Molecular and in silico analyses of the full-length isoform of usherin identify new pathogenic alleles in Usher type II patients

Université de Montpellier 1, Montpelhièr, Languedoc-Roussillon, France
Human Mutation (Impact Factor: 5.14). 08/2007; 28(8):781-9. DOI: 10.1002/humu.20513
Source: PubMed


The usherin gene (USH2A) has been screened for mutations causing Usher syndrome type II (USH2). Two protein isoforms have been identified: a short isoform of 1,546 amino acids and a more recently recognized isoform extending to 5,202 amino acids. We have screened the full length by genomic sequencing. We confirm that many mutations occur in the exons contributing solely to the longer form. USH2 is an autosomal recessive disorder and, in contrast to previous studies, both mutations were identified in 23 patients and a single mutation in 2 out of 33 patients. A total of 34 distinct mutated alleles were identified, including one complex allele with three variants and another with two. A total of 27 of these are novel, confirming that most mutations in usherin are private. Many of the mutations will lead to prematurely truncated protein but as there are a substantial number of missense variants, we have used in silico analysis to assess their pathogenicity. Evidence that they are disease-causing has been produced by protein alignments and three-dimensional (3D) structural predictions when possible. We have identified a previously unrecognized cysteine rich structural domain, containing 12 dicysteine repeats, and show that three missense mutations result in the loss of one of a pair of the defining cysteine-cysteine pairs.

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    • "Comprehensive diagnostic strategies for USH2A must include (1) sequencing of the 72 exons and their flanking intronic regions by Sanger or next-generation sequencing (NGS) to detect point mutations and small indels, (2) multiplex ligation-dependent probe amplification (MLPA) analysis, quantitative multiplex PCR of short fragments or microarray-based comparative genomic hybridization (aCGH) to detect large rearrangements, and (3) the screening of the deep intronic mutation c.7595-2144A>G [Le Quesne Stabej et al., 2012; Vaché et al., 2012; Steele-Stallard et al., 2013; Baux et al., 2014; Besnard et al., 2014]. Despite these extensive approaches, for 1.8%– 19% of clinically well-defined USH2 individuals, only one mutation in USH2A has been identified [Baux et al., 2007; Garcia-Garcia et al., 2011; Le Quesne Stabej et al., 2012; unpublished data]. Indeed, promoter and regulatory untranslated regions or intronic sequences are not routinely screened. "
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    ABSTRACT: Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy.
    Full-text · Article · Oct 2015 · Human Mutation
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    • "2006, 2011; Baux et al. 2007; Dreyer et al. 2008; Bonnet et al. "
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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.
    Full-text · Article · Jan 2014
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    • "USH2 accounts for well over one-half of all Usher cases and up to date, 3 genes are known to be involved in the pathogenesis of this clinical form: USH2A, GPR98 and DFNB31 [4], [5], [6], [7]. Mutations in the USH2A gene are responsible for the majority of USH2 cases [8], [9], [10] and are also responsible for atypical Usher syndrome and recessive non-syndromic RP [11], [12]. Two main isoforms have been described for this gene. "
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    ABSTRACT: Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndrome is clinically classified into three types: USH1, USH2 and USH3. USH2 accounts for well over one-half of all Usher cases and mutations in the USH2A gene are responsible for the majority of USH2 cases, but also for atypical Usher syndrome and recessive non-syndromic RP. Because medaka fish (Oryzias latypes) is an attractive model organism for genetic-based studies in biomedical research, we investigated the expression and function of the USH2A ortholog in this teleost species. Ol-Ush2a encodes a protein of 5.445 aa codons, containing the same motif arrangement as the human USH2A. Ol-Ush2a is expressed during early stages of medaka fish development and persists into adulthood. Temporal Ol-Ush2a expression analysis using whole mount in situ hybridization (WMISH) on embryos at different embryonic stages showed restricted expression to otoliths and retina, suggesting that Ol-Ush2a might play a conserved role in the development and/or maintenance of retinal photoreceptors and cochlear hair cells. Knockdown of Ol-Ush2a in medaka fish caused embryonic developmental defects (small eyes and heads, otolith malformations and shortened bodies with curved tails) resulting in late embryo lethality. These embryonic defects, observed in our study and in other ciliary disorders, are associated with defective cell movement specifically implicated in left-right (LR) axis determination and planar cell polarity (PCP).
    Full-text · Article · Sep 2013 · PLoS ONE
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