Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts. (December 2022)

• Record Type: Journal Article
• Authors: Huang, Di; Thompson, Jennifer A.; Chen, Shang-Chih; Adams, Abbie; Pitout, Ianthe; Lima, Alanis; Zhang, Dan; Jeffery, Rachael C. Heath; Attia, Mary S.; McLaren, Terri L.; Lamey, Tina M.; De Roach, John N.; McLenachan, Samuel; Aung-Htut, May Thandar; Fletcher, Sue; Wilton, Steve D.; Chen, Fred K.
• Is Part Of: Experimental eye research. Volume 225(2022)
• Publication Date: 2022-12
• Subjects: In vitro splice assay -- Stargardt disease -- ABCA4 splice variants -- Patient-derived fibroblasts -- Molecular analysis; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2022.109276
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: The ATP-binding cassette subfamily A member 4 gene ( ABCA4 )-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico . Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction. Highlights: What is already known on this topic – ABCA4 transcript can be detected in non-retinal tissues and cells, including fibroblasts. Fibroblast-based splice assay has previously been used to investigate the splicing defects of a limited number of ABCA4 mutations. What this study adds – ABCA4 transcripts from exons 13 to 50 in were readily amplified in Stargardt patient-derived fibroblasts, whilst exons 1 to 12 were absent. Approximately 77% of ABCA4 mutations in our Stargardt cohort reside in exons 13–50, consistent with that in LOVD (75%). Among these mutations, 10 out of 18 predicted splice mutations were confirmed to cause aberrant splicing of ABCA4 transcripts using fibroblast assay. Evidence of in vitro splicing defects contributes to establishing pathogenicity and broadens the mutation spectrum of Stargardt disease. How this study might affect research, practice or policy – Fibroblasts can provide a powerful platform for analysis of splice mutations. Retinal-like cells play a role in further validating the splice variants, assessing variants that cannot be evaluated using fibroblasts and variants where altered splicing is not evident in fibroblasts. Characterisation of splice variants will provide opportunities for developing therapeutic approaches that modulate ABCA4 pre-mRNA splicing.