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Julia Mohr, Nicola Gloeckle, Susanne Kohl, Tim Scheurenbrand, Andrea Sprecher, Antje Bernd, Eberhart Zrenner, Bernd Wissinger, Konstanze Hörtnagel; Genetic diagnostic testing in a large cohort of retinitis pigmentosa patients using panel-based next generation sequencing. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3368.
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Worldwide, a total of two million patients are affected by retinitis pigmentosa (RP). We used panel-based next generation sequencing (NGS) to study the underlying genetic heterogeneity of autosomal recessive and sporadic RP cases, and to evaluate its applicability in a diagnostic setting.
A cohort of 150 patients with sporadic or suspected autosomal recessive RP was selected. Using a customized enrichment panel including 105 genes involved in retinal disease, the samples were analyzed by NGS on a SOLiD 5500xl platform. Sequence reads analyzed by LifeScope software were mapped to the human reference genome. Annotation of the variants generated by LifeScope software package was performed using the Ensembl database, dbSNP and in-house variant databases. Indel calling was also supported. Variants were selected for further analysis, if the global minor allele frequency (dbSNP and Exome Variant Server) was below 5%. Validation of all mutations identified by NGS as well as analysis of underrepresented regions (<10 reads per base) was performed using Sanger sequencing. To ensure an ideal and fast analysis, all genes currently known to be associated with autosomal recessive RP were analyzed first. In unsolved cases the analysis was expanded to all remaining genes of the panel.
In 53% of patients we were able to identify the causative mutation(s), 12% of cases were inconclusive and 35% remained unsolved. We observed mutations in 39 different genes, with USH2A, EYS and RPGR being the most prevalent mutated genes. In 5 cases (3%) and in 11 cases (7%) we found causative mutations in X-linked RP genes and in autosomal dominant RP genes, respectively.
Like in other populations, we observed large genetic heterogeneity among our cohort of RP patients. Our study demonstrates that panel-based NGS is capable to uncover the principle genetic cause in more than 50% of cases with apparently sporadic and autosomal recessive RP. Interestingly, about 10% of cases showed a pathogenic mutation that is most likely incompatible with autosomal recessive inheritance. Therefore, genes associated with an autosomal dominant or X-linked form of RP must also be taken into account in patients with apparently sporadic RP which is of major relevance for genetic counseling.
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