BACKGROUND: Wilson's disease (WD) is an autosomal recessive inherited disease caused by ATP7B gene mutations. Some mutations in ATP7B are presumed to be pathogenic by altering pre-mRNA splicing, while most have not been functionally verified. This study aimed to perform functional studies to verify the pathogenicity of variants that may affect pre-mRNA splicing. METHODS: We recruited 42 pediatric patients who were clinically diagnosed with WD (Leipzig score ≥ 4) and underwent ATP7B gene sequencing. We leveraged in silico analysis and prioritized seven splice genic variants in ATP7B. Minigene assays were used to evaluate the effects of the selected variants on transcript splicing. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed on samples from several patients to verify the splicing alterations. RESULTS: This study screened 42 distinct mutations for their potential effects on splicing based on in silico analysis and functional verification. Five intronic variants (c.1286-1delG, c.1543 + 1G >
T, c.1708-1G >
C, c.1870-8A >
G, and c.2121 + 3A >
T) and one missense variant (c.2120A >
G) were proved to alter the splicing of ATP7B transcription by minigene assays. The transcript assays demonstrated splicing changes in vivo in patient PBMCs for c.3993 T >
G. The altered transcription products resulting from c.2570_2572del were confirmed by sequencing. CONCLUSIONS: This study adds experimental evidence to genetic diagnosis based on assessing the genetic defects of 42 pediatric WD patients and provides new insights into the pathogenicity of the splicing variants.