Abstract
BACKGROUND: ATP-binding cassette subfamily B member 4 (ABCB4) deficiency is associated with cholestatic liver disease primarily because of missense mutations, and many variants remain unidentified. Here, we validate the pathogenicity and mechanism of ABCB4 variants in clinical and in vitro trials, hypothesizing that these variants are responsible for impaired biliary function and contribute to the development of cholestatic liver diseases. AIM: To clarify the functional features and pathogenicity of ABCB4 variants. METHODS: Clinical data were collected from five patients with cholestatic liver disease that was initially not detected by routine examinations. Later, whole-exome sequencing confirmed ABCB4 variants and the patients were treated from January 2017 to December 2023. Pathogenic mechanisms were analyzed using bioinformatics tools, and a cell model in vitro was established to investigate ABCB4 mRNA expression, multidrug resistance protein 3 (MDR3) expression, cellular localization, and phosphatidylcholine secretion. Results were compared using Student's t-tests. RESULTS: Five missense variants (c.1757T>A, c.1865G>A, c.2362C>T, c.2777C>T and c.3250C>T), one intron variant (c.537-32G>T), and one synonymous (c.C504T) variant were identified. Three of the five patients had various degrees of cholestasis, two presented with liver cirrhosis, and all had elevated gamma-glutamyl transferase. Three of the four patients who underwent a liver biopsy had bile duct dilation, and one had gallstones. Two of the four patients had normal and reduced MDR3 immunohistochemical levels. Bioinformatic analysis indicated that these variants were likely pathogenic except c.C504T variant. None of the missense variants influenced subcellular MDR3 Localization in vitro. However, the c.1865G>A variant significantly decreased ABCB4 mRNA values, and all missense variants down-regulated phosphatidylcholine secretion. CONCLUSION: This study uncovered new ABCB4 variants and emphasized the pathogenic potential of specific variants. The findings from five patients provided insight into the pathogenic mechanisms underlying ABCB4-related diseases.