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Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes
Author(s) -
Le GuédardMéreuze Sandie,
Vaché Christel,
Baux David,
Faugère Valérie,
Larrieu Lise,
Abadie Caroline,
Janecke Andreas,
Claustres Mireille,
Roux AnneFrançoise,
TufferyGiraud Sylvie
Publication year - 2010
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21193
Subject(s) - biology , rna splicing , gene , genetics , splice , ex vivo , alternative splicing , splice site mutation , mutation , computational biology , exon , in vivo , rna
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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