A Targeted Next-Generation Sequencing Approach for Identification of the X-linked RP23 Gene

Abstract

Purpose: : We previously mapped a locus for a severe form of X-linked retinitis pigmentosa, RP23, to a 10.56 Mb interval on Xp22 which contains approximately 700 exons in 62 known and predicted genes. Conventional candidate gene screening did not identify the causative mutation, so we adopted a targeted next-generation sequencing (NGS) approach to determine the underlying molecular cause of RP23.

Methods: : We used a 2.1M Custom Array from Nimblegen to capture 19.2 Mb of the X-chromosome including known X-linked RP genes RPGR and RP2 and the entire RP23 genomic interval from DXS1223 and DXS7161 (10.56 Mb). In addition to an RP23 affected male, we also sequenced 2 control male samples. Sequence data were generated using 100 bp paired-end sequencing on an Illumina Genome Analyzer. Variants identified were filtered against common variants found in dbSNP and 1000 genomes.

Results: : NGS data confirmed the lack of RP2 and RPGR mutation. These genes were included to exclude sequence variation outside of the coding exons. We achieved mean depth of coverage of between 98-102 reads per base and sequence coverage between 83-88%. Within the RP23 interval we detected 6,827 variants. After filtering out known SNPs and low quality calls we identified 242 novel variants of which 119 were intragenic. The first GC rich exons of 3 genes were not captured and sequenced using this strategy.

Conclusions: : The NGS strategy confirmed our earlier candidate gene screening data, as no potentially pathogenic coding sequence variant was identified in previously screened genes. We are currently analysing the novel sequence variants that lie within or near annotated genes to evaluate their potential pathogenicity. Furthermore, RP2 and RPGR mutation negative X-linked families have been recruited for genetic mapping to identify additional RP23 families which may refine the locus and confirm potential pathogenicity of gene specific variants within the RP23 interval.

Keywords: retinal degenerations: hereditary • genetics • gene screening