Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics

Abstract

Purpose

Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands.

Methods

Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays.

Results

In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband.

Conclusion

Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

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Fig. 1: Distribution of different types of alleles and deep-intronic variants in ABCA4.
Fig. 2: Novel splice defects due to deep-intronic ABCA4 variants.
Fig. 3: Splice defects due to variants in ABCA4 introns 13 and 44.
Fig. 4: Novel heterozygous structural variants in ABCA4.

References

  1. 1.

    Carss KJ, Arno G, Erwood M, et al. Comprehensive rare variant analysis via whole-genome sequencing to determine the molecular pathology of inherited retinal disease. Am J Hum Genet. 2017;100:75–90.

  2. 2.

    Blacharski PA, Newsome DA. Bilateral macular holes after Nd:YAG laser posterior capsulotomy. Am J Ophthalmol. 1988;105:417–418.

  3. 3.

    Cornelis SS, Bax NM, Zernant J, et al. In silico functional meta-analysis of 5,962 ABCA4 variants in 3,928 retinal dystrophy cases. Hum Mutat. 2017;38:400–408.

  4. 4.

    Sangermano R, Khan M, Cornelis SS, et al. ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease. Genome Res. 2018;28:100–110.

  5. 5.

    Khan M, Cornelis SS, Khan MI, et al. Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease. Hum Mutat. 2019;40:1749–1759.

  6. 6.

    Schulz HL, Grassmann F, Kellner U, et al. Mutation spectrum of the ABCA4 gene in 335 Stargardt disease patients from a multicenter German cohort—impact of selected deep intronic variants and common SNPs. Invest Ophthalmol Vis Sci. 2017;58:394–403.

  7. 7.

    Bauwens M, Garanto A, Sangermano R, et al. ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants. Genet Med. 2019;21:1761–1771.

  8. 8.

    Braun TA, Mullins RF, Wagner AH, et al. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum Mol Genet. 2013;22:5136–5145.

  9. 9.

    Sangermano R, Garanto A, Khan M, et al. Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides. Genet Med. 2019;21:1751–1760.

  10. 10.

    Zernant J, Xie YA, Ayuso C, et al. Analysis of the ABCA4 genomic locus in Stargardt disease. Hum Mol Genet. 2014;23:6797–6806.

  11. 11.

    Bauwens M, De Zaeytijd J, Weisschuh N, et al. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum Mutat. 2015;36:39–42.

  12. 12.

    Bax NM, Sangermano R, Roosing S, et al. Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum Mutat. 2015;36:43–47.

  13. 13.

    Fadaie Z, Khan M, Del Pozo-Valero M, et al. Identification of splice defects due to noncanonical splice site or deep-intronic variants in ABCA4. Hum Mutat. 2019;40:2365–2376.

  14. 14.

    Maugeri A, van Driel MA, van de Pol DJR, et al. The 2588G -> C mutation in the ABCR gene is a mild frequent founder mutation in the western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am J Hum Genet. 1999;64:1024–1035.

  15. 15.

    Rivera A, White K, Stohr H, et al. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am J Hum Genet. 2000;67:800–813.

  16. 16.

    Jaakson K, Zernant J, Kulm M, et al. Genotyping microarray (gene chip) for the ABCR (ABCA4) gene. Hum Mutat. 2003;22:395–403.

  17. 17.

    Maia-Lopes S, Aguirre-Lamban J, Castelo-Branco M, Riveiro-Alvarez R, Ayuso C, Silva ED. ABCA4 mutations in Portuguese Stargardt patients: identification of new mutations and their phenotypic analysis. Mol Vis. 2009;15:584–591.

  18. 18.

    Albert S, Garanto A, Sangermano R, et al. Identification and rescue of splice defects caused by two neighboring deep-intronic ABCA4 mutations underlying Stargardt disease. Am J Hum Genet. 2018;102:517–527.

  19. 19.

    Charbel Issa P, Barnard AR, Herrmann P, Washington I, MacLaren RE. Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization. Proc Natl Acad Sci U S A. 2015;112:8415–8420.

  20. 20.

    Allocca M, Doria M, Petrillo M, et al. Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest. 2008;118:1955–1964.

  21. 21.

    Lu B, Malcuit C, Wang S, et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells. 2009;27:2126–2135.

  22. 22.

    Cremers FPM, Cornelis SS, Runhart EH, Astuti GDN. Author response: penetrance of the ABCA4 p.Asn1868Ile allele in Stargardt disease. Invest Ophthalmol Vis Sci. 2018;59:5566–5568.

  23. 23.

