Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics
Authors: Khan Mubeen , Cornelis Stéphanie S. , del Pozo-Valero Marta , Whelan Laura , Runhart Esmee H. , Mishra Ketan , Bults Femke , AlSwaiti Yahya , AlTabishi Alaa , De Baere Elfride , Banfi Sandro , Banin Eyal , Bauwens Miriam , Ben-Yosef Tamar , Boon Camiel J.F. , van den Born L. Ingeborgh , Defoort Sabine , Devos Aurore , Dockery Adrian , Dudakova Lubica , Fakin Ana , Farrar G. Jane , Ferraz Sallum Juliana Maria , Fujinami Kaoru , Gilissen Christian , Glavač Damjan , Gorin Michael B. , Greenberg Jacquie , Hayashi Takaaki , Hettinga Ymkje , Hoischen Alexander , Hoyng Carel B. , Hufendiek Karsten , Jägle Herbert , Kamakari Smaragda , Karali Marianthi , Kellner Ulrich , Klaver Caroline C.W. , Kousal Bohdan , Lamey Tina , MacDonald Ian M. , Matynia Anna , McLaren Terri , Mena Marcela D. , Meunier Isabelle , Miller Rianne , Newman Hadas , Ntozini Buhle , Oldak Monika , Pieterse Marc , Podhajcer Osvaldo L. , Puech Bernard , Ramesar Raj , Rüther Klaus , Salameh Manar , Salles Mariana Vallim , Sharon Dror , Simonelli Francesca , Spital Georg , Steehouwer Marloes , Szaflik Jacek P. , Thompson Jennifer A. , Thuillier Caroline , Tracewska Anna M. , van Zweeden Martine , Vincent Andrea L. , Zanlonghi Xavier , Liskova Petra , Stöhr Heidi , De Roach John , Ayuso Carmen , Roberts Lisa , Weber Bernard H.F. , Dhaenens Claire-Marie , Cremers Frans P.M. 
Published: October 2019

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). 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 that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.