Université Montpellier 1, UFR médecine, Montpellier, France.
Hum Mutat. 2010 Mar;31(3):347-55. doi: 10.1002/humu.21193.
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.
在 1 型和 2 型 Usher 综合征患者中进行分子诊断，导致在 MYO7A、CDH23、USH1C 和 USH2A 基因的非典型剪接位点发现了 21 个序列变异。为了实验确定这些替代物的剪接模式，这些替代物的剪接影响并不总是可通过现有软件预测的，进行了体外剪接分析。还使用分支点映射策略进一步研究了 USH2A 内含子 43 中的假定分支点突变。在测试的 21 个序列变异中的 16 个（76.2%）中证明了异常剪接。与外显子跳跃（37.5%）相比，突变更频繁地导致激活附近的隐秘剪接位点或使用新的剪接位点。这项研究使一个无义突变（c.7872G>A(p.Glu2624Glu) 在 CDH23 中）和四个错义突变（c.2993G>A(p.Arg998Lys) 在 USH2A 中，c.592G>A(p.Ala198Thr)，c.3503G>C[p.Arg1168Pro]，c.5944G>A(p.Gly1982Arg) 在 MYO7A 中）重新分类为剪接突变，而在其他四个病例中提供了结构/功能作用的线索：c.802G>A(p.Gly268Arg)，c.653T>A(p.Val218Glu)（USH2A），和 c.397C>T(p.His133Tyr)，c.3502C>T(p.Arg1168Trp)（MYO7A）。我们的数据提供了对 Usher 基因中剪接突变贡献的深入了解，并说明了为诊断目的准确定义其剪接结果的必要性。
