A Comprehensive Analysis of Novel Variations Associated with Bile Duct Cancer: Insights into Expression, Methylation, and 3D Protein Structure.

Cholangiocarcinoma is a rare but highly lethal cancer of the biliary epithelium, marked by heterogeneous molecular subtypes, unclear etiology, and poor five-year survival, highlighting the need for new diagnostic and therapeutic strategies; therefore, this study integrates genomic, transcriptomic, single-cell, methylomic, and molecular-dynamics data to pinpoint pathogenic variants. We performed an integrative multi-omics analysis of publicly available datasets. Somatic variants from 23 tumor samples in The Cancer Genome Atlas were annotated with 11 pathogenicity tools (AUC ≥ 0.86 across EVE, REVEL, SIFT, AlphaMissense, DEOGEN2 were the most stringent). Differential gene expression was assessed in matched bulk RNA-seq (tumor vs. non-tumor) using DESeq2 with Benjamini-Hochberg FDR correction. A single-cell RNA-seq dataset comprising 23,782 cells from an intrahepatic cholangiocarcinoma was clustered with marker genes identified by Wilcoxon rank-sum tests. Illumina 450 K methylation arrays (52 tumors, 12 normal livers) were analyzed with limma and DMRcate to detect differentially methylated probes and regions. AlphaFold3 models of wild-type and MAP2K1