Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

Authors

  • Sandie Le Guédard-Méreuze,

    1. Université Montpellier 1, UFR médecine, Montpellier, France
    2. INSERM, U827, Montpellier, France
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  • Christel Vaché,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • David Baux,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • Valérie Faugère,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • Lise Larrieu,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • Caroline Abadie,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • Andreas Janecke,

    1. Division of Clinical Genetics, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Austria
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  • Mireille Claustres,

    1. Université Montpellier 1, UFR médecine, Montpellier, France
    2. INSERM, U827, Montpellier, France
    3. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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  • Anne-Françoise Roux,

    1. CHU Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Génétique Moléculaire, Montpellier, France
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    • The last two authors contributed equally to this work.

  • Sylvie Tuffery-Giraud

    Corresponding author
    1. Université Montpellier 1, UFR médecine, Montpellier, France
    2. INSERM, U827, Montpellier, France
    • Laboratoire de Génétique Moléculaire et INSERM U827, IURC, Institut Universitaire de Recherche Clinique, 641 Avenue du Doyen Giraud, 34093 Montpellier Cedex 5, France
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    • The last two authors contributed equally to this work.


  • Communicated by Claude Ferec

Abstract

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. Hum Mutat 31:1–9, 2010. © 2010 Wiley-Liss, Inc.

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