Abstract
Purpose: :
Retinitis Pigmentosa (RP) is a clinically and genetically heterogeneous inherited retinal degenerative disease. Although the causative genes for approximately half of cases have been identified, it is currently difficult to test all of these and provide a genetic diagnosis for new patients. We have therefore developed a new high-throughput sequencing strategy to screen all known RP genes in a single assay.
Methods: :
A sequence capture array (Nimblegen) was designed to target exons and splice sites of 46 known non-syndromic RP genes. This was used to enrich these regions from the DNA of 5 RP patients; one with a known homozygous mutation in PDE6B, one compound heterozygote in CRB1 and three with unknown mutations. The enriched samples were sequenced using a Genome Analyzer (Illumina), either singly or in a pool of 4 per lane. The ~10 million reads from each run were aligned to reference sequences and sequence variants detected.
Results: :
In total, approximately 500 variants were detected, half of which were known SNPs. All previously known mutations and 11 additional non-synonymous variants were detected. The effect of non-synonymous variants on protein function was modelled and potential effects upon splicing of all novel variants assessed using the Human Splicing Finder (HSF) tool. The frequencies of potentially pathogenic variants were assessed in a control population.
Conclusions: :
Targeted sequence capture followed by next generation sequencing provides an effective approach to the parallel screening of multiple genes, enabling the detection of both known and novel mutations in disease-associated genes.
Keywords: gene screening • retinal degenerations: hereditary • genetics