A Multi-pronged Approach For Discovery Of Genes For Inherited Retinal And Macular Diseases: From Linkage Mapping To Exome Sequencing

Abstract

Purpose: : Insufficient family size often limits gene identification through conventional genetic mapping methods. Massively parallel sequencing of targeted regions and exome sequencing have greatly facilitated gene discovery in such families. In this present study, we combine inheritance analysis and exome sequencing in five families with inherited retinal diseases to identify the causal genes.

Methods: : We performed Affymetrix Nsp 250K SNP array genotyping in five families and homozygosity mapping in two of the five families with apparent autosomal recessive inheritance. Known candidates in regions of suggestive linkage were excluded by Sanger sequencing. To subsequently identify the disease genes using exome sequencing, whole genome libraries were prepared and enriched for exons using Agilent's SureSelect Human All Exon Kit. Sequencing of the captured fractions was performed on the Illumina’s Genome Analyzer IIx.

Results: : Genetic mapping identified suggestive linkage peaks and homozygosity regions at different chromosomes in all five families. In one Leber's congenital amaurosis family, homozygosity mapping identified regions at 9p24 and 14q11. Sequencing of the candidate gene RPGRIP1 identified a novel mutation, c.3490delA. Exome sequencing in four other families targeted 50 Mb at 188,260 exons from 18,560 genes. Read alignments were performed using Eland and BOWTIE. In search of causative gene, filters are being applied to exclude common variants identified in the dbSNP130 or HapMap databases as well as to select for variants identical-by-descent in the affected siblings. These investigations are currently underway.

Conclusions: : Our study illustrates that a combined approach of conventional mapping with exome sequencing can be a powerful tool in identifying causal genes in retinal diseases.

Keywords: genetics