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

Orphanet Journal of Rare Diseases

Maria Bitner-Glindzicz²  Andrew R Webster⁵  Anne-Francoise Roux⁴  Mireille Claustres⁴  Linda M Luxon³  Eva Lenassi⁵  Polona Le Quesne Stabej¹  Heather B Steele-Stallard¹ 

[1] UCL Institute of Child Health, London, UK;Great Ormond Street Hospital, London, UK;UCL Ear Institute, London, UK;Inserm, U827, Montpellier F-34000, France;Moorfields Eye Hospital, London, UK

关键词: Array CGH;    Multiplex ligation dependant probe amplification (MLPA);    Pseudoexon;    Duplication;    Deletion;    USH2A;    Usher syndrome;   

Others  :  863610
DOI  :  10.1186/1750-1172-8-122

 received in 2013-04-16, accepted in 2013-08-04,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Steele-Stallard et al.; licensee BioMed Central Ltd.

【 图 表 】

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