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

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. Fil: Khan, Mubeen. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Cornelis, Stéphanie S.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Del Pozo Valero, Marta. Hospital Universitario Fundación Jiménez Díaz; España. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Whelan, Laura. Trinity College; Estados Unidos Fil: Runhart, Esmee H.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Mishra, Ketan. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Bults, Femke. Radboud University Nijmegen Medical Centre; Países Bajos Fil: AlSwaiti, Yahya. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: AlTalbishi, Alaa. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: De Baere, Elfride. University of Ghent; Bélgica Fil: Banfi, Sandro. Seconda Universita Degli Studi Di Napoli; Italia Fil: Banin, Eyal. The Hebrew University of Jerusalem; Israel Fil: Bauwens, Miriam. University of Ghent; Bélgica Fil: Ben Yosef, Tamar. The Ruth And Bruce Rappaport Faculty Of Medicine; Israel Fil: Boon, Camiel J. F.. Leiden University. Leiden University Medical Center; Países Bajos Fil: van den Born, L. Ingeborgh. Rotterdam Ophthalmic Institute; Países Bajos Fil: Defoort, Sabine. Universite Lille; Francia Fil: Devos, Aurore. Universite Lille; Francia Fil: Dockery, Adrian. Trinity College; Estados Unidos Fil: Dudakova, Lubica. Charles University and General University Hospital; República Checa Fil: Fakin, Ana. Charles University and General University Hospital; República Checa Fil: Farrar, G. Jane. Trinity College; Estados Unidos Fil: Ferraz Sallum, Juliana Maria. Universidade Federal de Sao Paulo; Brasil Fil: Fujinami, Kaoru. UCL Institute of Ophthalmology; Reino Unido Fil: Gilissen, Christian. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Glavac, Damjan. University of Ljubljana; Eslovenia Fil: Gorin, Michael B.. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Greenberg, Jacquie. University of Cape Town; Sudáfrica Fil: Hayashi, Takaaki. The Jikei University School of Medicine; Japón Fil: Hettinga, Ymkje M.. Bartiméus Diagnostic Center for Complex Visual Disorders; Países Bajos Fil: Hoischen, Alexander. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hoyng, Carel B.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hufendiek, Karsten. University Eye Hospital Hannover Medical School; Alemania Fil: Jägle, Herbert. University Regensburg; Alemania Fil: Kamakari, Smaragda. OMMA Ophthalmological Institute of Athens; Grecia Fil: Karali, Marianthi. Seconda Universita Degli Studi Di Napoli; Italia Fil: Kellner, Ulrich. No especifíca; Fil: Klaver, Caroline C. W.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Kousal, Bohdan. Charles University and General University Hospital; República Checa Fil: Lamey, Tina M.. University of Western Australia; Australia Fil: MacDonald, Ian M.. University of Alberta; Canadá Fil: Matynia, Anna. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: McLaren, Terri L.. University of Western Australia; Australia Fil: Mena, Marcela D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Meunier, Isabelle. Université Montpellier II; Francia Fil: Miller, Rianne. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Newman, Hadas. Universitat Tel Aviv; Israel Fil: Ntozini, Buhle. University of Cape Town; Sudáfrica Fil: Oldak, Monika. No especifíca; Fil: Pieterse, Marc. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Puech, Bernard. Universite Lille; Francia Fil: Ramesar, Raj. University of Cape Town; Sudáfrica Fil: Rüther, Klaus. No especifíca; Fil: Salameh, Manar. No especifíca; Fil: Salles, Mariana Vallim. Universidade de Sao Paulo; Brasil Fil: Sharon, Dror. The Hebrew University of Jerusalem; Israel Fil: Simonelli, Francesca. Seconda Universita Degli Studi Di Napoli; Italia Fil: Spital, Georg. No especifíca; Fil: Steehouwer, Marloes. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Szaflik, Jacek P.. No especifíca; Fil: Thompson, Jennifer A.. No especifíca; Fil: Thuillier, Caroline. Universite Lille; Francia Fil: Tracewska, Anna M.. No especifíca; Fil: van Zweeden, Martine. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Vincent, Andrea L.. University of Auckland; Nueva Zelanda Fil: Zanlonghi, Xavier. No especifíca; Fil: Liskova, Petra. Charles University and General University Hospital; República Checa Fil: Stöhr, Heidi. Universitat Regensburg; Alemania Fil: De Roach, John N.. University of Western Australia; Australia Fil: Ayuso, Carmen. Hospital Universitario Fundación Jiménez Díaz; España Fil: Roberts, Lisa. University of Cape Town; Sudáfrica Fil: Weber, Bernhard H. F.. Universitat Regensburg; Alemania Fil: Dhaenens, Claire Marie. Universite Lille; Francia Fil: Cremers, Frans P. M.. Radboud University Nijmegen Medical Centre; Países Bajos