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Nicole Weisschuh, Anja-Kathrin Mayer, Nicola Gloeckle, Susanne Kohl, Saskia Biskup, Bernd Wissinger; Mutation screening in patients with retinal dystrophies using a Panel-based Next Generation Sequencing approach. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3268. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different forms of RD can be attributed to mutations in more than 100 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened 57 patients with different forms of RD (autosomal recessive and autosomal dominant Retinitis Pigmentosa, cone dystrophies, cone-rod dystrophies and Achromatopsia) in a research context for mutations in 105 RD-associated genes using hybridization-based enrichment and SOLiD-based NGS. Most of these patients had been tested negative upon screening for mutations in frequently affected genes applying Sanger Sequencing and/or high throughput mutation microarrays (Asper chips).
All putative disease-associated variants identified by our NGS approach were validated and tested for segregation with the phenotype in available family members by Sanger sequencing. In addition, exons with a coverage less than 10 reads of the NGS data were analyzed by Sanger sequencing. Whole exon deletions were validated by duplex PCR.
In total, we detected mutations in 20 different RD genes. Mutations explaining the disease phenotype were identified in 29 cases. In the remaining 28 patients, we did not detect sequence alterations that explain the disease phenotype. Among these, we found several autosomal recessive cases that carry sequence alterations in one of the frequently affected genes (e.g. USH2A or ABCA4), but lack a second clearly pathogenic variant. It cannot be excluded that deep intronic variants exist which were not detected due to the targeted enrichment of the exonic regions for NGS-based genetic testing. Another explanation is of course that these patients harbor mutations in yet unknown disease-genes. To elucidate this, we are currently analyzing the unsolved cases by whole-exome-sequencing.
To summarize, we used a targeted resequencing approach in a genetically pre-selected cohort of patients with RD and were able to identify causative mutations in 51% of cases. Non-solved cases are promising candidates for the identification of novel disease-genes using whole-exome sequencing.
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