Targeted Next Generation Sequencing As A Powerful Diagnostic Tool For RP

Abstract

Purpose: : Genetically heterogeneous disorders such as retinitis pigmentosa (RP) have been difficult to diagnose in a routine DNA diagnostic setting up till now. Available methods like the ASPER primer extension chips are only able to detect known variants, whereas the extreme heterogeneity of RP does not allow Sanger sequencing of all known RP genes. Next generation sequencing technologies now allow us to simultaneously sequence all known genes that are involved in RP.

Methods: : A customized 12-plex Nimblegen sequence capture array was developed to enrich all exons, splice junctions and UTRs of 111 known retinal dystrophy genes. This array was then used to enrich the determined gene package in 100 previously unsolved patients with isolated or autosomal recessive RP, for which ~50-60% of the causative genes are currently known. DNAs of 12 retinal dystrophy patients carrying 24 known disease causing variants served as controls. All 112 samples were sequenced on a Roche 454 GS FLX Titanium machine. Stringent bioinformatic data analysis and variant detection on average resulted in 4 variants per sample, which were validated using conventional Sanger sequencing and segregation analysis within available family members.

Results: : In 100 patients, a total of 397 variants were selected to be potentially pathogenic and were validated by Sanger sequencing. Up to now, 82% of the so far validated variants are confirmed. In addition to variants at basepair level, we could also identify larger homozygous and heterozygous pathogenic deletions. Preliminary data on the confirmation and segregation analysis of sequence variants in the corresponding families revealed the likely pathogenic mutations in at least 35 probands. As expected,~half of the cohort will carry a mutation in yet to be discovered genes that are not targeted on our array.

Conclusions: : Our data show that targeted next-generation sequencing combined with stringent bio-informatic data analysis is a time- and cost-effective strategy for the molecular genetic diagnosis of genetically heterogeneous disorders like RP.

Keywords: genetics • retinal degenerations: hereditary • mutations