Identification Of A Gene Mutation In An Arrp Family By Exome Analysis

Abstract

Purpose: : To identify the gene associated with early onset autosomal recessive retinitis pigmentosa (arRP) in a seven member consanguineous pedigree of European ancestry by exome sequencing.

Methods: : Phenotype of patients was evaluated using standard ophthalmic examination including fundus photography, ERG and fluorescein angiography. The exomes of two affected and one unaffected siblings were captured using NimbleGen SeqCap EZ exome capture probes. Library preparation and cluster generation protocols from the manufacturer were optimized to reduce PCR cycles and increase library complexity. Sequencing was done on Illumina GAIIx sequencer. Sequence reads were mapped to the human reference hg18 genome with Bowtie using a tiered mapping strategy. Single nucleotide variants (SNV) identification and annotation were performed with SAMtools, SeattleSeq and PolyPhen. Segregation and control sample analysis with PCR and Sanger sequencing confirmed SNVs. Expression profile of the corresponding gene was obtained in silico and with RT-PCR, westernblot analysis and immunohistochemistry.

Results: : Exons genome-wide were resequenced with an average depth of 40x (range 3-100). More than 95% of the covered region had >7x coverage. A total of 5401 synonymous and 4496 non-synonymous SNPs were identified in each individual. Nine novel homozygous changes were observed to be common between the two affected siblings. These changes were found either in the heterozygous state or absent in their unaffected sibling. None of these changes were observed in known SNP databases. Segregation and control sample analysis and the expression profile of the gene identified one novel, splice site mutation that is associated with arRP in this pedigree. The arRP gene encodes a secreted protein expressed in RPE, brain and embryonic tissue. Additional analysis is currently in progress to establish the pathogenicity of the novel mutation.

Conclusions: : We used exome sequencing to identify a homozygous potentially pathogenic mutation likely involved in causing arRP in a consanguineous pedigree. Understanding the impact of the variant observed will establish the molecular basis of arRP in this pedigree.

Keywords: genetics • retinal degenerations: hereditary • mutations