Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited dominant autosomal disorder caused by defects in the mismatch repair (MMR) mechanism that increases the risk of cancer in the colon and endometrium. The MSH2 gene is part of MMR, and when defective, can terminate its function, leading to an increased mutation rate and a higher development for HNPCC. Not all mutations lead to a defective MSH2, but to determine which single nucleotide polymorphism (SNP) is critical, an investigation was lead on how mutations in the gene MSH2 affects the mutation rate in yeast. Using three E.coli strains to produce more plasmids, PCR and restriction enzyme digestion was enacted to verify the identities of the empty vector, wild type and mutated MSH2 plasmids. Yeast cells were then transformed with the mutated plasmid carrying the mutation G693D, which was analyzed using ApE and Clustal Omega. After completing an FOA assay, the MSH2 alleles grew, signifying a high mutation rate and a likely mutation that can lead to the arise of HNPCC because G693D causes a defective MMR system. Classifying which mutations are harmful and which are benign can aid in detecting patients that have a higher risk for HNPCC.

Hereditary Nonpolypopsis Colorectal Cancer (HNPCC) is an inherited autosomal dominant genetic condition with an increased risk for colon or endometrial cancer. HNPCC is caused by a mutation in one of the genes associated with Mismatch Repair mechanism (MMR), such as MSH2. MMR repairs the single point mutations that are left uncorrected after DNA replication. This project aims to determine if a missense mutation in MSH2 affects the MMR function. The project begun with the molecular characterization of the mutation in MSH2. After plasmid purification, PCR, restriction digestion, and bioinformatic analysis, the mutation in MSH2 was determined to be H669Y. The mutated MSH2 was used to transform msh2 mutant yeast. After transformation, the mutation rate was measured with FOA assay. Our data indicated that H669Y mutation in MSH2 affected the MMR function. Data generated from this research project will allow clinics to look out for those specific alleles or mutations that put patients at risk of colorectal cancer.

Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or Lynch Syndrome is a genetic condition that is known to increase the risk of colon cancer. HNPCC accounts for about 5% of all colon cancer cases. HNPCC is caused by mutations in DNA mismatch-repair (MMR) genes such as MSH2. The MSH2 protein forms a heterodimer with other subunits such as MSH3 and MSH6 to perform MMR. This research aimed to test the effects of a single nucleotide polymorphism (SNP) in the MSH2 gene on MMR. Plasmid DNA from E. coli was extracted from cultures containing an empty vector (sp0), a wild type MSH2 gene (sp1), and a mutant msh2 gene (sp2). The identity of the plasmids were confirmed by restriction enzyme digestions, polymerase chain reaction (PCR), and gel electrophoresis. After aligning the wild type and mutant MSH2 protein sequences, the mutation was characterized as H658Y. This mutation is in a region that could potentially affect protein-protein interactions with other subunits that are necessary for MMR. Yeast were then transformed with the plasmid vectors and monitored for their growth and mutation rates. Methods from this project can be applied to other genetic disorders in an attempt to determine potential causes of other genetic diseases.

Hereditary NonPolyposis Colorectal Cancer (HNPCC) is an inherited condition, resulting from a mutation in the MSH2 gene, which encodes a protein essential for the DNA mismatch repair mechanism (MMR). Defects in MMR cause higher mutation rates leading to uncontrolled cell growth and tumor formation. However, it is unclear which mutations such as Single Nucleotide Polymorphisms (SNP) in MSH2 leads to dysfunctional MSH2. Some mutations could maintain a normal function of MSH2, while others can impair its function. In this study, we compared the mutation rates of yeast, our model organism, with a wild-type MSH2 gene, a mutated msh2 gene (G693D), or transformed with an empty vector, using a yeast FluoroOrotic Acid assay. If the mutation impairs the function of MSH2, the mutation rate of yeast should increase and can be pathogenic. If the mutation does not impair the function of MSH2, the mutation rate of yeast should remain as low as the wild-type yeast, and can be considered pseudo-wild-type. We predict that the mutation will impair the function of MSH2, increasing the mutation rate compared to the wild-type. This research may help determine whether an individual carrying a particular SNP has a higher probability of developing HNPCC.