Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing

Abstract

Purpose: To determine whether 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 is 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 RP, 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 with the 454GS FLX Titanium (Roche Diagnostics, Indianapolis, IN) and GAIIx (Illumina/Solexa, San Diego, CA) platforms.

Results: An average sequence depth of 70× and 125× was obtained for the 454GS FLX and GAIIx platforms, respectively. More than 9000 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 with traditional di-deoxy capillary electrophoresis sequencing of additional family members and control subjects. Five disease-causing mutations (24%) were identified in the 21 families.

Conclusion: 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.

Figure 1.

Coverage from 454GS FLX sequencing of individual samples (Roche Diagnostics, Indianapolis, IN). The fraction of targeted positions (∼250 kbp total) covered at 0× (red), 1× (green), 10× (light blue), and 20× (dark blue) are shown.

Figure 2.

Flow chart of variant analysis.

Figure 3.

Five families with identified pathogenic mutations. (A) VCH010. The p.A153V mutation in KLHL7 was present in all three affected family members tested. (B) VCH012. All five tested affected members of this family had the R838C mutation in GUCY2D (C) VCH017. Four affected members of this family were either heterozygous or hemizygous for the RPGR G738X mutation which was not present in the one unaffected family member tested. (D) VCH018. THE RPGR G65D mutation was present in seven affected members or female carriers in this family and absent from the one unaffected spouse tested. (E) VCH037. The c.946-1 splice site mutation in PRPF31 segregated with disease in the three family members tested. *Individuals tested in this study.

Figure 4.

Prevalence of mutations in genes causing dominant RP. Pathogenic mutations have been identified in 148 of the 230 adRP cohort families including the five families reported in this study. The mutation remains to be identified in 82 (35%) of the families.