ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Abstract

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The 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 the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The 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 data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. 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 IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

Keywords: ABCA4; Stargardt disease; branchpoint variant; midigene splice assay; whole genome sequencing.

FIGURE 1

Pedigrees of two unrelated individuals analyzed in this study. Arrows indicate the proband in each family.

FIGURE 2

Ophthalmic features of compound heterozygous retinopathy cases carrying c.6480-35A>G. Fundus autofluorescence (upper panel), OCT (middle panel), and color fundus (lower panel) for left (OS) and right (OD) eyes. (A) Proband A:II-6 carrying c.699_768+341del p.(Gln234Phefs*5) as the second allele. (B) Proband B:II-1 carrying c.1958G>A p.(Arg653His) as the second allele. Years, yrs.

FIGURE 3

In silico prediction scores of the c.6480-35A>G variant. Schematic representation of the intron 47–exon 48 boundary sequence of ABCA4, branchpoint, and splice prediction in the wild-type (WT; upper panel) and c.6480-35A>G (MUT; lower panel) situation. The branchpoint algorithm predicts the abolishment of the branchpoint. SpliceAI predicts a 0.47 increase in the probability of activation of a cryptic acceptor site in intron 47 (47 nt upstream of the canonical acceptor site).

FIGURE 4

Overview of midigene assay results of variant c.6480-35A>G in HEK293T cells. (A) Schematic representation of wt midigene (BA29_WT) where the position of the variant is indicated by an arrow. (B) Gel image of RT-PCR products of wild-type and mutant constructs. The rhodopsin exon 5 (RHO ex5) RT-PCR was used as a control for transfection efficiency. Schematic representation of the three RT-PCR products identified in the gel. Wt midigene reveals the expected 378 nt wt fragment (Fragment 2) and the exon 47 skipping fragment (Fragment 3). Mutant midigene reveals a partial intron 47 inclusion of 45 nt 5ʹ (Fragment 1) and 30.4% of the remaining wt fragment (Fragment 2). Fiji software was used for a semi-quantification of the fragments in the mutant construct. (C) Sanger sequence analysis of the RT-PCR fragments. The chromatograms show the breakpoints in all fragments. * Heteroduplex fragment.