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Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

ABSTRACT: BACKGROUND:Deletion-insertion (delins) variants in the retina-specific ATP-binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. METHODS:Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient-derived fibroblasts was analyzed by RT-PCR to evaluate splicing behavior of the ABCA4 variants. RESULTS:Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early-onset cone-rod dystrophy to late-onset macular dystrophy. This variant resulted in a 56-nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C-terminus would be truncated from the ABCA4 protein. CONCLUSION:This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient-derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment.

SUBMITTER: Huang D

PROVIDER: S-EPMC7336727 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

Huang Di D Thompson Jennifer A JA Charng Jason J Chelva Enid E McLenachan Samuel S Chen Shang-Chih SC Zhang Dan D McLaren Terri L TL Lamey Tina M TM Constable Ian J IJ De Roach John N JN Aung-Htut May Thandar MT Adams Abbie A Fletcher Sue S Wilton Steve D SD Chen Fred K FK

Molecular genetics & genomic medicine 20200423 7

<h4>Background</h4>Deletion-insertion (delins) variants in the retina-specific ATP-binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data.<h4>Methods</h4>Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RN ...[more]

PMID: 32627976

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Project description:BackgroundABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping.MethodsA STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids.ResultsLate onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids.ConclusionsExon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.

Dataset Information

Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

Publications

Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

Similar Datasets

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