 .INTS13 plays a crucial role in spermatogenesis in Drosophila melanogaster, as demonstrated by studies showing spermatocyte arrest during prophase of meiosis I in Drosophila knockout models 98 .Additionally, germline expression of mouse Asun (Ints13) rescued sterility and dynein mislocalization in Asun mutant flies 99 .Three variants in this gene are reported in NDD patients (Table 2).
PTHLH (parathyroid hormone like hormone MIM 168470) is associated with brachydactyly 123 and explains this phenotype in the patient DCP308811.Conversely, bi-allelic variants of IPO8 (importin 8 MIM 605600) have been linked to cardiovascular defects, skeletal anomalies, and immune dysregulation 124 .However, we excluded both genes from our study as their phenotypes were unrelated to KS and ID.
We propose that, despite not being encompassed by small CNVs we used in comparative genomic mapping, the expression levels of the two ID candidate genes DENND5B, and ETFBKMT could be altered due to position effect 125,126 .This likely explains the observed NDD phenotypes, including dystonia, global DD, growth delay, motor delay, etc., in one DECIPHER proband DCP288321 (Fig. 2A and Table 3).
To substantiate their pathogenicity, we also confirmed the high expression of our candidate genes in five different human tissues (i.e.brain, fetal brain, muscle, ovary, and testis) relevant to the phenotype of KS and NDD (Fig. 4B).
During the preparation of this manuscript, genome sequencing was performed on this same Patient 1 to map the translocation breakpoint.The analysis indicated that the deletion of RMST could potentially be a cause of KS due to loss of function, although this conclusion was based on an erroneous assumption that this chromosome translocation is balanced 12 .RMST physically interacts with SOX2 127 , a transcription factor known to regulate neural fate, and aids in the binding of SOX2 to the promoter of target genes important in neurogenesis 127 .Notably, SOX2 (SRY-box transcription factor 2, MIM 184429) is a known disease gene for hypogonadotropic hypogonadism and combined pituitary hormone deficiency 128 .Additionally, RMST has been associated with rhabdomyosarcoma and melanoma 129 .
However, the ostensible pathogenicity of RMST in KS remains to be confirmed, as this Patient 1 has an unbalanced translocation accompanied by an additional 4.7 Mb microdeletion that we identified.Moreover, no mutations of this gene were found in the 48 KS patients we recruited.This case underscores the necessity and significance of aCGH or sequencing analysis in individuals with disease-associated, apparently balanced translocations to rule out cryptic microdeletions.Simultaneously, our study highlights the benefits of the integrated usage of karyotype analysis, aCGH, and sequencing for a comprehensive approach to phenotypic assessment.
In summary, our study revealed that an apparently balanced translocation t(7;12)(q22;q24) 1,11,12 in Patient 1 is actually unbalanced, and the 4. 7 Mb cryptic deletion at 12p11.21-12p11.23likely explains the phenotype of KS and ID in the patient carrying these two unrelated chromosomal rearrangements.Through in silico comparative genomic mapping with 14 additional CNVs in this genomic region, we identified one potential KS candidate gene (TSPAN11), seven candidate genes for NDD (TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B, and ETFBKMT), and four candidate genes for KS with ID (INTS13, REP15, PPFIBP1, and FAR2).The candidacy of these genes was further supported by their high-level expression pattern in the relevant human tissues.We propose that some dosage-sensitive genes in this genomic region might contribute to sexual and/or cognitive impairment in patients with KS, ID, or both, as indicated by the probabilities of dosage sensitivity (STK38L: pHaplo/pTriplo 0.77/0.94,DENND5B: 0.96/0.96,PPFIBP1: 0.81/0.66) 35.This suggests that both increased or decreased expression can result in related deleterious phenotypes.To generate supporting evidence for this hypothesis, performing RT-qPCR or western blot analyses of candidate genes that are present in both the deletion and duplication regions will be crucial.
Considering the well-known phenomenon of heterogeneous neurodevelopmental phenotypes caused by mutations in the same gene 130 , our candidate genes at 12p11.21-12p11.23present an opportunity to identify NDD disease genes from NGS databases containing a myriad of autosomal dominant or de novo variants of uncertain significance (VUSs).

