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Shirel Weiss, Eran Eyal, Mali Salmon-Divon, Yoram Cohen, Nitza Goldenberg-Cohen; Carriers of degenerative retinal diseases can be easily identified using target capture next generation sequencing. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3371. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degeneration ranks high among the many genetic causes of blindness. Mutations in multiple genes are responsible for a wide variety of retinal dystrophy phenotypes, such as autosomal recessive Stargardt disease, cone-rod dystrophy and retinitis pigmentosa. Here, we report on the development of a new test to find all possible disease-associated variants in the coding sequences of 25 recessive degenerative retinal diseases, based on target enrichment and next generation sequencing (NGS).
372 exonic regions from 25 target genes were enriched by hybrid capture, sequenced by NGS to a depth of up to 0.36 gigabases, and assessed with stringent bioinformatic filters.
Two DNA samples of control subjects not known to suffer from retinal degeneration or any other eye disease were investigated, and 1 mutation diagnosed Leber Congenital Amuorosis (LCA) patient. An average target coverage of 179x (ranging from 151-218x) was achieved. 66.6% (45-93%) of nucleotides had at least 7x coverage, and 59.6% (39-86%) had at least 20x coverage. The enrichment factor which is the ratio of the coverage of the targeted region versus the coverage of the genome outside the target region was found to be 2331 (1768-3068). In the initial experiment we found, 191 (153-229) SNPS and 8 (5-11) indels ,138 (136-141) with median coverage > 37x, 95.5% of these (151-230) have been previously reported in dbSNP (v.132). 21 (19-24) aberrations were found in coding regions, of them 10 (9-12) resulted in changes of the protein sequence. Using the more sensitive blast alignment algorithm, we detected 10bps deletion in the CRB1 gene of the LCA patient located in a deeply covered region.
Given the difficulties in clinical diagnosis of similar phenotypes, this method proved efficient. It revealed the specific genetic mutations underlying retinal diseases, potentially lowering the costs of testing, and enabling broad screening for carriers and affected patients. If made available to the general population, NGS may be an economical and superior method of diagnosis, genetic consultation and treatment for patients.
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