Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.

The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

An Overview of the Genetics of ABCA4 Retinopathies, an Evolving Story.

Stargardt disease (STGD1) and ABCA4 retinopathies (ABCA4R) are caused by pathogenic variants in the ABCA4 gene inherited in an autosomal recessive manner. The gene encodes an importer flippase protein that prevents the build-up of vitamin A derivatives that are toxic to the RPE. Diagnosing ABCA4R is complex due to its phenotypic variability and the presence of other inherited retinal dystrophy phenocopies. ABCA4 is a large gene, comprising 50 exons; to date > 2000 variants have been described. These include missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of variants in ABCA4. However, in a significant proportion of patients with an ABCA4R phenotype, a second variant in ABCA4 is not identified. This could be due to the presence of yet unknown variants, or hypomorphic alleles being incorrectly classified as benign, or the possibility that the disease is caused by a variant in another gene. This underlines the importance of accurate genetic testing. The pathogenicity of novel variants can be predicted using in silico programs, but these rely on databases that are not ethnically diverse, thus highlighting the need for studies in differing populations. Functional studies in vitro are useful towards assessing protein function but do not directly measure the flippase activity. Obtaining an accurate molecular diagnosis is becoming increasingly more important as targeted therapeutic options become available; these include pharmacological, gene-based, and cell replacement-based therapies. The aim of this review is to provide an update on the current status of genotyping in ABCA4 and the status of the therapeutic approaches being investigated.