Non-USH2A mutations in USH2 patients

CHU Montpellier, Laboratoire de Génétique Moléculaire, Montpellier, France.
Human Mutation (Impact Factor: 5.14). 03/2012; 33(3):504-10. DOI: 10.1002/humu.22004
Source: PubMed


We have systematically analyzed the two known minor genes involved in Usher syndrome type 2, DFNB31 and GPR98, for mutations in a cohort of 31 patients not linked to USH2A. PDZD7, an Usher syndrome type 2 (USH2) related gene, was analyzed when indicated. We found that mutations in GPR98 contribute significantly to USH2. We report 17 mutations in 10 individuals, doubling the number of GPR98 mutations reported to date. In contrast to mutations in usherin, the mutational spectrum of GPR98 predominantly results in a truncated protein product. This is true even when the mutation affects splicing, and we have incorporated a splicing reporter minigene assay to show this, where appropriate. Only two mutations were found which we believe to be genuine missense changes. Discrepancy in the mutational spectrum between GPR98 and USH2A is discussed. Only two patients were found with mutations in DFNB31, showing that mutations of this gene contribute to only a very small extent to USH2. Close examination of the clinical details, where available, for patients in whom no mutation was found in USH2A, GPR98, or DFNB31, showed that most of them had atypical features. In effect, these three genes account for the vast majority of USH2 patients and their analysis provide a robust pathway for routine molecular diagnosis.

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Available from: Thomas Besnard,
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    • "Mutations in three genes, USH2A [13], GPR98 [14], and DFNB31/WHRN [15] have been identified as disease-causing for USH2. USH2A is the most frequently mutated gene in USH2 patients and mutations in USH2A alone account for 85% of USH2 while mutations in GPR98 account for 6% of USH2 in the French and other Caucasian populations [16], [17]. Mutations in one gene CLRN1 are found causative for USH3 [18]. "
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    ABSTRACT: Background Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II. Methods Whole exome sequencing followed by expanded familial validation by Sanger sequencing. Results We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98. Conclusion Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.
    PLoS ONE 09/2014; 9(9). DOI:10.1371/journal.pone.0107326 · 3.23 Impact Factor
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    • "Furthermore, large rearrangements have been described in MYO7A, CDH23, GPR98, USH2A and, particularly, in PCDH15, and their detection requires array-CGH studies and/or multiplex ligation-dependent probe amplification (see USHbases). Taken together, these strategies allow a reliable diagnosis for Usher patients with a mutation detection rate of about 90% for USH1 and USH2 patients (Roux et al. 2011; Besnard et al. 2012). A genotyping microarray commercially available (Cremers et al. 2006) allows rapid screening for hundreds of previously identified variations in nine USH genes (Vozzi et al. 2011), but its application in clinical diagnosis is hampered by a very low detection rate as most USHcausing DNA alterations are private or restricted to one or two families (see USHBases). "
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    ABSTRACT: We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of 71 patients including Usher patients previously screened by Sanger sequencing plus newly referred patients was studied. Ninety-eight percent of the variants previously identified by Sanger sequencing were found by next-generation sequencing (NGS). NGS proved to be efficient as it offers analysis of all relevant genes which is laborious to reach with Sanger sequencing. Among the 13 newly referred Usher patients, both mutations in the same gene were identified in 77% of cases (10 patients) and one candidate pathogenic variant in two additional patients. This work can be considered as pilot for implementing NGS for genetically heterogeneous diseases in clinical service.
    01/2014; 2(1):30-43. DOI:10.1002/mgg3.25
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    • "Previous work has identified such deletions through haplotype analysis, but to be comprehensive this requires the individual to carry informative variants in every exon. More recent methodologies have looked for deletions and duplications using array comparative genomic hybridization (array CGH) and multiplex ligation dependent probe amplification (MLPA) [10,14,15]. These methods have not yet been utilised to detect deletions and duplications in USH2A in USH2 and atypical individuals. "
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    ABSTRACT: 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. 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. 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. 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.
    Orphanet Journal of Rare Diseases 08/2013; 8(1):122. DOI:10.1186/1750-1172-8-122 · 3.36 Impact Factor
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