Patients and methods
Human patients.This study was approved by the Augusta University Institutional Review Board (IRB) and was conducted following the principles outlined in the American Society of Human Genetics Code of Ethics.Participants were recruited by endocrinologists, gynecologists, or clinical geneticists for clinical characterization and genetic studies.Informed consent, approved by the Augusta University IRB, was obtained from all participants before conducting the genetic studies.The cohort comprised 48 American probands with KS or IHH and other accompanying minor phenotypes (24 women, 24 men).We isolated DNA from each participant's peripheral blood, and subsequent mutation screening of positional candidate genes identified at both genomic breakpoints was performed.Notably, all 48 individuals tested negative for ANOS1 and FGFR1 variants.The KS diagnosis was based on the presence of IHH, which is characterized by delayed or absent pubertal maturation, along with low serum gonadotropins and sex steroids.Additionally, a smell deficit was identified from the patient's medical history and/or formal smell testing.

Clinical reports.
Patient 1-4.7 Mb del (12)(p11.21p11.23),t(7;12)(q21.13;q23.1)dn.A detailed description of patient DGAP032, who presented with a balanced de novo reciprocal translocation, 46,XY,t(7;12)(q22;q24)dn, was previously published in 1990 1 .In summary, this 44-year-old Chippewa/French man exhibited hypogonadotropic hypogonadism, based upon low levels of FSH, LH, and testosterone, along with sparse pubic hair, small testes (<1 cm), olfactory deficiency, skeletal and cranial anomalies, and ID.He sought medical attention at the age of 22 years due to delayed sexual development.During the evaluation, normal 17-hydroxycorticosteroids and abnormally low 17-ketosteroids and gonadotropin levels were observed.The epiphyseal centers of most long bones and the spine were not yet closed, indicating delayed bone maturation.The bone age of the hand was estimated to be around 12 years, and the metacarpals appeared shortened and clubbed distal ends, especially the 4 th right metacarpal, indicative of brachydactyly.In addition, a sharply outlined foramen near the internal occipital protuberance of the occipital bone was noted.In 1984, lymphocyte chromosome studies demonstrated an apparently reciprocal translocation, t(7;12)(q22;q24), but upon further molecular analysis, it was revised as t(7;12)(q21.13;q23.1)dn(Fig. 1A) 11 .Clinical signs of KS were not observed in his five full sisters, one full brother, two half-brothers, or one half-sister.
Patient 2-500 kb dup (12)(p11.23).Patient 2 (50943) is a 36-year-old man with a medical history including ID, DD, autism, and dyslexia.He was born with a birth weight of 3500 g.At 48 months of age, his IQ was assessed to be between 30 and 50.He achieved certain milestones independently, sitting at around 11 months, walking at 27 months, becoming toilet-trained by 2 years, and uttering his first words at 30 months.However, he also faced challenges such as learning disability, language delay, and speech delay.As he grew older, at 29 years of age, he was noted to frequently speak loudly and demand things with incomprehensible associations.While his tantrums were primarily verbal and seldom physical, they could be intense.He was extremely restless with no tics or stuttering, often repetitively using words and parrots "yes".He spoke very loudly.Although no apparent physical abnormalities were observed, hypertelorism and a slightly thicker lower lip were noted.He did not have seizures, bone anomalies, facial dysmorphism, or shortened fingers or toes (Fig. 5).However, a brain MRI displayed an arachnoid cyst.Further analysis using aCGH on genomic DNA revealed a de novo 500 kb duplication at 12p11.(12)(p11.22p11.23).Patient 3 (31606) is a 10-year-old female with a history of DD, speech delay, expressive language delays, and attention deficit hyperactivity disorder (ADHD).At 3 days old, she had chronic diarrhea and struggled with poor growth or weight gain.Over the course of her early life, she was hospitalized four times between 52 days and 5 months of age due to failure to thrive and chronic diarrhea.Despite thorough testing, no conclusive diagnosis was reached during an 18-day hospitalization.Her chronic dehydration resulted from her body's inability to absorb essential nutrients effectively as fluids rapidly passed through her system.A second gastroenterology opinion was sought, but no further testing was conducted.Dur-  Patient 5-215 kb dup (12)(p11.23).Patient 5 (DCP295472) is 12 years and 5-month-old Caucasian male diagnosed with ASD and learning disability.He was born at full-term by spontaneous vaginal delivery, with a weight of 3.810 kg (76th centile), length of 50 cm (38th centile), and head circumference of 34 cm (22nd centile).He suffered from repetitive ear infections in childhood and underwent a surgery for ear tubes.He began walking at 24 months, but with language delay associated with a global DD and impaired motor skills.At the age of 2, neurobehavioral concerns arose, including repetitive movements, stereotypy, difficulties regulating emotions, and limited facial expressions.He also developed sleeping disorders, waking up early at 3-4 am, which were treated with melatonin.Food selectivity was observed, and at 10 years of age, his weight was 30.8 kg (median) with a height of 135 cm (median) and head circumference of 53 cm (median).Right eyelid ptosis and dysmorphic features, such as bilateral downward palpebral fissures, frontal hair spike, lower lip eversion were noted.Although an ophthalmic surgery was scheduled, the patient never attended the anesthesiologist appointment.Additionally, he also presented with clinodactyly of the 5 th finger on his right hand (Fig. 5).He attended a regular schooling at Abbotsford Virtual School.As part of his treatment plan, he was prescribed methylphenidate and melatonin.The patient underwent a chromosome Fragile X analysis and aCGH as part of the diagnostic tests.She exhibited behavioral challenges including temper tantrums and a short attention span.Currently in the 3rd grade, she has mild ID and learning difficulties, with a poor attention span and easy distractibility.She received a special education with adapted curricular.She was seen in a neuropediatric clinic for recurrent headaches, which improved after discontinuing methylphenidate treatment.She is also under care in an endocrinology clinic for obesity.She has normal stature, normal head circumference and no dysmorphic features.She presents with a large thumb, shortening of the IV and V metacarpals and metatarsals, and tapering fingers without clinodactyly or syndactyly.She has short toes and a short 4 th metatarsal bone (Fig. 5).Her father with the same microdeletion had some learning difficulties and has similar foot abnormalities.However, he completed the 12th grade, as did his brothers, who were not tested for aCGH.

