Journal article
Using induced pluripotent stem cell-derived retinal pigment epithelial cells to model splicing defects of ABCA4 c.5461-10T > C detected in an Australian Stargardt disease cohort
Investigative Ophthalmology and Visual Science, Vol.62(8), Art. 3294
2021
Abstract
Purpose : Stargardt disease (STGD1, OMIM: 248200) is mainly caused by missense, frameshifting or nonsense mutations in the ATP-binding cassette transporter gene, ABCA4. However, sequence variants that alter splicing are also pathogenic. Herein, we describe an in vitro investigation of aberrant splicing in ABCA4 variants detected in a STGD1 cohort using patient-derived fibroblast-based assay. In addition, retinal pigment epithelium (RPE) cells differentiated from patient-derived induced pluripotent stem cells (iPSC) were used to further validate such splicing errors.
Methods : A cohort of 68 patients clinically diagnosed with STGD1 were recruited in this study. Genomic DNA obtained from recruited STGD1 patients was analysed by a commercial Stargardt/Macular dystrophy screening panel, targeting all exons of ABCA4 and flanking intronic regions, as well as already-known deep-intronic variants of ABCA4. Fibroblasts were propagated from 68 patients, total RNA was extracted and ABCA4 transcript structure was analysed by RT-PCR. The iPSCs reprogrammed from 2 patients carrying heterozygous c.[5461-10T>C;5603A>T] alleles were differentiated into RPE cells and the ABCA4 transcripts re-examined by RT-PCR.
Results : A total of 73 unique ABCA4 alleles were identified. Biallelic ABCA4 variants were detected in 66 patients (66/68, 97.06%) and 2 patients (2/68, 2.94%) had a single ABCA4 variant detected. Only exons 13-50 of ABCA4 could be readily amplified from fibroblast RNA. In this region, 9 out of 55 (16.36%) variants, carried by 19 patients (28%), resulted in aberrant splicing. The most prevalent splice variant, c.5461-10T>C, is complexed with c.5603A>T and carried heterozygously by 7 patients (10%). This variant results in mature ABCA4 mRNA transcripts missing exon 39, or exons 39 and 40. The splicing defect was also evident in patient-derived iPSC-RPE cells.
Conclusions : Patient-derived fibroblasts are useful for identifying ABCA4 splicing variants affecting exons 13-50. The iPSC-RPE cells provide a feasible platform for further validating retina-specific splice variants of ABCA4 that may be amendable to splice intervention therapies.
Details
- Title
- Using induced pluripotent stem cell-derived retinal pigment epithelial cells to model splicing defects of ABCA4 c.5461-10T > C detected in an Australian Stargardt disease cohort
- Authors/Creators
- D. Huang (Author/Creator)J.A. Thompson (Author/Creator)S. McLenachan (Author/Creator)S-C Chen (Author/Creator)D. Zhang (Author/Creator)R.C. Heath Jeffery (Author/Creator)M. Attia (Author/Creator)T.L. McLaren (Author/Creator)T.M. Lamey (Author/Creator)J.N. De Roach (Author/Creator)M.T. Aung-Htut (Author/Creator)A. Adams (Author/Creator)S. Fletcher (Author/Creator)S. Wilton (Author/Creator)F.K. Chen (Author/Creator)
- Publication Details
- Investigative Ophthalmology and Visual Science, Vol.62(8), Art. 3294
- Publisher
- Association for Research in Vision and Ophthalmology
- Identifiers
- 991005545410707891
- Murdoch Affiliation
- Centre for Molecular Medicine and Innovative Therapeutics
- Language
- English
- Resource Type
- Journal article
- Additional Information
- 2021 ARVO Annual Meeting abstract
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