{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T05:39:19Z","timestamp":1774935559601,"version":"3.50.1"},"reference-count":99,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T00:00:00Z","timestamp":1763683200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u2265 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\u2013Hochberg 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 MAP2K1R49C were subjected to 50 ns all-atom molecular-dynamics simulations in GROMACS; conformational shifts were quantified by RMSD\/RMSF and stability tested with FoldX5. Twenty-four tumor-specific missense variants were detected. The four highest-confidence pathogenic substitutions (EVE, REVEL, SIFT, AlphaMissense, DEOGEN2) occurred in TUBB3, FLNC, ABCA1, and MAP2K1. Bulk RNA-seq confirmed significant dysregulation of these genes and enrichment of extracellular-matrix organization, cytoskeletal remodeling, MAPK signaling, and cholesterol-efflux pathways. Single-cell analysis resolved 23 transcriptionally distinct clusters; proliferative malignant cholangiocytes selectively over-expressed ABCA1 and MAP2K1, indicating tumor-cell specificity. Methylome profiling identified 148,928 DMPs and 7040 DMRs; promoter hypomethylation of TUBB3 and ABCA1 correlated with their transcriptional activation. Substituting Arg-49 with Cys in MAP2K1 dismantles the Arg-centred hydrogen-bond\/salt-bridge cluster, reduces hydrophobic packing, and, corroborated by 50 ns MD (Welch\u2019s t = \u221258.06, p = 3.17 \u00d7 10\u2212230) and FoldX5 (\u0394\u0394G = +2.3 kcal mol\u22121), significantly destabilises the protein, manifesting as higher backbone RMSD and increased local flexibility relative to wild type. This multi-omics, public data-driven synthesis delineates a coherent network of genomic, epigenomic, transcriptomic, and structural vulnerabilities, offering a rational framework for therapeutic targeting of cholangiocarcinoma. This study reveals novel bile duct-associated variations that expand our understanding of cholangiocarcinoma pathogenesis and provide potential targets for precision medicine approaches.<\/jats:p>","DOI":"10.3390\/ijms262311244","type":"journal-article","created":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T08:59:46Z","timestamp":1763715586000},"page":"11244","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Comprehensive Analysis of Novel Variations Associated with Bile Duct Cancer: Insights into Expression, Methylation, and 3D Protein Structure"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5359-4357","authenticated-orcid":false,"given":"Alper","family":"B\u00fclb\u00fcl","sequence":"first","affiliation":[{"name":"Department of Biostatistics and Bioinformatics, Institute of Health Sciences, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5886-3269","authenticated-orcid":false,"given":"Gizel","family":"Gerdan","sequence":"additional","affiliation":[{"name":"Department of Genome Studies, Institute of Health Sciences, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0853-9740","authenticated-orcid":false,"given":"Cansu","family":"Portakal","sequence":"additional","affiliation":[{"name":"Department of Genome Studies, Institute of Health Sciences, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-3550-3276","authenticated-orcid":false,"given":"Sudenaz","family":"Bajrami","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Faculty of Engineering and Natural Sciences, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7263-2969","authenticated-orcid":false,"given":"Cemaliye","family":"Boylu Akyerli","sequence":"additional","affiliation":[{"name":"Department of Genome Studies, Institute of Health Sciences, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"},{"name":"Department of Medical Biology, School of Medicine, Ac\u0131badem Mehmet Ali Ayd\u0131nlar University, \u0130stanbul 34638, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1038\/s41572-021-00300-2","article-title":"Cholangiocarcinoma","volume":"7","author":"Brindley","year":"2021","journal-title":"Nat. Rev. Dis. Prim."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1474","DOI":"10.1038\/ng.2806","article-title":"Exome sequencing identifies distinct mutational patterns in liver fluke\u2013related and non-infection-related bile duct cancers","volume":"45","author":"Ong","year":"2013","journal-title":"Nat. Genet."},{"key":"ref_3","unstructured":"(2025, July 02). Statistics About Bile Duct Cancer|Cholangiocarcinoma Stats. Available online: https:\/\/www.cancer.org\/cancer\/types\/bile-duct-cancer\/about\/key-statistics.html."