Genomic Fragment Capture and Pyrophosphate Sequencing for Retinitis Pigmentosa Gene Discovery

Abstract

Purpose: : Retinitis Pigmentosa (RP) is an inherited disease of photoreceptors that is likely to be caused by more than 100 different genes. Dozens of these genes remain to be discovered and many of these will each be responsible for less than one percent of RP cases. This experiment was designed to use Roche/NimbleGen genomic fragment capture followed by pyrophosphate sequencing to identify disease-causing mutations in known RP genes as well as in genes not previously associated with this disease.

Methods: : Custom Roche/NimbleGen sequence capture arrays were generated with sequence corresponding to the coding sequences and proximal promoter regions of 215 genes. One hundred twenty-three of these genes have been previously associated with human retinal disease while the remainder were judged to be good candidates for involvement in RP because of their retinal expression, their homology to known disease genes, or both. The sequence data were analyzed using custom software written by the authors. Plausible disease-causing mutations were confirmed with conventional automated DNA sequencing.

Results: : Four RP patients were screened for mutations with this approach. An average of 84,657 reads of 231 bp in length were obtained from each patient in this experiment. Out of the 2914 exons included in the array design, 2878 were represented in the resulting sequence data. Plausible disease-causing mutations were identified in all four patients. In one patient, two correctly segregating disease-causing mutations were found in a known RP gene. The putative mutations observed in the other three patients were found in the heterozygous state and additional evaluation of these genes is underway in an attempt to identify a second disease allele.

Conclusions: : Genomic sequence capture and pyrophosphate sequencing enable one to evaluate several million nucleotides of genomic sequence in a single experiment which is a significant advantage for extensively heterogeneous diseases like RP. A disadvantage of this broad approach is that as the number of genes sampled extends into the hundreds the likelihood of observing a clinically irrelevant heterozygous carrier state of a true disease-causing allele becomes very high.

Keywords: genetics • gene screening • gene/expression