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



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.


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.


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.


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.


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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).

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

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