Figure 1 .
Figure 1.(A) Ideogram illustrating the revised t(7;12)(q21.13;q23.1)dnkaryotype in patient 1, DGAP032.After breakage of two chromosomes, the reciprocal exchange of chromosome segments between chromosomes 7 and 12 has taken place, generating two derivative chromosomes in the patient with two horizontal gray bars at the breakpoint positions.On chromosome 12, the deleted cryptic segment at 12p11.21-12p11.23identified was depicted as a horizontal yellow bar.(B) Physical mapping of the 12q23 translocation breakpoint of DGAP032 by FISH and Southern blot hybridization.Diagram shows breakpoint refined by FISH and Southern blot.For FISH, two BAC clones RP11-492N15 and CTD-2235H23 spanning the breakpoint, which is represented as a dashed vertical red line, were identified, and shown as red bars.The breakpoint was further narrowed to 3.5 kb between CTD-2268E11 and CTD-2542D2 shown as black bars.Southern blot analysis using Blot 4 with the probe KS-7 identified aberrant fragments of patient DNA digested with five different restriction enzymes (DraI, BbVI, MboI, PvuII, and HaeIII, Fig. 3B).The breakpoint was refined to 492 bp between the centromeric end of HaeIII and the telomeric end of DraI, which was then isolated with suppression PCR and sequenced.The breakpoint at 12q23.1 is located in intron 2 of RMST (NR_152618.1).

Figure 2 .
Figure 2. (A) Cryptic 4.7 Mb heterozygous deletion encompassing 29 genes located from 12p11.21 to 12p11.23.Eight heterozygous CNV cases from the DECIPHER database are denoted by DCP along with six heterozygous CNVs (patients 2-7) we recruited.These 14 CNVs are encompassed in Patient 1 to help narrow down the candidate gene region by in silico comparative genomic mapping.Red bars represent deletions, whereas blue bars represent duplications.One gene in green is a candidate for KS, whereas seven genes in brown are NDD candidates.Four genes in purple are chosen as candidates for KS combined with ID.Arrow indicates transcriptional direction of each gene.(B) FISH with two BAC clones spanning the breakpoints at 12q23.1 and 7q21.13,respectively.BAC clone RP11-492N15 shows normal signals on normal chromosome 12 and split signals on both derivative chromosomes 7 and 12. BAC clone CTD-2325L19 with normal signals on normal chromosome 7 and split signals on both derivative chromosomes 7 and 12.

