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Silvia Albert, Riccardo Sangermano, Nathalie Bax, Miriam Bauwens, Ingeborgh Van Den Born, Elfride De Baere, Alejandro Garanto, Rob Collin, Carel C B Hoyng, Frans P Cremers; Identification and validation of intronic ABCA4 mutations in Stargardt patients by using induced pluripotent stem cell-derived photoreceptor progenitor cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6590. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The ABCA4 gene is associated with retinal disease in 95% of persons with autosomal recessive (ar) Stargardt disease (STGD1) and ~30% of cases with ar cone rod dystrophy (CRD). Non-canonical ABCA4 splice mutations are frequently observed in STGD1, although the effect at the RNA level in many cases is poorly understood. In about 30% of persons with STGD1, only one or no known ABCA4 mutations were identified. Heterozygous deletions and a subset of deep-intronic variants have previously been detected in some cases. The analysis of patient-derived ABCA4 mRNA is not straightforward, as the ABCA4 gene is poorly expressed in patient-derived peripheral blood and fibroblast cells. This study systematically searched for the ‘missing mutations’ and investigated the effects of non-canonical splice variants and deep-intronic variants with unclear functional effects.
To identify deep-intronic ABCA4 variants, Haloplex-based locus sequencing was performed in 35 mono-allelic maculopathy cases. After applying splice site prediction programs, the effect of putative splice variants was tested in vitro by cloning 1-kb fragments into minigenes, later transfected into HEK293T cells. Fibroblasts of 11 cases were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into photoreceptor progenitor cells (PPCs). The effects of a frequent non-canonical splice variant, c.5461-10T>C, and previously identified and novel deep-intronic variants on mRNA splicing, were analysed by reverse transcription (RT)-PCR of PPC mRNA and by in vitro assays with minigene constructs.
Haloplex sequencing revealed rare deep-intronic variants which potentially activate cryptic splice sites in 10 cases. Eleven STGD1 or arCRD patient-derived fibroblasts and two controls were reprogrammed into iPSCs and differentiated into PPCs. The c.5461-10T>C variant was shown to result in skipping of exon 39 or exon 39/40 in PPCs, effectively resulting in a null allele. PPC mRNA analysis provided preliminary evidence for abnormal RNA splicing due to deep-intronic variants.
The c.5461-10T>C variant was shown to completely inactivate ABCA4 function, which renders it the most frequent severe ABCA4 variant. Locus sequencing, minigene splice assays and RT-PCR analysis of PPCs revealed several variants that potentially disrupt ABCA4 mRNA splicing.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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