Exon Capture and Next Generation Sequencing for Genetic Diagnosis of Inherited Retinal Degenerations

Abstract

Purpose: : Inherited retinal degenerations (IRDs) are among the most genetically diverse disorders, with over 160 disease genes identified to date. It is predicted that many more IRD disease genes remain to be discovered, since the identified mutations are estimated to account for ~50% of patients with these disorders. Clinical genetic testing for IRDs is improving, but is still limited to a subset of identified disease genes. We tested the use of exon capture and next generation sequencing technologies to improve genetic diagnosis for IRDs, and to facilitate identification of new IRD disease genes.

Methods: : Genomic DNA from 25 patients with recessive IRDs who did not have their disease gene identified through clinically available genetic testing was used for these studies. Exons from 153 known IRD disease genes and 596 candidate genes were selectively enriched using a custom Agilent SureSelect system. The enriched exons were sequenced on an Illumina Genome Analyzer IIx using single and pair-end 120bp reads. Sequence data was aligned to the genome using BWA, and SNPs, indels, as well as structural variations were detected from SAM alignments.

Results: : Mean sequence coverage of the captured exons ranged from 100-600 fold per patient. 400 to 550 non-synonymous SNPs and indels were identified for each patient, of which 18-116 were unique for each patient in the dataset. Filtering for homozygous or compound heterozygous variants reduced the number of candidate pathogenic genes to 0-12 per patient. Segregation analyses generated genetic diagnoses for 14 of the 25 patients. In several cases, pathogenic mutations were identified in IRD disease genes typically associated with phenotypes distinct from those displayed by the affected patients. For example, a 7 year old with chorioretinal atrophy similar in appearance to gyrate atrophy was found to have two null mutations in NRL, a gene usually associated with dominant retinitis pigmentosa.

Conclusions: : Selective exon capture and next generation sequencing techniques can facilitate genetic diagnosis of patients with IRDs. Testing IRD patients for mutations in all IRD disease genes identified pathogenic mutations in genes not typically associated with the phenotypes displayed by the affected patients. These results suggest that the disease genes identified to date may account for a larger percentage of patients with IRDs than previously estimated.

Keywords: retinal degenerations: hereditary • genetics • gene screening