Zhou Xiaoying, Zhou Wei, Wang Chunli, Wang Lan, Jin Yu, Jia Zhanjun, Liu Zhifeng, Zheng Bixia
Department of Gastroenterology, Children's Hospital of Nanjing Medical University, Nanjing, China.
Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China.
Front Genet. 2021 Feb 25;11:592611. doi: 10.3389/fgene.2020.592611. eCollection 2020.
Next-generation sequencing is effective for the molecular diagnosis of genetic diseases. However, the identification of the clinical significance of synonymous variants remains a challenge. Our previous study showed that some synonymous variants in gene produced splicing disruptions, leading to Wilson disease (WD). To test the hypothesis that synonymous variants of cause abnormal splicing by disrupting authentic splice sites or splicing regulatory elements, we used computational tools and minigene assays to characterize 253 naturally occurring gene synonymous variants in this study. Human Splicing Finder (HSF) and ESE Finder 3.0 were used to predict the impact of these rare synonymous variants on pre-mRNA splicing. Then, we cloned 14 different wild-type Minigene_ATP7B_ex constructs for minigene assay, including 16 exons of gene. After computational prediction, 85 candidate variants were selected to be introduced into the corresponding Minigene_ATP7B_ex constructs for splicing assays. Using this two-step procedure, we demonstrated that 11 synonymous variants in ExAc database (c.1620C>T, c.3888C>T, c.1554C>T, c.1677C>T, c.1830G>A, c.1875T>A, c.2826C>A, c.4098G>A, c.2994C>T, c.3243G>A, and c.3747G>A) disrupted RNA splicing , and two (c.1620C>T and c.3243G>A) of these caused a complete exon skipping. The results not only provided a reliable experimental basis for the genetic diagnosis of WD patients but also offered some new insights into the pathogenicity of synonymous variants in genetic diseases.
下一代测序技术在遗传性疾病的分子诊断中具有重要作用。然而，同义变异临床意义的判定仍是一大挑战。我们之前的研究表明，某基因中的一些同义变异会导致剪接中断，进而引发威尔逊病（WD）。为验证同义变异通过破坏真实剪接位点或剪接调控元件导致异常剪接这一假说，我们在本研究中运用计算工具和小基因分析对253个天然存在的该基因同义变异进行了特征分析。利用人类剪接预测软件（HSF）和外显子剪接增强子预测软件3.0来预测这些罕见同义变异对前体mRNA剪接的影响。随后，我们克隆了14种不同的野生型小基因_ATP7B_ex构建体用于小基因分析，其中包含该基因的16个外显子。经过计算预测后，挑选出85个候选变异引入相应的小基因_ATP7B_ex构建体中进行剪接分析。通过这两步实验流程，我们证实了ExAc数据库中的11个同义变异（c.1620C>T、c.3888C>T、c.1554C>T、c.1677C>T c.1830G>A、c.1875T>A、c.2826C>A、c.4098G>A、c.2994C>T、c.3243G>A和c.3747G>A）破坏了RNA剪接，其中两个（c.1620C>T和c.3243G>A）导致了整个外显子跳跃。这些结果不仅为WD患者的基因诊断提供了可靠的实验依据，还为遗传性疾病同义变异的致病性研究提供了新的见解。
