Identification and functional analysis of novel deep intronic and structural ABCA4 variants in 876 Stargardt disease cases

Abstract

Purpose : The ABCA4 gene, implicated in Stargardt disease (STGD1), is the most frequently mutated maculopathy gene. Despite ABCA4 being identified 24 years ago, for thousands of cases the mutations causing STGD1 remain unknown. Previously, 1054 unsolved STGD and STGD-like probands were analyzed using single molecule molecular inversion probes (smMIPs), revealing bi-allelic ABCA4 variants in 448 probands, including 13 novel causal deep-intronic variants (DIVs) in 18 alleles, and 11 novel structural variants (SVs) in 16 alleles (Khan et al. GenetMed, 22:1235-1246, 2020). Here, we aim to shed further light on the missing heritability of STGD1 by analyzing another large cohort of genetically unsolved STGD probands.

Methods : 876 STGD and STGD-like patients were collected from 27 collaborators worldwide, 725 probands carried no or one ABCA4 variant and 151 probands carried two alleles, one of which was p.Asn1868Ile or p.Gly1961Glu, which are sometimes found in cis with DIVs. The complete 128-kb ABCA4 gene was sequenced using previously designed smMIPs. The effect of putative splice defects was assessed through in vitro midigene splice assays in HEK293T cells. The breakpoints of SVs were determined by junction PCR and Sanger sequencing.

Results : ABCA4 sequence analysis revealed two causal alleles in 436 of 876 probands (49.8%), and 13 known (in 63 alleles) and 15 novel (in 20 alleles) causal DIVs. In vitro splice assays revealed, among others, a DIV (c.4539+1964G>T) leading to the in-frame insertion of a pseudoexon, a near exon variant (c.859-25A>G) leading to complex splicing defects and a splice variant (c.4667+5G>T) that is part of a novel complex allele. In addition, 4 novel and 1 known SV were identified and characterized in 5 alleles.

Conclusions : Similar to a previous ABCA4 sequencing study of STGD and STGD-like cases, in which we genetically solved 448/1054 (42.5%) probands, we were able to genetically solve 49.8% of the probands. The total number of novel DIVs and SVs, i.e. 24 in 872 alleles (2.8%) is similar to the previous study in which we discovered 34 in 896 alleles (3.8%), indicating that there still are many rare DIVs and SVs to be discovered in Caucasian STGD1 cases. The identification of 83 DIVs in 81 probands suggests these persons may be eligible for splice modulation therapies when available.

This is a 2021 ARVO Annual Meeting abstract.