CDKN1B variant prioritization reveals Y74N as a highly destabilizing mutation and identifies regulatory SNPs linked to structural and transcriptional disruption

The CDKN1B -encoded tumor suppressor protein p27 Kip1 is a key regulator of the G1-S cell cycle checkpoint, and its dysregulation is implicated in human malignancies. However, a comprehensive in silico characterization of both coding and non-coding variants in this gene remains limited. This study presents an integrated computational investigation of deleterious coding and non-coding single nucleotide polymorphisms (SNPs) in CDKN1B through functional prediction, structural analysis, molecular docking, molecular dynamics simulation, and regulatory characterization. A non-redundant set of 689 non-synonymous SNPs (nsSNPs), retrieved from dbSNP, Ensembl, and gnomAD, underwent a two-tier functional screening using nine algorithms via dbNSFP v4.5a, followed by Meta-SNP, PhD-SNPg, CScape, SuSPect, and PolyPhen-2.0. Protein stability was evaluated using I-Mutant 2.0, MUPRO, I-Stable, DynaMut, Align-GVGD, and INPS, while structural and functional effects were analyzed by MutPred2.0 and HOPE. Three-dimensional models generated by AlphaFold2 and refined with GalaxyRefine were docked against the cyclin E-CDK2 complex (PDB: 7KJS) using HDOCK. Selected variants and FTsite-predicted ligand-binding residues were further subjected to 200ns molecular dynamics simulations using GROMACS. Non-coding analysis of 456 untranslated region (UTR) and 715 intronic SNPs was conducted using RegulomeDB, PolymiRTS, GTEx, the GWAS Catalog, and RNAfold. The two-tier screening identified 16 high-confidence deleterious variants. Molecular dynamics simulations were performed on four representative variants (P69S, F64S, W60C and Y74N), selected based on docking characteristics and localization within the predicted interaction interface. All mutation sites were localized within the helical core region (residues 37–89), critical for CDK2 interaction. Molecular docking demonstrated aberrant binding geometries in all mutants relative to the wild type, while molecular dynamics simulations identified Y74N as the most conformationally disruptive variant (RMSD ~ 2.8–3.1 nm). Non-coding analysis prioritized 74 putative regulatory variants based on RegulomeDB annotations and supporting regulatory evidence. The 3′UTR variant rs34330 was associated with systemic lupus erythematosus and was predicted to alter mRNA secondary structure, whereas the intronic variant rs3093735 showed supporting eQTL evidence in lymphoblastoid tissue, suggesting a potential association with reduced CDKN1B expression. Overall, the findings suggest that deleterious CDKN1B coding and non-coding variants may influence protein structure, molecular interactions, and regulatory mechanisms. Among the coding variants, Y74N exhibited the greatest structural perturbation during molecular dynamics simulations, while several non-coding variants displayed regulatory and disease-associated signatures that warrant further experimental investigation.