Detection Of A Dominant-acting Mutation In RPE65 Using Whole-genome Linkage Mapping And Whole-exome Sequencing

Abstract

Purpose: : To use linkage mapping, whole-exome capture and next-generation sequencing to identify the disease-causing gene and mutation in a large four-generation Irish family (TCD-G) with autosomal dominant retinitis pigmentosa (adRP).

Methods: : DNA and phenotypic information was collected from family TCD-G. All genes and loci known to cause adRP were excluded using dideoxy sequencing and linkage testing against mapped loci. Family members were genotyped using the Affymetrix Genome-wide SNP Array 6.0, followed by multipoint linkage analysis and calculation of LOD scores. Exome sequencing of 3 affected and 1 unaffected family members was done using the Agilent SureSelect whole-exome capture kit followed by paired-end sequencing on a Illumina/Solexa GAIIx platform. Dideoxy sequencing was used for candidate gene screening, variant confirmation, and to screen regions not covered by exome sequencing.

Results: : Linkage mapping identified an 8.8 Mb region on 1p with a multipoint LOD score of 3.6. Of the 50 known genes in this region, 11 of the best candidates (including RPE65 and PDE4B) were sequenced directly. Exome capture was performed simultaneously and potential disease-causing variants from the linkage region were identified. Additional sequencing was done to cover coding regions not adequately covered by exome sequencing. The only protein variant identified in the linkage region present in all 20 affected family members and not present in unaffected controls was a novel amino acid substitution in exon 14 of RPE65 - Asp477Gly. This variant was not present in CEPH controls or in 342 Irish controls but was subsequently found in a second family.

Conclusions: : Mutations in RPE65 are known to cause both recessive Leber congenital amaurosis (LCA) and recessive retinitis pigmentosa. The novel mutation identified in this study is the first report of a dominant-acting mutation in patients with retinitis pigmentosa.

Keywords: retinal degenerations: hereditary • gene mapping • linkage analysis