},{"key":"ref_4","first-page":"229","article-title":"Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"74","author":"Bray","year":"2024","journal-title":"CA Cancer J. Clin."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1134","DOI":"10.1053\/j.gastro.2009.02.038","article-title":"Burden of digestive diseases in the United States Part III: Liver, biliary tract, and pancreas","volume":"136","author":"Everhart","year":"2009","journal-title":"Gastroenterology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"200","DOI":"10.37349\/etat.2022.00079","article-title":"Genomic alterations in cholangiocarcinoma: Clinical significance and relevance to therapy","volume":"3","author":"Carotenuto","year":"2022","journal-title":"Explor. Target. Antitumor Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1038\/s41586-023-06045-0","article-title":"A Genomic Mutational Constraint Map Using Variation in 76,156 Human Genomes","volume":"625","author":"Chen","year":"2024","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1038\/s41586-021-04043-8","article-title":"Disease variant prediction with deep generative models of evolutionary data","volume":"599","author":"Frazer","year":"2021","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/nprot.2015.123","article-title":"SIFT missense predictions for genomes","volume":"11","author":"Vaser","year":"2016","journal-title":"Nat. Protoc."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"W201","DOI":"10.1093\/nar\/gkx390","article-title":"DEOGEN2: Prediction and interactive visualization of single amino acid variant deleteriousness in human proteins","volume":"45","author":"Raimondi","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1016\/j.ajhg.2016.08.016","article-title":"REVEL: An ensemble method for predicting the pathogenicity of rare missense variants","volume":"99","author":"Ioannidis","year":"2016","journal-title":"Am. J. Hum. Genet."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/s41597-024-03327-8","article-title":"Analysis of AlphaMissense data in different protein groups and structural context","volume":"11","author":"Tordai","year":"2024","journal-title":"Sci. Data"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1038\/cdd.2017.169","article-title":"How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression?","volume":"25","author":"Aubrey","year":"2018","journal-title":"Cell Death Differ."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"111","DOI":"10.7150\/ijbs.23230","article-title":"The role of TGF-\u03b2\/SMAD4 signaling in cancer","volume":"14","author":"Zhao","year":"2018","journal-title":"Int. J. Biol. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1807","DOI":"10.1126\/science.1164382","article-title":"An integrated genomic analysis of human glioblastoma multiforme","volume":"321","author":"Parsons","year":"2008","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1668","DOI":"10.1126\/science.272.5268.1668","article-title":"Human homolog of patched, a candidate gene for the basal cell nevus syndrome","volume":"272","author":"Johnson","year":"1996","journal-title":"Science"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1002\/(SICI)1097-0215(19980812)77:4<522::AID-IJC8>3.0.CO;2-W","article-title":"Functionally inactivating point mutation in the tumor-suppressor IRF-1 gene identified in human gastric cancer","volume":"77","author":"Nozawa","year":"1998","journal-title":"Int. J. Cancer"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1038\/nrc3106","article-title":"RAS oncogenes: Weaving a tumorigenic web","volume":"11","author":"Grabocka","year":"2011","journal-title":"Nat. Rev. Cancer"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1097\/JTO.0b013e3181d35236","article-title":"MEK1 and AKT2 mutations in Japanese lung cancer","volume":"5","author":"Sasaki","year":"2010","journal-title":"J. Thorac. Oncol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1007\/s00439-009-0704-z","article-title":"Accelerated, decline, in, lung, function, in, cigarette, smokers, is, assoc, iated with TP53\/MDM2 polymorphisms","volume":"126","author":"Hancox","year":"2009","journal-title":"Hum. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/nature10866","article-title":"IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype","volume":"483","author":"Turcan","year":"2012","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"608","DOI":"10.1186\/s40001-025-02876-x","article-title":"Unveiling the multifaceted role of the FLNC gene: Implications for cancer diagnosis and prognosis","volume":"30","author":"Ni","year":"2025","journal-title":"Eur. J. Med. Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1006\/geno.1995.9961","article-title":"Complete structural organization of the human alpha 1(V) collagen gene (COL5A1): Divergence from the conserved organization of other characterized fibrillar collagen genes","volume":"29","author":"Takahara","year":"1995","journal-title":"Genomics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e2114994119","DOI":"10.1073\/pnas.2114994119","article-title":"Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules","volume":"119","author":"LaFrance","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"G501","DOI":"10.1152\/ajpgi.00212.2022","article-title":"Myosin 5b is required for proper localization of the intermicrovillar adhesion complex in the intestinal brush border","volume":"323","author":"Dooley","year":"2022","journal-title":"Am. J. Physiol.\u2013Gastrointest. Liver Physiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1002\/ar.23330","article-title":"Type V collagen in health, disease, and fibrosis","volume":"299","author":"Mak","year":"2016","journal-title":"Anat. Rec."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1945","DOI":"10.1002\/humu.24452","article-title":"A loss-of-function cysteine mutant in fibulin-3 (EFEMP1) forms aberrant extracellular disulfide-linked homodimers and alters extracellular matrix composition","volume":"43","author":"Woodard","year":"2022","journal-title":"Hum. Mutat."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/S0167-4781(99)00078-0","article-title":"Interferon regulatory factor 1 tryptophan 11 to arginine point mutation abolishes DNA binding","volume":"1446","author":"Eason","year":"1999","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3157","DOI":"10.1101\/gad.11.23.3157","article-title":"Dual role of the Smad4\/DPC4 tumor suppressor in TGF\u03b2-inducible transcriptional complexes","volume":"11","author":"Liu","year":"1997","journal-title":"Genes Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2200","DOI":"10.1093\/hmg\/ddl145","article-title":"Calcium-sensing receptor dimerizes in the endoplasmic reticulum: Biochemical and biophysical characterization of CASR mutants retained intracellularly","volume":"15","author":"Pidasheva","year":"2006","journal-title":"Hum. Mol. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/0014-5793(88)81361-9","article-title":"Family of human Na+,K+-ATPase genes. Structure of the gene for the catalytic subunit (alpha III-form) and its relationship with structural features of the protein","volume":"233","author":"Ovchinnikov","year":"1988","journal-title":"FEBS Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"22008","DOI":"10.1074\/jbc.274.31.22008","article-title":"Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells","volume":"274","author":"Voskoboinik","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1016\/j.oraloncology.2009.02.003","article-title":"PTCH1 mutations in odontogenic keratocysts: Are they related to epithelial cell proliferation?","volume":"45","author":"Pan","year":"2009","journal-title":"Oral Oncol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1002\/humu.21377","article-title":"Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders","volume":"32","author":"Gremer","year":"2011","journal-title":"Hum. Mutat."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1038\/ejhg.2008.256","article-title":"Spectrum of MEK1 and MEK2 gene mutations in cardio-facio-cutaneous syndrome and genotype\u2013phenotype correlations","volume":"17","author":"Dentici","year":"2009","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"eaar6795","DOI":"10.1126\/scisignal.aar6795","article-title":"Activating mutations in MEK1 enhance homodimerization and promote tumorigenesis","volume":"11","author":"Yuan","year":"2018","journal-title":"Sci. Signal."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1111\/his.12497","article-title":"Tubulin \u03b2-III: A novel immunohistochemical marker for intrahepatic peripheral cholangiocarcinoma","volume":"65","author":"Zen","year":"2014","journal-title":"Histopathology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1200\/JCO-24-01383","article-title":"SWOG S1815: A Phase III Randomized Trial of Gemcitabine, Cisplatin, and Nab-Paclitaxel Versus Gemcitabine and Cisplatin in Newly Diagnosed, Advanced Biliary Tract Cancers","volume":"43","author":"Shroff","year":"2025","journal-title":"J. Clin. Oncol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Choi, J.H., and Thung, S.N. (2024). Recent advances in pathology of intrahepatic cholangiocarcinoma. Cancers, 16.","DOI":"10.20944\/preprints202403.0198.v1"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2397","DOI":"10.1002\/hep.26762","article-title":"Desmoplastic