| ROS (Reactive oxygen species) are produced by normal cellular metabolism, ionizing radiation or environmental exposure to transition metals, various chemical oxidants and free radicals. Excessive ROS levels can cause structural and functional damage to proteins, lipids and DNA, causing cell dysfunction through disturbing the balance of anti-oxidative and oxidative level. DNA bases are particularly susceptible to oxidation mediated by ROS. The most abundant oxidation product is8-OHdG (8-hydroxy-2’-deoxyguanosine), which plays a key role in carcinogenesis and aging. The presence of8-oxoG is often used as a cellular biomarker to indicate the extent of oxidative stress.8-OxoG can mispair with adenine during DNA replication, leading to G:C to T:A transversions. hMTH1(human mutT homolog1), hOGG1(human8-oxoguanine glycosylase1) and MUTYH (human mutY homolog) genes constitute the8-oxoG repair pathway.Type2diabetes mellitus (T2DM) is a multifactorial metabolic disease characterized by insulin resistance and/or abnormal insulin secretion. Reactive oxygen species (ROS) are involved in the development of insulin resistance,β-cell dysfunction, impaired glucose tolerance and, ultimately, T2DM and its complications. Single nucleotide polymorphisms (SNPs) in DNA repair genes may alter the function of their encoded proteins and reduce DNA repair capacity, which may lead to genetic instability and increased susceptibility to age-related diseases. The researches on the association of DNA repair genes variants and the risk of T2DM could make great contribution to explore the genetic pathogen and find the new way in the prevention of T2DM.In this study, we screened for the polymorphism variants in hMTH1, hOGG1and MUTYH on the risk of T2DM in the Chinese population in an age-/gender-matched case-control study. Meanwhile, individual DNA samples of T2DM cases suspected to be mosaic when screening the hOGG1variants. We made further confirmation of the mosaicism and investigated its influence on the association studies.Part One:Detection of mosaicism for the polymorphic variants in the5’-UTR of hOGG1and the effects of mosaicism on the association-based investigations Background and ObjectiveMosaicism refers to the presence of genetically distinct cell lines within an organism or a tissue. Somatic mosaicism exists in distinct populations of somatic cells and commonly arises as a result of somatic mutations, mainly in early embryonic development. We explored the nature of the phenomenon that sequencing of the mutant alleles in individuals showed weak peaks and its effect on association-based studies, when we investigated the association of polymorphism variants c.-53G>C (rs56387615), c.-23A>G (rs1801129) and c.-18G>T (rs1801126) in the5’-UTR of hOGG1and the risk of T2DM in the Chinese population.Methods1. We screened for the genotypes of functional variants in the5’-UTR of the hOGG1gene in a case-control study by High Resolution Melting (HRM) technique.2. The heterozygotes were submitted to direct sequencing to determine the genotypes of these polymorphism variants.3. DNA samples that demonstrated low mutant peaks in sequencing figures were subsequently cloned and sequenced.4. Pyrosequencing technique and an artificial mosaicism model were established to confirm the existence of somatic mosaicism. Results1. Upon detection of the polymorphisms c.-53G>C, c.-23A>G, and c.-18G>T in the hOGG1gene, we found that mosaicism was present in3/28(10.71%),7/51(13.73%), and1/44(2.27%) patients respectively, who were carriers of these single nucleotide variations, by cloning and sequence analysis.2. Pyrosequencing technique and an artificial mosaicism model confirmed the above results.3. When mosaicism is included in the statistical analysis, our results demonstrate that the heterozygote frequency of c.-23A>G is significantly higher in T2DM patients than in healthy subjects (OR:2.173;95%CI:1.329-3.554). There were no significant differences in the c.-53G>C or c.-18G>T polymorphism between the patients and controls. A significant difference in A/G genotype frequency of c.-23A>G was observed between the male T2DM patients and controls (OR:2.828;95%CI:1.404-5.697). By contrast, no significant difference was identified between the female patients and controls. Additionally, A/G heterozygosity was present at higher frequencies in the T2DM patients <60years or≥60years old than in the parallel controls (OR:1.974,95%CI:1.071-3.640; OR:2.573,95%CI:1.121-5.908, respectively).4. When mosaicism is excluded from the analysis, the frequency of the SNP detection in the5’-UTR of hOGGl in T2DM patients differs, particularly in c.-23A>G. No significant difference existed between the T2DM patients and controls when the analysis was stratified by age, in neither the <60years nor≥60years groups.ConclusionWe screened for the variants of the5’-UTR of the hOGG1gene in T2DM and healthy controls. Somatic mosaicism was identified by cloning and sequence analysis and pyrosequencing among the polymorphisms in the5’-UTR of hOGG1, which can affect the detection of the polymorphism. It will be important to consider the existence of mosaicism when performing SNP association-studies for disease risk. Part Two:Combined analysis of polymorphism variants in hMTHl, hOGGl and MUTYH genes on the risk of type2diabetes in the Chinese population Background and ObjectiveROS are considered to play a role in the development of T2DM and its complications. hMTH1, hOGG1and MUTYH constitute the8-oxoG repair pathway. In this study, we screened for the polymorphism variants Val83Met (c.247G>A, rs4866) in hMTH1; the total variants in c.-53G>C, c.-23A>G and c.-18G>T in the5’-UTR of hOGG1; and AluYb8insertion in MUTYH (AluYb8MUTYH, rs10527342) and investigated their synergistic effect on the risk of T2DM in the Chinese population.Methods1. The genotypes of hMTH1c.247G>A and hOGG15’-UTR variant were determined by HRM technique.2. The heterozygotes of hOGGl5’-UTR were submitted to direct sequencing to determine the genotypes of these polymorphism variants.3. The AluYb8MUTYH variant genotypes were determined by electrophoresis.Results1. Our results showed that the c.247G>A variant in the hMTHl gene increased the risk of T2DM in>55years of age groups (OR=1.579;95%CI:1.029-2.421).2. The set of c.-53G>C, c.-23A>G and c.-18G>T variants detected in the5’UTR of the hOGG1gene were associated with an increased risk of T2DM (OR=1.507,95%CI:1.122-2.024).3. AluYb8insertion in the MUTYH gene was associated with an increased risk of T2DM (OR=1.229,95%CI:1.030-1.466).4. Combined analysis of the variations among the three genes suggested that c.247G>A variant in hMTH1combined with AluYb8MUTYH variant had a synergistic effect on increasing the risk of T2DM (OR=1.635;95%CI:1.147-2.330). This synergy was also observed between the variants in the5’-UTR of the hOGG1and the AluYb8MUTYH variant (OR=1.804;95%CI:1.254-2.595).ConclusionPolymorphism variants in hMTH1, hOGG1and MUTYH genes were associated with the risk of type2diabetes in the Chinese population. Our results suggest, for the first time, the combined effects of AluYb8MUTYH with either hMTHl c.247G>A or variants in the5’-UTR of the hOGGl on the risk of T2DM. |
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