Study On The Association Between DNA Repair Gene Variants And Risk Of Cancer, And The Molecular Mechanism Of These Variations On The Gene Function

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. DNA repair gene damage is an important cause of cancer. Mismatch repair (MMR) and base excision repair (BER) are two important components of DNA repair system.This paper focused on the variants of hMSH2gene in MMR system and hMUTYH gene in BER system and risk of cancer and molecular mechanism.Part One:The foundation of hMSH2/hMSH6protein interaction system, and the study of the functional evaluation of hMSH2missense mutationsGerm-line mutations in the DNA mismatch repair (MMR) gene HMSH2is a frequent cause of hereditary nonpolyposis colorectal cancer(HNPCC) and about one-third of these mutations are missense. Several missense mutations in hMSH2have frequently been detected in East Asian patients with suspected HNPCC, but their pathogenic role has not been extensively assessed. The aim of this study was to perform functional analysis of these variants and examine their association with colorectal cancer in East Asian patients.Nine missense mutations, MSH2c.505A>G, c.518T>G, c.1168C>T, c.1223A>G, C.1255C>A, c.1261C>A, c.1886A>G, c.2108C>A, c.2516A>G, and one frameshift mutation hMSH2c.l664delA were evaluated in this study.We first used gene engineering to construct recombinant plasmids pGADT7-hMSH2, pGBKT7-hMSH6from template plasmids pFastBacl-hMSH2, pFastBacl-hMSH6. Then we used site-directed mutagenesis to construct10mutated pGADT7-hMSH2plasmids. And we have constructed recombinant pGBKT7plasmids with different hMSH6domains:pGBKT7-hMSH6-N-terminal domain, MutS Ⅰ-Ⅳ, MutS Ⅰ-Ⅴ, MutS Ⅱ-Ⅳ, MutS Ⅱ-Ⅴ, MutSIII-IV, MutSⅢ-Ⅴ.We used yeast two-hybrid assay to analyze hMSH2/hMSH6protein interaction in yeast Y187and AH109. We found that the AH109two-hybrid transformants pGADT7-hMSH2/pGBKT7-hMSH6-MutS Ⅱ-Ⅴ, pGADT7-hMSH2/pGBKT7-hMSH6-MutSⅢ-Ⅴ could grow on the histidine-deficient medium. This showed that hMSH6-MutS-Ⅴ, MutSⅢ-Ⅴ could interact with hMSH2in yeast AH109. Thus, we used yeast two-hybrid transformants pGADT7-hMSH2/pGBKT7-hMSH6-MutS Ⅱ-Ⅴ to construct hMSH2/hMSH6protein interaction system, and evaluate nine hMSH2variants. We found that the yeast two-hybrid transformants hMSH2c.505A>G, c.1168C>T,C.1255C>A,c.1261C>Agrew transformantnormal, s hMSH2c.1223A>G, c.1886A>G, c.2108C<A,c.2516A>G grew slow, transformants hMSH2c.518T>G and c.1664delA couldn’t grow in the histidine-deficient medium in yeast AH109compared wild-type hMSH2.For further study, we used gene engineering to construct recombinant plasmids pCMV-Myc-hMSH2and pCMV-HA-hMSH6. We used plasmids pCMV-Myc-hMSH2and pCMV-HA-hMSH6to co-transfect293T cell. Western blot result showed that293T cell with plasmid pCMV-Myc-hMSH2transfection successfully expressed hMSH2protein in cell. Unfortunately the detection of hMSH6protein was prohibited by the false protein band of Western blot.We further integrated the literature on the functional analysis of the9mutations, including conservative site analysis, analysis of amino acid change, protein structure modeling, and case-control analysis. The functional evaluation of nine mutations but hMSH2c.1255C>A was consistent with our yeast two-hybrid assay.Inclusion, we have preliminarily established the hMSH2/hMSH6protein interaction system using yeast two-hybrid assay, and functional evaluated9missense mutations of hMSH2gene. hMSH2c.1223A>G, c.1886A>G, C.2108C>A,c.2516A>G have partly impacted the hMSH2/hMSH6protein interaction, and may partly affected the hMSH2function. hMSH2c.518T>G have prohibited the hMSH2/hMSH6protein interaction, and was a pathological mutation. hMSH2c.505A>G, C.1168C>T, c.1255C>A, c.1261C>A haven’t affected hMSH2/hMSH6protein interaction, and were innocuous variants.Part Two:The risk of AluYb8MUTYH and the breast and gastric cancers in the Chinese populationOxidative stress is defined as the excessive production of reactive oxygen species in the presence of diminished antioxidant substances. The most frequently reported oxidative DNA damage is8-hydroxy-2’deoxyguanosine (8-OHdG), and an increased8-OHdG level has been demonstrated in cancer patients. Oxidized DNA bases are substrates for several overlapping repair pathways, mainly the base excision repair (BER) system. Several DNA glycosylases are associated with the BER system. Human MutY glycosylase homolog (MUTYH) is specifically involved in the removal of adenines mismatched with8-OHdG resulting from DNA replication errors and DNA recombination. Cooperating with8-oxoG glycosylase (hOGGl) and human MutT homolog, the MUTYH protein can protect the cell from the mutagenic effects of8-OHdG. The germline mutations of MUTYH, which can lead to autosomal recessive colorectal adenomatous polyposis and cancers, were discovered recently.A common polymorphism of the AluYb8insertion in the MUTYH gene (AluYb8MUTYH), which led to the increase of oxidative DNA damage and acceleration of chronic diseases, was previously detected. Considering the relationship between carcinogenesis and oxidative stress, an investigation was held on whether the common variant of the MUTYH gene increases the risk for gastric and breast cancers.The AluYb8MUTYH allele frequencies of545breast cancer patients and762gastric cancer patients were analyzed and compared with that of the healthy control group using the Chi-square test. Genomic DNA specimens from the investigated population were tested by polymerase chain reaction in agarose gel electrophoresis. According to the insertion absence or presence of the variant segment, the patterns for the AluYb8MUTYH genotypes were classified as a homozygous of absence/absence (A/A) and presence/presence (PIP) or a heterozygous of absence/presence (A/P).The variant allele frequency (insertion present, P) was inclined to be enhanced in breast cancer patients as compared with the normal female controls (46.8%versus43.3%), and also, in gastric cancer patients, as compared with the general normal controls (45.1%versus43.9%). However, a significantly different P allele frequency was only detected between the early-onset breast cancer patients (<55years old) and their counterpart female controls (46.6%versus40.9%, p=0.042; OR=1.26,95%CI,1.01-1.56), as well as between the early-onset gastric cancer patients and their respective controls (49.2%versus41.3%, p=0.042; OR,1.37;95%CI,1.02-1.85). Comparisons on the genotypes of AluYb8MUTYH show that this variation of MUTYH has also a significantly higher prevalence in the early-onset cancer patients, either in breast or gastric cancer patients, than that in their counterpart controls.The AluYb8MUTYH allele frequency can be associated with the early-onset breast and gastric cancer in the Chinese population. Probably, there is importance in screening the carriers with the susceptibility alleles to evaluate their risk of breast and gastric cancer for further research.