Interpreting rare missense variants in ABCB4 using ACMG recommended in silico tools, bespoke prediction algorithm...

Intrahepatic Cholestasis of Pregnancy (ICP) is the most common pregnancy-related liver disorder. ABCB4 heterozygous variants are implicated in ICP, but interpretation of rare variants remains challenging, leading to many variants of uncertain significance. This study compares predictions from multiple in silico tools on nine heterozygous missense variants identified in women with ICP. Using the Genomics England Research Environment, 253 women with ICP and whole-genome sequencing data were analysed. Variants with minor allele frequency < 0.05 were filtered, and nine rare missense variants were assessed using SIFT, PolyPhen, CADD, Vasor (ABCB4-specific), and AlphaMissense. Only 44% (4/9) of variants had consistent classification across all tools. Published functional studies often conflicted with predictions. For example, T175A showed no detectable effect in HepG2/HEK293 cells but was classified as likely pathogenic by Vasor and benign by AlphaMissense. Similarly, N510S impacted protein stability functionally and was deemed likely pathogenic by Vasor but benign by AlphaMissense. In silico tools show conflicting predictions for ABCB4 rare variants, highlighting the difficulty of classification without functional or segregation data as well as the heavy reliance on computational predictions.