Screening for Duplications, Deletions and a Common Intronic Mutation Detects 35% of Second Mutations in Patients With USH2A Monoallelic Mutations on Sanger Sequencing

Orphanet J Rare Dis. 2013 Aug 8;8:122. doi: 10.1186/1750-1172-8-122.

Abstract

Background: Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G.

Methods: Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints.

Results: Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome.

Conclusions: We found that 8 of 23 (35%) of 'missing' mutations in Usher type 2 probands with only a single heterozygous USH2A mutation detected with Sanger sequencing could be attributed to deletions, duplications or a pathogenic deep intronic variant. Future mutation detection strategies and genetic counselling will need to take into account the prevalence of these types of mutations in order to provide a more comprehensive diagnostic service.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Comparative Genomic Hybridization
  • Extracellular Matrix Proteins / genetics*
  • Family
  • Female
  • Fibroblasts
  • Gene Deletion*
  • Gene Duplication*
  • Humans
  • Introns / genetics
  • Male
  • Multiplex Polymerase Chain Reaction
  • Mutation*
  • Sequence Analysis, DNA / methods
  • Usher Syndromes / genetics*
  • Usher Syndromes / pathology

Substances

  • Extracellular Matrix Proteins
  • USH2A protein, human

Supplementary concepts

  • Usher syndrome, type 2A