非常规剪接位点变异越来越被认为是USH2A相关疾病的相关原因。非综合征性常染色体隐性遗传性视网膜色素变性和Usher综合征2型。许多非规范剪接位点变异已经在公共数据库中报道,但是对pre-mRNA剪接的影响仅在功能上得到了这些变体的一个子集的验证。在这项研究中,我们旨在通过评估一组选定的USH2A非规范剪接位点变异来扩展有关剪接事件的知识,并研究其潜在的致病性.基于四个剪接预测工具选择11个非规范剪接位点变体。产生10种不同的USH2A构建体,并在HEK293T细胞中进行小基因剪接测定。观察到所有11种变体对pre-mRNA剪接的影响。各种事件,比如外显子跳跃,观察到双外显子跳跃和部分外显子跳跃,8个受试变体对剪接有完全影响,因为未检测到常规剪接的mRNA.我们证明了USH2A中的非经典剪接位点变异是相关疾病的遗传病因的重要因素。这种类型的变异通常不应该在遗传筛选中被忽略。在USH2A相关疾病以及其他遗传性疾病中。此外,具有这些特定变异的病例现在可以接受决定性的基因诊断.
Non-canonical splice site variants are increasingly recognized as a relevant cause of the USH2A-associated diseases, non-syndromic autosomal recessive retinitis pigmentosa and Usher syndrome type 2. Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH2A non-canonical splice site variants and to study their potential pathogenicity. Eleven non-canonical splice site variants were selected based on four splice prediction tools. Ten different USH2A constructs were generated and minigene splice assays were performed in HEK293T cells. An effect on pre-mRNA splicing was observed for all 11 variants. Various events, such as exon skipping, dual exon skipping and partial exon skipping were observed and eight of the tested variants had a full effect on splicing as no conventionally spliced mRNA was detected. We demonstrated that non-canonical splice site variants in USH2A are an important contributor to the genetic etiology of the associated disorders. This type of variant generally should not be neglected in genetic screening, both in USH2A-associated disease as well as other hereditary disorders. In addition, cases with these specific variants may now receive a conclusive genetic diagnosis.