A central focus of cancer genetics is the study of mutations that are causally implicated in tumorigenesis. The identification of such causal mutations not only provides insight into cancer biology but also presents anticancer therapeutic targets and diagnostic markers. Mutations in the MSH2 gene whose products participate in DNA mismatch repair underlie the increased risk of cancer in families with hereditary nonpolyposis colon carcinoma (HNPCC). The deleterious effects of missense mutations are commonly attributed to their impact on primary amino acid sequence and protein structure. We sought to predict the deleterious nsSNPs using evolutionary-based and structurebased approach. Of the 29 nsSNPs, 9 nsSNPs (31%) were identified to be deleterious by SIFT, 10 nsSNPs (35%) were considered to be damaging by PolyPhen and these nsSNPs may have an affect on the tertiary structure of proteins and their functionality. The PupaSuite tool predicted the phenotypic effect of SNPs on the structure and function of the affected protein. In addition we showed that has-mir-135b upregulates the expression of allele G3099 and not that of A3099 using miRBase. To further investigate the possible causes of disease at molecular level, we mapped the deleterious nsSNPs to 3D protein structure encoded by MSH2 gene and which is at amino acid position Y43C for nsSNP (with id rs1800093). An analysis of solvent accessibility and secondary structure was also performed to understand the impact of a mutation on protein function and stability. Our computational study also demonstrates the presence of other deleterious mutations in the MSH2 gene that may affect the expression and function of proteins with possible roles in colon cancer. The data presented here show that this novel bioinformatics approach to classifying cancer-associated variants is robust and can be used for large-scale analysis. Copyright © 2009 American Scientific Publishers All rights reserved.