Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing

Abstract

BackgroundUsher 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.MethodsForty-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.ResultsEight 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.ConclusionsWe 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.

Partial USH2A deletions contribute to Usher syndrome in Denmark

“…No duplications were identified. Steele et al 22 identified also USH2A deletions with similar frequency, as deletions were identified in 5% (7/121) unselected USH2 patients.…”

“…21,22 Investigation of PCDH15 revealed two deletions of 55 kb (exon 3 including the flanking introns) and 82 kb (exon 4-6 including the flanking introns), respectively, 21 whereas investigation of USH2A revealed one duplication (from exon 4 to exon 13) and five deletions (exon 4, exons 22-23, exon 27, exon 40 or exon 70 respectively). 22 Here we used MLPA to screen a cohort of 50 Danish USH patients (20 USH1 and 30 USH2) for exon deletions and duplications in PCDH15 and USH2A.…”

Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: mutation spectrum and new genotype-phenotype correlation

“…Braun and co-workers were first to provide evidence for the possibility of intronic variants near rare alternate splice junctions that could be pathogenic by increasing the probability of mis-splicing at these sites [65]. Deep intronic mutations leading to truncated proteins have also been described in Usher syndrome and LCA [66, 67]. This need to be further investigated in our 6 patients.…”

Comprehensive Molecular Screening in Chinese Usher Syndrome Patients

“…21,22 Moreover, several studies have demonstrated that copy number variants (CNVs) of some USH genes account for certain proportions of mutations. 23,24 Most of these CNVs are heterozygous and cannot be detected by Sanger sequencing or APEX. These kinds of genomic DNA rearrangements must be detected by other techniques, such as real-time quantitative polymerase chain reaction (q-PCR), multiplex ligation-dependent probe amplification (MLPA), or comparative genomic hybridization (a-CGH).…”

“…These kinds of genomic DNA rearrangements must be detected by other techniques, such as real-time quantitative polymerase chain reaction (q-PCR), multiplex ligation-dependent probe amplification (MLPA), or comparative genomic hybridization (a-CGH). 23 Next-generation sequencing (NGS) technologies developed in recent years can perform whole genome; whole exome; and targeted exome sequencing (TES). 25 Several studies have proven TES as a highefficiency method of molecular diagnosis of USH, with a detection rate of around 70%.…”