PURPOSE: To determine if massively parallel next-generation DNA sequencing offers rapid and efficient detection of disease-causing mutations in patients with monogenic inherited diseases. Retinitis pigmentosa (RP) is a challenging application for this technology because it is a monogenic disease in individuals and families, but highly heterogeneous in patient populations. RP has multiple patterns of inheritance, with mutations in many genes for each inheritance pattern and numerous, distinct, disease-causing mutations at each locus; further, many RP genes have not been identified yet.
METHODS: Next-generation sequencing was used to identify mutations in pairs of affected individuals from 21 families with autosomal dominant retinitis pigmentosa, selected from a cohort of families without mutations in "common" RP genes. One-thousand amplicons targeting 249,267 unique bases of 46 candidate genes were sequenced using the 454GS FLX Titanium (Roche) and Illumina (Solexa) GAIIx platforms.
RESULTS: An average sequence depth of 70X and 125X was obtained for the 454 and Solexa platforms respectively. Over 9,000 sequence variants were identified and analyzed to assess the likelihood of pathogenicity. One-hundred-twelve of these were selected as likely candidates and tested for segregation using traditional di-deoxy capillary electrophoresis sequencing of additional family members and controls. Five disease-causing mutations (24%) were identified in the 21 families.
DISCUSSION: This project demonstrates that next-generation sequencing is an effective approach for detecting novel, rare mutations causing heterogeneous monogenic disorders such as RP. With the addition of this technology, disease-causing mutations can now be identified in 65% of autosomal dominant RP cases.