Frans P.M. Cremers, PhD
Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands
We employed 3,866 single molecule Molecular Inversion Probes to sequence the complete 128-kb ABCA4 gene implicated in 1,054 genetically unsolved Stargardt disease (STGD) and STGD-like probands. We found bi-allelic causal variants in 448 probands . Structural variant analysis revealed 11 distinct heterozygous deletions in 16 probands. Interestingly, 14 different known causal deep-intronic variants and 13 different putative causal deep-intronic variants were identified in 117 of 896 alleles. We previously established 31 overlapping midigene (insert sizes 4.7-11.5 kb) splicing constructs containing wild-type sequences of 48 of the 50 ABCA4 exons . The 13 novel deep-intronic variants were introduced into midigene constructs, and wild-type and mutant constructs were transfected into HEK293T cells. After 2 days, RNA was extracted and analyzed using RT-PCR. Based on these in vitro splice assays, the novel deep-intronic variants were found to result in pseudoexon (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 overlapping PE insertions that were accompanied by flanking exon deletions.
ABCA4 variants show different effects, i.e. they can be severe, moderaty severe, or mild. Usually, early-onset STGD1 cases carry two severe variants and classical STGD1 cases carry a severe and a mild allele, or two moderately severe alleles . Late-onset STGD1 cases often carry a severe allele and a mild-incomplete penetrant allele, i.e. p.Asn1868Ile . The allele combinations severe/p.Asn1868Ile and severe/p.Gly1961Glu were found more often in females than males (1.7 : 1 and 2.1 : 1, respectively), suggesting non-monogenic STGD1 in ~25% of the cases [4,5]. The different severities of ABCA4 variants complicate genetic counselling. To provide a more accurate prediction of the risk that STGD1 probands have offspring that also develop STGD1, we collected all cases that carried bi-allelic ABCA4 variants(see: www.lovd.nl/ABCA4), and classified ~1,685 unique ABCA4 variants into different severity categories. Based on the allele frequencies of these variants in an ethnically adjusted gnomAD dataset, we calculated the risk that when a STGD1 proband and a healthy spouse will have a child, that child will also develop STGD1. This was calculated for all combinations of variant severities. The risk of STGD1 was estimated at ~1 in 34 for offspring of a STGD1 case carrying severe/severe alleles and ~1 in 60 for offspring of a STGD1 case carrying severe/mild alleles (Cornelis, Runhart et al. Submitted). ABCA4 sequence analysis of the unaffected spouse would reduce these risks significantly assuming that no causal variants are identified. The results of our study greatly facilitate genetic counselling in at risk families.
 Khan et al. Genet. Med. 22:1235-1246, 2020
 Sangermano et al. Genome Res. 28:100-110, 2018
 Cremers et al. Prog. Retin. Eye Res. 79:100861, 2020
 Runhart et al. Invest. Ophthalm. Vis. Sci., 59:3220-3231, 2018
 Runhart et al. Jama Ophthalm. 138:1035-1042, 2020