Figure 3 .
Figure 3. (A) Restriction map of a 7.3 kb HindIII genomic fragment (97,460,797-97,468,119/hg38) containing the breakpoint on 12q23.BamHI, HincII, HpaI and SnaBI do not have restriction sites on this map and only the relevant restriction sites in relation to the fragments detected by shown probes are indicated.Positions of the PCR-derived probes KS-1 to KS-7 used for the breakpoint mapping by Southern analysis are indicated below.Note that the breakpoint is located 2.7 kb upstream of the region narrowed by FISH.(B) Genomic DNA blots hybridized with probes from the 12q23 breakpoint region.Each lane contains genomic DNA digested with the designated restriction enzymes from either DGAP032 (P) or a normal control (C).Additional bands in the P lanes indicate novel restriction junction fragments generated by the interchromosomal exchange.The hybridization probe KS-7 detected aberrant bands indicated by red arrows containing breakpoints at 12q23.The numbers next to normal restriction fragment seen in both patient and control lanes indicate the size of genomic restriction fragment in bp.

Figure 4 .
Figure 4. (A) Sequences of the junction fragment composed of two different chromosomes at the translocation breakpoints.Sequence comparison of the normal chromosomes 7 and 12 with der(7) and der(12) at the breakpoints junctions.Three bp sequence CTC from chr12 is deleted at the junction of der(7), and a 17 bp insertion was found at the junction of the der(12).(B) Transcript levels of INTS13, TM7SF3, STK38L, ARNTL2, PPFIBP1, REP15, FAR2, ERGIC2, TMTC1, TSPAN11, and DENND5B in five different human tissues (i.e.brain, fetal brain, muscle, ovary and testis) were determined by RT-qPCR.Varying levels of expression of these candidate genes were detected in different tissue samples.

Figure 5 .
Figure 5. Facial and limb pictures of individuals with CNVs at 12p11.21-12p11.23.P2: Patient 2 on Table 1 (a) hypertelorism and slightly thicker lower lip (b) borderline posteriorly rotated right ear, (c) palms showing special form and disrupted vertical creases.P4: Patient 4 on Table1shows (a) microcephaly at 1 year (b) lateral view of right face at 3 years 10 months (c) hypoplasia of the vertical palmar flexion creases (PFC) on the right palm (d) the vertical PFC looks short and the transverse proximal looks with some tendency to Sydney line on the left palm (e) tapering fingers (f) a minor syndactyly between toes 2 and 3 (g) small toes and a questionable gap between toes 1 and 2. P5: Patient 5 on Table1shows (a) small forehead, right eyelid ptosis, bilateral downward palpebral fissures, frontal hair spike, lower lip eversion (c) tapering fingers and bilateral short 5th fingers with clinodactyly (d) the transverse proximal PFC has tendency to join the transverse distal, a variant of the PFC.P6: Patient 6 on Table1shows (a,b) short 4th toes likely due to short 4th metatarsals on both feet (c,d) dorsal view of tapering fingers with short 4th and 5th fingers in both hands (e) the transverse distal palmar flexion crease is rather short and doesn't start at the ulnar margin on both palms (f) dorsal view of both hands showing bilateral short 4th fingers and short 5th fingers especially at right.P7: Patient 7 on Table1shows (a) essentially no dysmorphism (b,c) microcephaly (d) tapering fingers (e,f) clinodactyly of the 5th toes and a gap between toes 1 and 2 in the left foot.

Table 2 .
Twelve autosomal dominant positional candidate genes identified by in silico CNV mapping at 12p11.21-12p11.23from telomeric to centromeric direction.They include one gene for KS, seven genes for NDDs, and four genes for KS coupled with NDDs.NDD denotes neurodevelopmental disorder, ID intellectual disability, HH hypogonadtropic hypogonadism, ASD autism spectrum disorder, KS Kallmann syndrome.
).It is important to note that the possibility of mutations in other causative genes for KS in this patient cannot be excluded.

