ABCA4 c.6480-35A>G, a novel branchpoint variant associated with cone-rod dystrophy and macular dystrophy

Abstract

Purpose : Inherited retinal dystrophies (IRDs) can be caused by variants in more than 270 genes. ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt Disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of ABCA4 reported variants affects RNA splicing. In most cases it is necessary to perform a functional assay to determine the effect of these variants. Here, we identified an ABCA4 intron 47 variant, c.6480-35A>G, in a Spanish proband with CRD and we assessed its pathogenicity by in vitro functional analysis.

Methods : Whole genome sequencing (WGS) was performed in 64 Spanish probands with IRDs. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. In silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48.

Results : Through the analysis of WGS from a cohort of 64 cases, we identified two variants in ABCA4 in one proband. A previously described deletion, c.699_768+342del p.(Gln234Phefs5), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that both variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at the position c.6480-47. Midigene splice assays revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of unresolved IRD-cases.

Conclusions : The variant c.6480-35A>G disrupts a predicted branchpoint, leading to inclusion of 47nt in the mRNA resulting in protein truncation. This variant was found in two unrelated individuals from Spanish descent. The combination of this moderate variant and the severe deletion underlies CRD in one proband and leads to macular dystrophy with the moderately severe missense variant in the second proband. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.