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Abstract
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 ATP7B gene produced splicing disruptions, leading to Wilson disease (WD). To test the hypothesis that synonymous variants of ATP7B cause abnormal splicing by disrupting authentic splice sites or splicing regulatory elements, we used computational tools and minigene assays to characterize 253 naturally occurring ATP7B 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 in vitro minigene assay, including 16 exons of ATP7B 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 in vitro, 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.
Highlights
Wilson disease (OMIM, #277900, WD) is an autosomal, recessively inherited disorder characterized by the dramatic accumulation of intracellular hepatic copper with subsequent hepatic and neurologic abnormalities (Xie and Wu, 2017)
A total of 253 rare synonymous variants that presented in Exome Aggregation Consortium (ExAc) database or were previously reported in literature were included in this study (El-Mougy et al, 2014; Dong et al, 2016; Qian et al, 2019)
Based on the predicted results synonymous mutations into the following three groups for Minigene Assays: (1) 18 variants that may destroy a variety of Exonic splicing enhancers (ESEs), (2) 46 variants that may produce new exonic splicing silencers (ESSs), and (3) 21 variants that may affect donor or acceptor site splicing
Summary
Wilson disease (OMIM, #277900, WD) is an autosomal, recessively inherited disorder characterized by the dramatic accumulation of intracellular hepatic copper with subsequent hepatic and neurologic abnormalities (Xie and Wu, 2017). Loss-of-function mutations in ATPase copper-transporting beta gene (ATP7B) are responsible for WD. The ATP7B gene (OMIM, ∗606882) is located on chromosome 13q14.3 encoding copper-transporting ATPase 2 (ATP7B), Splicing Characterization of Synonymous Variants in ATP7B acting as a plasma membrane copper-transport protein. At present (Hedera, 2019), genetic screening of ATP7B gene is widely used in the diagnosis of WD, which can greatly benefit the clinical diagnosis and management of patients (Li et al, 2019). Synonymous variants yield from mutation screening with uncertain clinical significance (VUS). The functional and clinical interpretations pose a challenge for genomic diagnosis of WD
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