stroma and cholangiocarcinoma: Clinical implications and therapeutic targeting","volume":"59","author":"Sirica","year":"2014","journal-title":"Hepatology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41419-024-06873-6","article-title":"Myofibroblasts derived type V collagen promoting tissue mechanical stress and facilitating metastasis and therapy resistance of lung adenocarcinoma cells","volume":"15","author":"Zhu","year":"2024","journal-title":"Cell Death Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"557","DOI":"10.7314\/APJCP.2013.14.1.557","article-title":"Gene expression profiling of intrahepatic cholangiocarcinoma","volume":"14","author":"Subrungruang","year":"2013","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1111\/liv.15800","article-title":"Filamin A is involved in human intrahepatic cholangiocarcinoma aggressiveness and progression","volume":"44","author":"Vitali","year":"2024","journal-title":"Liver Int."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.18632\/oncotarget.2645","article-title":"Filamin C, a dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells","volume":"6","author":"Qiao","year":"2014","journal-title":"Oncotarget"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"68242","DOI":"10.18632\/oncotarget.11921","article-title":"Quantitative proteomics reveals FLNC as a potential progression marker for the development of hepatocellular carcinoma","volume":"7","author":"Qi","year":"2016","journal-title":"Oncotarget"},{"key":"ref_46","first-page":"5173","article-title":"Downregulation of ABCA1 and ABCG1 transporters by simvastatin in cholangiocarcinoma cells","volume":"18","author":"Seeree","year":"2019","journal-title":"Oncol. Lett."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3827","DOI":"10.1245\/s10434-014-3828-x","article-title":"Genomic profiling of intrahepatic cholangiocarcinoma: Refining prognosis and identifying therapeutic targets","volume":"21","author":"Zhu","year":"2014","journal-title":"Ann. Surg. Oncol."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Quinn, L.M., Haldenby, S., Antzcak, P., Fowler, A., Bullock, K., Kenny, J., Gilbert, T., Andrews, T., Diaz-Nieto, R., and Fenwick, S. (2023). Genomic profiling of idiopathic peri-hilar cholangiocarcinoma reveals new targets and mutational pathways. Sci. Rep., 13.","DOI":"10.1038\/s41598-023-33096-0"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1038\/s41416-022-01903-6","article-title":"Randomised, Phase II study of selumetinib, an oral inhibitor of MEK, in combination with cisplatin and gemcitabine chemotherapy for patients with advanced biliary tract cancer","volume":"127","author":"Doherty","year":"2022","journal-title":"Br. J. Cancer"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e152670","DOI":"10.1172\/JCI152670","article-title":"Multicenter randomized phase II trial of atezolizumab with or without cobimetinib in biliary tract cancers","volume":"131","author":"Yarchoan","year":"2021","journal-title":"J. Clin. Investig."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"4017","DOI":"10.1200\/JCO.2024.42.16_suppl.4017","article-title":"A randomized phase 2 study of combination atezolizumab and varlilumab (CDX-1127) with or without addition of cobimetinib in previously treated unresectable biliary tract cancer (ETCTN 10476)","volume":"42","author":"Heumann","year":"2024","journal-title":"J. Clin. Oncol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1056\/NEJMoa2206834","article-title":"Futibatinib for FGFR2-rearranged intrahepatic cholangiocarcinoma","volume":"388","author":"Goyal","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1038\/ng.2764","article-title":"The cancer genome atlas pan-cancer analysis project","volume":"45","author":"Weinstein","year":"2013","journal-title":"Nat. Genet."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"e164","DOI":"10.1093\/nar\/gkq603","article-title":"ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data","volume":"38","author":"Wang","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1581","DOI":"10.1038\/ng.3703","article-title":"M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity","volume":"48","author":"Jagadeesh","year":"2016","journal-title":"Nat. Genet."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/s13073-022-01120-z","article-title":"MetaRNN: Differentiating rare pathogenic and rare benign missense SNVs and InDels using deep learning","volume":"14","author":"Li","year":"2022","journal-title":"Genome Med."