Functional inactivation of MDR3 caused by a homozygous ABCB4 missense variant leading to liver failure

Progressive familial intrahepatic cholestasis (PFIC) is a rare hereditary liver disorder that is caused by defective hepatobiliary transport. Variants in ATP binding cassette 4 ( ), encoding phosphatidylcholine floppase MDR3, are a frequent cause; however, many remain classified as variants of uncertain significance (VUS), limiting molecular diagnosis. Here, we functionally characterized a previously reported homozygous ABCB4 missense variant (c.431G>A, p.(Arg144Gln)) without experimental evidence of pathogenicity. An in silico analysis using the ABCB4-specific prediction tool Vasor indicated a high probability of pathogenicity (0.88). Structural modeling suggested that Arg144Gln disrupted key electrostatic interactions essential for MDR3 membrane anchoring. Immunofluorescence analyses demonstrated markedly reduced membrane localization with residual cytoplasmic retention, consistent with complete loss of protein function. In conclusion, the ABCB4 p.(Arg144Gln) variant causes functional inactivation of MDR3 and represents a novel pathogenic mutation. Combined genetic, structural, and functional analyses are valuable tools for characterizing variants of uncertain significance in ABCB4-associated cholestatic liver disease.