Table 3 .
Summary of the eight heterozygous DECIPHER CNV cases at 12p11.21-12p11.23.This suggests that duplications and deletions may be used to further refine the candidate gene region at 12p12.11-12p11.
ing infancy, she exhibited aversion to textured foods, necessitating feeding therapy.She was also diagnosed with congenital sucrose-isomaltase deficiency (CSID).She managed the milestone of walking at the age of 15 months, but her inability to crawl led to the need for occupational therapy.Additionally, she required social/ emotional therapy.Early childhood education and speech therapy were implemented, and she later attended a mainstream school at 4.5 years old.aCGHanalysisrevealed a 750 kb duplication at 12p11.22-p11.23,resulting in 46,XX,arr[hg 38](chr12:27,157,806-27,907,534)x3.Patient 4-1.94 Mb dup(12)(p11.22).Patient 4 (022821) is a four-year-old white male with a medical history of learning disability, dyslexia, hypotonia, language delays, and delayed speech.Born at full-term, via normal spontaneous delivery, his developmental milestones were delayed, with crawling at 7 months, sitting alone at 9 months, and walking unassisted at 2 years of age.Due to lack of strength in his abdomen, he needed some time to be able to sit alone.He began taking his first steps at 26 months, and he has been receiving occupational therapy (OT) since the age of 30 months.At 15 months, a neurologist diagnosed him with microcephaly (OFC 43 cm, approximately -3SD for age), short stature, DD, and impaired motor skills.Additionally, he has syndactyly and tapering fingers.At age 2 years, visual evoked potential testing detected some visual asymmetry.Maternal CMV infection and other intrauterine infections were ruled out.Though attentive to his surroundings, he showed no attempt to communicate.At 3 years and 10 months, it was discovered that he had moderate hearing loss in the left ear and mild hearing loss in the right ear (Fig.5).An EEG performed at age 23 months showed normal results.Chromosomal analysis was normal, but a SurePrint-Ga Human Genome Kit Agilent aCGH (4x180K) revealed a 1.94Mb duplication, 46,XY,arr[hg 38](chr12:28,047,313-29,990,575)x3 in 12p11.22.The patient presents characteristics of ASD, such as walking on tiptoe, repetitive movements, and obsession with spinning objects.
Vol:.(1234567890) Scientific Reports | (2023) 13:12984 | https://doi.org/10.1038/s41598-023-40037-4www.nature.com/scientificreports/ The chromosomal analysis showed a paternally inherited 215 kb duplication, 46,XY,arr[hg 38] (chr12:27,400,730-27,615,518)x3 pat at 12p11.23 and 407 kb deletion at 2q13, arr[hg 38] (chr2: 108,684,076-109,090,916)x1 pat.The father also presented with anxiety disorder, similar to his affected son, but follow-up with the father was limited.Patient 6-2.18 Mb del(12)(p11.21p11.22).Patient 6 (DCP370033) is an 11-year-old Caucasian girl with a history of DD, speech delay, learning difficulties, and ADHD.She is the first child of a healthy and non-consanguineous couple.The pregnancy was uncomplicated, and she was born full term by spontaneous vaginal delivery with a birth weight of 3 kg (99th centile).She has two younger sisters, one of whom has a unilateral third finger brachydactyly, as reported by her mother.The patient sat independently at approximately 7 months, started walking at 22 months, and began speaking her first words late.At age 5 years, speech delay with articulation problems became evident.Psychological tests revealed a clear discrepancy between performance and verbal capacities.
The father's brothers despite having more severe learning difficulties, are capable of living independently.aCGH(CGX-HD180K by PerkinElmer®) revealed a paternally inherited 2.18 Mb deletion-arr[hg38] 12p11.21p11.22(chr12:28,414,984-30,598,365)x1patanda 49 kb duplication with unknown inheritance at Xp22.33, arr[hg38] (chrX:1,259,698-1,308,697)x3.Patient 7-652 kb dup(12)(p11.22).Patient 7 (DCP293962) is a 48-year-old Caucasian male with a history of DD, dyslexia and ID with poor academic performance.He was born full term by spontaneous vaginal delivery with an average birth weight.His neonatal period was unremarkable.He achieved normal gross and fine motor milestones and displayed average social interactions during childhood.In early childhood, he was diagnosed with dyslexia and has speech delay.He could not read and can barely write his name.He left school in grade 8 and has been on a disability pension since then because he had difficulties holding a job.Despite his challenges, he does not have any disruptive or aggressive behavior.At the age of 42, he married and had the following physi-