    Runhart EH, Sangermano R, Cornelis SS, et al. The common ABCA4 variant p.Asn1868Ile shows nonpenetrance and variable expression of Stargardt disease when present in trans with severe variants. Invest Ophthalmol Vis Sci. 2018;59:3220–3231.

  24. 24.

    Zernant J, Lee W, Collison FT, et al. Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration. J Med Genet. 2017;54:404–412.

  25. 25.

    Hafez M, Hausner G. Convergent evolution of twintron-like configurations: one is never enough. RNA Biol. 2015;12:1275–1288.

  26. 26.

    Zernant J, Lee W, Nagasaki T, et al. Extremely hypomorphic and severe deep intronic variants in the ABCA4 locus result in varying Stargardt disease phenotypes. Cold Spring Harb Mol Case Stud. 2018;4:a002733.

  27. 27.

    Nassisi M, Mohand-Said S, Andrieu C, et al. Prevalence of ABCA4 deep-intronic variants and related phenotype in an unsolved “one-hit” cohort with Stargardt disease. Int J Mol Sci. 2019;20:5053.

  28. 28.

    Runhart EH, Valkenburg D, Cornelis SS, et al. Late-onset Stargardt disease due to mild, deep-intronic ABCA4 alleles. Invest Ophthalmol Vis Sci. 2019;60:4249–4256.

  29. 29.

    Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem. 2010;79:181–211.

  30. 30.

    McVey M, Lee SE. MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet. 2008;24:529–538.

  31. 31.

    Vissers LE, Bhatt SS, Janssen IM, et al. Rare pathogenic microdeletions and tandem duplications are microhomology-mediated and stimulated by local genomic architecture. Hum Mol Genet. 2009;18:3579–3593.

  32. 32.

    Bacolla A, Wells RD. Non-B DNA conformations, genomic rearrangements, and human disease. J Biol Chem. 2004;279:47411–47414.

  33. 33.

    Fingert JH, Eliason DA, Phillips NC, Lotery AJ, Sheffield VC, Stone EM. Case of Stargardt disease caused by uniparental isodisomy. Arch Ophthalmol. 2006;124:744–745.

  34. 34.

    Riveiro-Alvarez R, Valverde D, Lorda-Sanchez I, et al. Partial paternal uniparental disomy (UPD) of chromosome 1 in a patient with Stargardt disease. Mol Vis. 2007;13:96–101.

  35. 35.

    Liehr T. Cytogenetic contribution to uniparental disomy (UPD). Mol Cytogenet. 2010;3:8.

  36. 36.

    Rivolta C, Berson EL, Dryja TP. Paternal uniparental heterodisomy with partial isodisomy of chromosome 1 in a patient with retinitis pigmentosa without hearing loss and a missense mutation in the Usher syndrome type II gene USH2A. Arch Ophthalmol. 2002;120:1566–1571.

  37. 37.

    Roosing S, van den Born LI, Hoyng CB, et al. Maternal uniparental isodisomy of chromosome 6 reveals a TULP1 mutation as a novel cause of cone dysfunction. Ophthalmology. 2013;120:1239–1246.

  38. 38.

    Thompson DA, Gyurus P, Fleischer LL, et al. Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci. 2000;41:4293–4299.

  39. 39.

    Thompson DA, McHenry CL, Li Y, et al. Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively. Am J Hum Genet. 2002;70:224–229.

  40. 40.

    Wiszniewski W, Lewis RA, Lupski JR. Achromatopsia: the CNGB3 p.T383fsX mutation results from a founder effect and is responsible for the visual phenotype in the original report of uniparental disomy 14. Hum Genet. 2007;121:433–439.

  41. 41.

    Souzeau E, Thompson JA, McLaren TL, et al. Maternal uniparental isodisomy of chromosome 6 unmasks a novel variant in TULP1 in a patient with early onset retinal dystrophy. Mol Vis. 2018;24:478–484.

  42. 42.

    Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, et al. Predicting splicing from primary sequence with deep learning. Cell. 2019;176:535–48 e524.

