Identification of Homozygous and Hemizygous Genomic Deletions that Cause Inherited Retinal Degenerations by Analyzing Whole Exome Sequencing Data

Abstract

Purpose: Inherited retinal degenerations (IRDs) are a common cause of visual disturbance with a high clinical and genetic heterogeneity. Various mutations in more than 200 genes were identified and / or mapped causing this disease. Over the last few years, the development of next-generation sequencing techniques such as whole exome sequencing (WES) contributed to the discovery of many of these genes. While effective in identification of localized sequence changes, large deletions may be overlooked when analyzing WES data by conventional methods. The aim of the current study was to utilize WES data in order to identify large deletions that include at least one exon in known IRD genes.

Methods: Patients diagnosed with different IRDs underwent a comprehensive ophthalmic evaluation, including ophthalmic ancillary tests. Index cases were initially screened for known mutations, and when found negative, WES was performed using the NimbleGen V2 paired-end kit and Illumina HiSeq2000 (Otogenetics). An analysis of exon coverage data (taking into account the mean coverage and standard deviation of each of the 185,636 exons in the human genome) was performed.

Results: We analyzed data of 81 WES samples from index patients with IRDs. By calculating the average coverage for all exons in the human genome, we were able to identify homozygous or hemizygous deletions of at least one exon in 6 families (7.5%). In one family we identified a single-exon deletion in EYS, in two families we identified deletions of three consecutive exons in MYO7A and NPHP4, in one family we identified a 4-exon deletion in RPGR, in one family we identified a deletion of eight consecutive exons in RPGR, and in one family with the original diagnosis of retinitis pigmentosa we identified a multi-gene deletion on the X-chromosome, including the CHM gene. By using PCR-walking analysis, we were able to identify the borders of two of the deletions and to screen our set of patients for the presence of additional patients with either heterozygous or homozygous deletions.

Conclusions: To the best of our knowledge, this is the first study in which WES data were systematically analyzed to identify large deletions in IRD genes. Our analysis indicates that large deletions (including at least one exon) are relatively frequent (about 7.5 % in our cohort) and should be part of the routine analysis of WES data