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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    • "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.
    01/2014; 2(1):30-43. DOI:10.1002/mgg3.25
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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).
    PLoS ONE 09/2013; 8(9):e74995. DOI:10.1371/journal.pone.0074995 · 3.23 Impact Factor
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    • "Usher syndrome is a genetically heterogeneous autosomal recessive disorder characterized by early onset sensorineural hearing loss and later onset retinitis pigmentosa (RP). Mutations in the USH2A gene are the most common cause of Usher syndrome type I (Aller et al., 2006; Baux et al., 2007; DePristo et al., 2011) and are also a common cause of non-syndromic RP (McGee et al., 2010; Vaché et al., 2012). The combination of hearing loss and retinitis pigmentosa in Usher syndrome creates an unusual opportunity for the development of effective gene replacement therapy. "
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    ABSTRACT: Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:
    eLife Sciences 08/2013; 2(2):e00824. DOI:10.7554/eLife.00824 · 9.32 Impact Factor
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