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Acknowledgements

We thank Ellen Blokland, Duaa Elmelik, Emeline Gorecki, Marlie Jacobs-Camps, Charlene Piriou, Mariateresa Pizzo, and Saskia van der Velde-Visser for technical assistance. We thank Béatrice Bocquet, Dominique Bonneau, Krystyna H. Chrzanowska, Hélene Dollfus, Isabelle Drumare, Monika Heusipp, Takeshi Iwata, Beata Kocyła-Karczmarewicz, Atsushi Mizota, Nobuhisa Nao-i, Adrien Pagin, Valérie Pelletier, Rafal Ploski, Agnieszka Rafalska, Rosa Riveiro, Malgorzata Rydzanicz, Blanca Garcia Sandoval, Kei Shinoda, Francesco Testa, Kazushige Tsunoda, Shinji Ueno, and Catherine Vincent-Delorme for their cooperation and ascertaining STGD1 cases. We thank Rolph Pfundt for his assistance in exome sequencing data analysis. We are grateful to the Eichler and Shendure labs (Department of Genome Sciences, University of Washington), for assistance with the initial MIP protocol. We thank the European Reference Network (ERN)-EYE and European Retinal Disease Consortium (ERDC) networks, the Japan Eye Genetics Consortium, and the East Asian Inherited Retinal Disease Society.

This work was supported by the RetinaUK, grant number GR591 (to F.P.M.C.); a Fighting Blindness Ireland grant, grant number FB18CRE (to F.P.M.C., G.J.F.); a Horizon 2020, Marie Sklodowska-Curie Innovative Training Network entitled European Training Network to Diagnose, Understand and Treat Stargardt Disease; Frequent Inherited Blinding Disorder-StarT (813490) (to E.D.B., F.P.M.C., S.B., G.J.F.); Foundation Fighting Blindness USA, grant number PPA-0517-0717-RAD (to F.P.M.C.); the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C.); and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds and the Stichting Blinden-Penning that contributed through Uitzicht 2016-12 (to F.P.M.C.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden that contributed through UitZicht 2014-13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C.). This work was also supported by Groupement de Coopération Sanitaire Interrégional G4 qui réunit les Centres Hospitaliers Universitaires Amiens, Caen, Lille et Rouen (GCS G4) and by the Fondation Stargardt France (to C.-M.D.), Federal Ministry of Education and Research (BMBF), grant numbers 01GM0851 and 01GM1108B (to B.H.F.W.), programs SVV 260516, UNCE 204064, and PROGRES-Q26/LF1 of the Charles University (to B.K., L.D., P.L.). This work was supported by grant AZV NU20-07-00182 (to P.L., B.K. and L.D.). The work of A.D. was supported by Fighting Blindness Ireland, Health Research Board of Ireland and the Medical Research Charities Group (MRCG-2016-14) (to G.J.F.). This work was supported by grant AZV NU20-07-00182 (to P.L., B.K., L.D.). This work was also supported by the Ghent University Research Fund (BOF15/GOA/011), by the Research Foundation Flanders (FVO) G0C6715N, by the Hercules foundation AUGE/13/023 and JED Foundation (to E.D.B.). M.B. was PhD fellow of the FWO and recipient of a grant of the funds for Research in Ophthalmology (FRO). E.D.B. is Senior Clinical Investigator of the FWO (1802215N; 1802220N). The work of M.D.P-.V. is supported by the Conchita Rábago Foundation and the Boehringer Ingelheim Fonds. The work of C.A. is supported by grants PI16/0425 from ISCIII partially supported by the European Regional Development Fund (ERDF), RAREGenomics-CM (CAM, B2017/BMD-3721), ONCE, and Ramon Areces Foundation. This work was supported by the Peace for Sight grant (to D.S., A.A.). The work of L.R. and R.R. was supported by Retina South Africa and the South African Medical Research Council (MRC). This work was also supported by the Foundation Fighting Blindness, grant/award number BR‐GE‐0214–0639‐TECH and BRGE‐0518–0734‐TECH (to T.B.-Y., D.S., H.N.); the Israeli Ministry of Health, grant/award number 3‐12583Q4 (to T.B.-Y., D.S., H.N.); Olive Young Fund, University Hospital Foundation, Edmonton (to I.M.M.); the National Science Center (Poland) grant number N N402 591640 (5916/B/P01/2011/40) (to M.O.); and UMO-2015/19/D/NZ2/03193 (to A.M.T.). This work was supported by the Italian Fondazione Roma (to S.B., F.S.), the Italian Telethon Foundation (to S.B.), and the Ministero dell’Istruzione del l’Università e della Ricerca (MIUR) under PRIN 2015 (to S.B., F.S.). M.B.G. and A.M. were supported by the Daljit S. and Elaine Sarkaria Charitable Foundation. The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants.

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Correspondence to Frans P. M. Cremers PhD.

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Khan, M., Cornelis, S.S., Pozo-Valero, M.D. et al. Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics. Genet Med (2020). https://doi.org/10.1038/s41436-020-0787-4

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Keywords

  • ABCA4
  • Stargardt disease
  • smMIPs
  • deep-intronic variants
  • structural variants

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