Genomewide search for genetic modifiers in patients with Leber congenital amaurosis using whole exome sequencing

Abstract

Purpose: Leber congenital amaurosis (LCA) is inherited childhood blindness. LCA accounts for 6% of all retinal dystrophies, and mutations in at least 20 genes have so far been associated with LCA. Although recognizable phenotypes have been delineated for many of the genotypes, there remains unexplained variation in severity within some genotypes and this can be independent of age and biochemical properties of the mutants. Sequence analysis of known LCA genes have identified more than one potentially disease causing variants in individual LCA patients, implicating the involvement of modifier genes in generating clinical differences. In this study, we have applied whole exome sequencing (WES) for elucidating a complete spectrum of modifier alleles in LCA caused by RPE65 mutations.

Methods: We first performed WES on four members of a LCA family and identified novel segregating variants in two unlinked genes, RPE65 and CHD7. RPE65 mutations are known to cause LCA, whereas mutations in CHD7 cause CHARGE syndrome. This observation guided us to explore whether mutations in more than one gene (i.e., RPE65) were involved in clinical manifestation of the LCA phenotype. Thus in an unbiased approach for modifier search, we performed WES on additional 37 LCA patients, including 4 sib-pairs, with known RPE65 mutations. We initiated the analysis by filtering for genes with rare second-site modifier alleles in two or more patients. Further prioritization of candidates involved employing bioinformatic tools for predicting the pathogenicity of variants, and screening of human retinal and RPE RNA-seq data for highly expressed retinal genes. Finally, we looked for targets of NRL and CRX, using ChIP-seq data.

Results: In the first set of analysis involving the family and four sib-pairs, we have identified potential modifiers among several known retinal degeneration genes (ERCC6, FAM161A, NPHP4, USH2A and MYO7A) as well as in novel genes (e.g. CHD7, SETD5, DSCAML1). To obtain a complete spectrum of modifiers, we are currently analyzing the exome sequencing data of simplex cases carrying discrete RPE65 mutations and LCA patients carrying the same mutation but exhibiting differences in disease severity.

Conclusions: Our study provides a genome-wide approach for examining rare alleles that may contribute to LCA pathogenesis and should help in elucidating differences in disease expression among patients.

Keywords: 539 genetics • 696 retinal degenerations: hereditary • 604 mutations