Copy-number variation is an important contributor to the genetic causality of inherited retinal degenerations

Abstract

Purpose: Despite substantial progress in sequencing, current strategies can genetically solve only approximately 55-60% of inherited retinal degeneration (IRD) cases. This can be partially attributed to elusive mutations in the known IRD genes, which are not easily identified by the targeted next-generation sequencing (NGS) or Sanger sequencing approaches. We hypothesized that copy-number variations (CNVs) are a major contributor to the elusive genetic causality of IRDs.

Methods: Twenty-eight cases previously unsolved with a targeted NGS were investigated with whole-genome single-nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays.

Results: Deletions in the IRD genes were detected in 5 of 28 families, including a de novo deletion. We suggest that the de novo deletion occurred through nonallelic homologous recombination (NAHR) and we constructed a genomic map of NAHR-prone regions with overlapping IRD genes. In this article, we also report an unusual case of recessive retinitis pigmentosa due to compound heterozygous mutations in SNRNP200, a gene that is typically associated with the dominant form of this disease.

Conclusions: CNV mapping substantially increased the genetic diagnostic rate of IRDs, detecting genetic causality in 18% of previously unsolved cases. Extending the search to other structural variations will probably demonstrate an even higher contribution to genetic causality of IRDs.Genet Med advance online publication 13 October 2016.

Figure 1. Deletion mapping in families OGI-046 and OGI-023

A) A heterozygous deletion of 20 genes on chr2q12.1 found in family OGI-046 by CGH and SNP arrays, and validated by SNPs found in WES B) Pedigree of family OGI-046 with mutant allele segregation indicated. C) Fine mapping of the deletion by qPCR in family OGI-046. D) Pedigree of family OGI-023 with mutant allele segregation indicated. E) Fine mapping of the deletion by qPCR in family OGI-023. Each primer pair in the qPCR experiments was normalized to two reference genes GPR15 and ZNF80, where the error bars represent standard deviation between these results.

Figure 2. Deletion mapping in family OGI-086

A) Pedigree of family OGI-086 with mutant allele segregation. B) Fine mapping of the deletion by qPCR with deleted genes indicated (error bars represent standard deviation between two normalization genes GPR15 and ZNF80). C) PCR amplification across the breakpoints. D) Sanger sequencing electropherogram showing the precise position of the deletion.

Figure 3. Deletion mapping in family OGI-014

A) Pedigree of family OGI-014 with mutant allele segregation. B) Composite fundus photograph of the left eye of patient OGI-014-038. C) Fine mapping of the deletion by qPCR with four deleted exons of EYS indicated (error bars represent standard deviation between two normalization genes GPR15 and ZNF80).

Figure 4. Non-allelic homologous recombination prone regions and IRD genes

NAHR-prone regions (green bars) were computationally predicted using the following criteria: >10kb long regions of >95% identity, with intervening sequence between 50 kb and 10 Mb and not spanning the centromere. Direct and inverted repeats are included. IRD genes overlapping the NAHR-prone regions are indicated as red arrows. Only the chromosomes, where NAHR-prone regions and IRD genes overlap are presented. The full genome-wide map of NAHR-prone regions is presented in Table S2. The asterisk denotes IRD whole-gene CNVs reported in this manuscript for the first time.