Targeted High-Throughput DNA Sequencing for Gene Discovery in Retinitis Pigmentosa

Abstract

Purpose: : To use next-generation sequencing techniques, including microarray-based target selection and ultra high-throughput sequencing, to identify genes and mutations causing autosomal dominant retinitis pigmentosa (adRP) in a cohort of 88 families without apparent mutations in known adRP genes.

Methods: : AdRP patients and families selected for study are from a larger cohort of families that have been screened for mutations in genes known to cause adRP. The selected families do not have apparent mutations in known genes and are suitable for segregation analysis and follow-up studies of potential mutations. Pull-down microarrays incorporating known and candidate genes causing retinal degeneration, both full-length genomic sequences and coding sequences only, will be used to isolate targeted regions. Sequencing is being done on 454 Life Sciences sequencers. Evaluation of potential pathogenic variants includes comparison to known variants and mutations, bioinformatic analysis, segregation testing, and functional studies.

Results: : The first-generation microarray contains over 600 genes including all genes known to cause inherited retinal diseases (RetNet genes), human homologs of genes known to cause retinal disease in other animals, retina cilia-associated genes, and other abundant photoreceptor-expressed genes. A "training set" of 50 retinal disease genes are being sequenced in a subset of 20 families using conventional PCR and 454 sequencing to provide validation for subsequent large-scale targeted sequencing of the 600 genes at 50X depth. Data storage and analysis is based on protocols established for other large-scale sequencing projects such as the "cancer genome".

Conclusions: : New developments in DNA sequencing technology will produce exceptionally large data sets for identification of genes and mutations in patients and families with Mendelian diseases including inherited retinopathies. AdRP offers a model system for identifying pathogenic variants in extensive genomic sequences. This will contribute to a better understanding of the causes of adRP and provide experience in integrating genomic sequencing into clinical practice.

Keywords: genetics • retinal degenerations: hereditary • gene screening