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Detailed analysis of an enriched deep intronic ABCA4 variant in Irish Stargardt disease patients.

ABSTRACT: Over 15% of probands in a large cohort of more than 1500 inherited retinal degeneration patients present with a clinical diagnosis of Stargardt disease (STGD1), a recessive form of macular dystrophy caused by biallelic variants in the ABCA4 gene. Participants were clinically examined and underwent either target capture sequencing of the exons and some pathogenic intronic regions of ABCA4, sequencing of the entire ABCA4 gene or whole genome sequencing. ABCA4 c.4539 + 2028C > T, p.[= ,Arg1514Leufs*36] is a pathogenic deep intronic variant that results in a retina-specific 345-nucleotide pseudoexon inclusion. Through analysis of the Irish STGD1 cohort, 25 individuals across 18 pedigrees harbour ABCA4 c.4539 + 2028C > T and another pathogenic variant. This includes, to the best of our knowledge, the only two homozygous patients identified to date. This provides important evidence of variant pathogenicity for this deep intronic variant, highlighting the value of homozygotes for variant interpretation. 15 other heterozygous incidents of this variant in patients have been reported globally, indicating significant enrichment in the Irish population. We provide detailed genetic and clinical characterization of these patients, illustrating that ABCA4 c.4539 + 2028C > T is a variant of mild to intermediate severity. These results have important implications for unresolved STGD1 patients globally with approximately 10% of the population in some western countries claiming Irish heritage. This study exemplifies that detection and characterization of founder variants is a diagnostic imperative.

SUBMITTER: Whelan L

PROVIDER: S-EPMC10256698 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Detailed analysis of an enriched deep intronic ABCA4 variant in Irish Stargardt disease patients.

Whelan Laura L Dockery Adrian A Stephenson Kirk A J KAJ Zhu Julia J Kopčić Ella E Post Iris J M IJM Khan Mubeen M Corradi Zelia Z Wynne Niamh N O' Byrne James J JJ Duignan Emma E Silvestri Giuliana G Roosing Susanne S Cremers Frans P M FPM Keegan David J DJ Kenna Paul F PF Farrar G Jane GJ

Scientific reports 20230609 1

Over 15% of probands in a large cohort of more than 1500 inherited retinal degeneration patients present with a clinical diagnosis of Stargardt disease (STGD1), a recessive form of macular dystrophy caused by biallelic variants in the ABCA4 gene. Participants were clinically examined and underwent either target capture sequencing of the exons and some pathogenic intronic regions of ABCA4, sequencing of the entire ABCA4 gene or whole genome sequencing. ABCA4 c.4539 + 2028C > T, p.[= ,Arg1514Leufs ...[more]

PMID: 37296172

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Project description:PurposeThe purpose of this study was to investigate the contribution and natural progression of ABCA4 deep intronic variants (DIVs) among a Chinese Stargardt disease (STGD) cohort.MethodsFor unsolved STGD probands, DIVs in ABCA4 were detected by next-generation sequencing, and splicing effects were evaluated by in silico tools and validated through minigene experiments. Comprehensive ocular examinations, especially fundus changes, were carried out and analyzed. These and long-term follow-up data were compared with data of patients carrying biallelic coding variants of ABCA4.ResultsSeven DIVs in ABCA4 were identified in 18 of 40 (45.0%) unsolved STGD probands, involving 2 novel and 5 known variants. Four DIVs were confirmed to effect splicing through minigene assay. The c.161-395G>A was the most prevalent DIV allele (30.6%, 11/36). In the early 5-year duration, localized maculopathy was predominant, accounting for 51.9% (14/27) of fundus recordings. Expanded macular lesion within the vascular arch, with or without flecks, was observed in 75.0% (12/16) of recordings beyond the 5-year duration, whereas generalized retinal dystrophy was rarely observed. Compared with those in the non-DIV group, the patients in the DIV group manifested milder fundus change at all disease stages (P < 0.05). Follow-up visits utilizing wide-field fundus autofluorescence (FAF) further validated the slower development of lesions. Optical coherence tomography angiography (OCTA) documented a gradual reduction in perfusion in each layer's capillaries and high-reflective deposits below the sub-RPE layer.ConclusionsDIVs contribute to nearly half of STGD cases with missing heritability, totally occupying 7.8% of all STGD families. Based on optimized grading criteria, patients with DIV alleles manifested localized macular lesions with slow progress. The amount of residual correctly spliced mRNA might play a role, suggesting that adding or enhancing normal ABCA4 expression might be a potential approach of intervention.

Project description: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.

Dataset Information

Detailed analysis of an enriched deep intronic ABCA4 variant in Irish Stargardt disease patients.

Publications

Detailed analysis of an enriched deep intronic ABCA4 variant in Irish Stargardt disease patients.

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