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Silvia Albert, Riccardo Sangermano, Nathalie Bax, Susanne Roosing, L. I. van den Born, Anke den Engelsman-van Dijk, Angelique Ramlal, Edwin M Stone, Carel C B Hoyng, Frans Cremers; Towards the identification of deep-intronic ABCA4 mutations in Stargardt patients by using induced pluripotent stem cell-derived photoreceptor progenitor cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3174.
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© ARVO (1962-2015); The Authors (2016-present)
We previously found that ~40% of persons with autosomal recessive Stargardt disease (STGD1) carry only one, and ~10% no ABCA4 mutations. We hypothesized that the lacking mutations are either heterozygous deletions not detected by exon PCR and Sanger sequencing, or located deep in the introns, most likely affecting ABCA4 mRNA. We have shown previously that ABCA4 mRNA is expressed at very low levels in lymphoblasts and fibroblasts but robustly in photoreceptor cells that have differentiated from human stem cells. We propose to perform sequence analysis of ABCA4 mRNA isolated from photoreceptor progenitor cells (PPCs) that were differentiated from reprogrammed patient-specific induced pluripotent stem cells (iPSCs).
We performed Sanger sequencing of the ABCA4 promoter region in a cohort of 45 patients with STGD1 or STGD1 with cone-rod dystrophy phenotypes carrying one ABCA4 mutation, multiplex ligation-dependent probe amplification (MLPA) of the 50 ABCA4 exons in 27 cases of the cohort, and Sanger sequencing of 20 weak deep-intronic splice sites in 45 cases. Skin biopsy fibroblasts were cultured from eight STGD1 patients and reprogrammed into iPSCs. iPSCs were differentiated into PPCs, and the presence of the ABCA4 transcript was evaluated by quantitative RT-PCR (qRT-PCR) and immunocytochemistry (ICC). The ABCA4 transcript was also analysed by RT-PCR and Sanger sequencing to identify insertions or deletions.
MLPA and Sanger sequencing revealed heterozygous deletions in two maculopathy families, deep-intronic variants in six families, and a combination of a heterozygous deletion and a deep-intronic variant in one family. Patient-derived fibroblasts were successfully reprogrammed into iPSCs, and differentiated into PPCs, in seven and five probands, respectively. qRT-PCR and ICC of the cultured cells confirmed their differentiation into PPCs. The ABCA4 transcripts produced revealed the presence of several abnormal cDNA fragments.
Heterozygous deletions and deep-intronic variants were identified in nine of 45 (20%) maculopathy families illustrating that deep-intronic variants are a significant cause of macular disease. To identify additional deep-intronic variants, we successfully obtained cultures of PPCs that strongly expressed ABCA4 after four weeks of differentiation.
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