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1471-2164-14-S3-S3","article-title":"Identifying Mendelian disease genes with the variant effect scoring tool","volume":"14","author":"Carter","year":"2013","journal-title":"BMC Genom."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"D1143","DOI":"10.1093\/nar\/gkad989","article-title":"CADD v1. 7: Using protein language models, regulatory CNNs and other nucleotide-level scores to improve genome-wide variant predictions","volume":"52","author":"Schubach","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Kramer, O. (2016). Machine Learning for Evolution Strategies, Springer.","DOI":"10.1007\/978-3-319-33383-0"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1186\/s13073-020-00803-9","article-title":"dbNSFP v4: A comprehensive database of transcript-specific functional predictions and annotations for human nonsynonymous and splice-site SNVs","volume":"12","author":"Liu","year":"2020","journal-title":"Genome Med."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"W83","DOI":"10.1093\/nar\/gkw199","article-title":"g: Profiler\u2014A web server for functional interpretation of gene lists (2016 update)","volume":"44","author":"Reimand","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Kanehisa, M. (2002, January 15). The KEGG database. Proceedings of the \u2018In Silico\u2019 Simulation of Biological Processes: Novartis Foundation Symposium 247, London, UK.","DOI":"10.1002\/0470857897.ch8"},{"key":"ref_63","first-page":"D498","article-title":"The reactome pathway knowledgebase","volume":"48","author":"Jassal","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"D1207","DOI":"10.1093\/nar\/gkaa1043","article-title":"The human phenotype ontology in 2021","volume":"49","author":"Gargano","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"D613","DOI":"10.1093\/nar\/gkaa1024","article-title":"WikiPathways: Connecting communities","volume":"49","author":"Martens","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"2498","DOI":"10.1101\/gr.1239303","article-title":"Cytoscape: A software environment for integrated models of biomolecular interaction networks","volume":"13","author":"Shannon","year":"2003","journal-title":"Genome Res."},{"key":"ref_67","first-page":"882","article-title":"sva: Surrogate variable analysis","volume":"3","author":"Leek","year":"2019","journal-title":"R Package Version"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: A Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"6087","DOI":"10.1038\/ncomms7087","article-title":"Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma","volume":"6","author":"Sia","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1089\/omi.2011.0118","article-title":"clusterProfiler: An R package for comparing biological themes among gene clusters","volume":"16","author":"Yu","year":"2012","journal-title":"OMICS"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1002\/wics.147","article-title":"ggplot2","volume":"3","author":"Wickham","year":"2011","journal-title":"Wiley Interdiscip. Rev. Comput. Stat."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.omto.2022.09.008","article-title":"ScRNA-seq and bulk RNA-seq reveal the characteristics of ferroptosis and establish a risk signature in cholangiocarcinoma","volume":"27","author":"Yao","year":"2022","journal-title":"Mol. Ther. Oncolytics"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13059-017-1382-0","article-title":"SCANPY: Large-scale single-cell gene expression data analysis","volume":"19","author":"Wolf","year":"2018","journal-title":"Genome Biol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/0098-3004(93)90090-R","article-title":"Principal components analysis (PCA)","volume":"19","author":"Mackiewicz","year":"1993","journal-title":"Comput. Geosci."},{"key":"ref_76","doi-asserted-by":"crossref","unstructured":"McInnes, L., Healy, J., and Melville, J. (2018). Umap: Uniform manifold approximation and projection for dimension reduction. arXiv.","DOI":"10.21105\/joss.00861"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-019-41695-z","article-title":"From Louvain to Leiden: Guaranteeing well-connected communities","volume":"9","author":"Traag","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"2091","DOI":"10.1002\/hep.30493","article-title":"Integrative analysis defines distinct prognostic subgroups of intrahepatic cholangiocarcinoma","volume":"69","author":"Goeppert","year":"2019","journal-title":"Hepatology"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s12859-018-2096-3","article-title":"A framework for analyzing DNA methylation data from Illumina Infinium HumanMethylation450 BeadChip","volume":"19","author":"Wang","year":"2018","journal-title":"BMC Bioinform."