title: Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes.
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.
authors.affiliation: Université Montpellier 1, UFR médecine, Montpellier, France.
authors.forename: Sandie
authors.initials: S
authors.lastname: Le Guédard-Méreuze
authors.forename: Christel
authors.initials: C
authors.lastname: Vaché
authors.forename: David
authors.initials: D
authors.lastname: Baux
authors.forename: Valérie
authors.initials: V
authors.lastname: Faugère
authors.forename: Lise
authors.initials: L
authors.lastname: Larrieu
authors.forename: Caroline
authors.initials: C
authors.lastname: Abadie
authors.forename: Andreas
authors.initials: A
authors.lastname: Janecke
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Roux
authors.forename: Sylvie
authors.initials: S
authors.lastname: Tuffery-Giraud
mesh_terms: D000465:Algorithms; D017398:Alternative Splicing; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005091:Exons; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D052245:Usher Syndromes / Q000235:genetics*