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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.

A Comprehensive Analysis and Splicing Characterization of Naturally Occurring Synonymous Variants in the ATP7B Gene