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1093\/bioinformatics\/btw691","article-title":"Preprocessing, normalization and integration of the Illumina HumanMethylationEPIC array with minfi","volume":"33","author":"Fortin","year":"2017","journal-title":"Bioinformatics"},{"key":"ref_81","unstructured":"Hansen, K.D., Aryee, M., and Timp, W. (2025, June 06). minfiData: Example Data for the Illumina Methylation 450 k Array. R Package Version 0.54.0. Available online: https:\/\/bioconductor.org\/packages\/minfiData."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"Ritchie","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"e109","DOI":"10.1093\/nar\/gkab637","article-title":"Calling differentially methylated regions from whole genome bisulphite sequencing with DMRcate","volume":"49","author":"Peters","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"3257","DOI":"10.1093\/bioinformatics\/btz096","article-title":"mCSEA: Detecting subtle differentially methylated regions","volume":"35","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_85","doi-asserted-by":"crossref","unstructured":"Hahne, F., and Ivanek, R. (2016). Visualizing genomic data using Gviz and bioconductor. Statistical Genomics: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-4939-3578-9_16"},{"key":"ref_86","unstructured":"Wickham, H., and Wickham, M.H. (2025, July 30). Package \u2018Stringr\u2019. Available online: https:\/\/github.com\/tidyverse\/stringr."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1093\/protein\/8.2.127","article-title":"LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions","volume":"8","author":"Wallace","year":"1995","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"1950","DOI":"10.1002\/prot.22711","article-title":"Improved side-chain torsion potentials for the Amber ff99SB protein force field","volume":"78","author":"Piana","year":"2010","journal-title":"Proteins"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"020001","DOI":"10.4279\/pip.020001","article-title":"Structural and dynamic properties of SPC\/E water","volume":"2","author":"Campo","year":"2010","journal-title":"Pap. Phys."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"4711","DOI":"10.1021\/jacs.8b13613","article-title":"Biomolecular Solvation Structure Revealed by Molecular Dynamics Simulations","volume":"141","author":"Wall","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"ref_93","doi-asserted-by":"crossref","unstructured":"Aier, I., Varadwaj, P.K., and Raj, U. (2016). Structural insights into conformational stability of both wild-type and mutant EZH2 receptor. Sci. Rep., 6.","DOI":"10.1038\/srep34984"},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H","article-title":"LINCS: A linear constraint solver for molecular simulations","volume":"18","author":"Hess","year":"1997","journal-title":"J. Comput. Chem."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0021889802020502","article-title":"How root-mean-square distance (rmsd) values depend on the resolution of protein structures that are compared","volume":"36","author":"Carugo","year":"2003","journal-title":"J. Appl. Crystallogr."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"2319","DOI":"10.1002\/jcc.21787","article-title":"MDAnalysis: A toolkit for the analysis of molecular dynamics simulations","volume":"32","author":"Denning","year":"2011","journal-title":"J. Comput. Chem."},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"e1298","DOI":"10.1002\/wcms.1298","article-title":"Using PyMOL as a platform for computational drug design","volume":"7","author":"Yuan","year":"2017","journal-title":"Wiley Interdiscip. Rev. Comput. Mol. Sci."},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.csbj.2018.01.002","article-title":"FoldX as protein engineering tool: Better than random based approaches?","volume":"16","author":"Rudat","year":"2018","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1063\/1.457480","article-title":"Error estimates on averages of correlated data","volume":"91","author":"Flyvbjerg","year":"1989","journal-title":"J. Chem. Phys."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/23\/11244\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T09:03:36Z","timestamp":1763715816000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/23\/11244"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,21]]},"references-count":99,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ijms262311244"],"URL":"https:\/\/doi.org\/10.3390\/ijms262311244","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,21]]}}}