Study Of The Genetic Association Between The Polymorphism Of Metallothionein Gene And Type 2 Diabetes Mellitus

Recent years, the incidence of diabetes mellitus (DM) has been growing rapidly in the world with the development of living standard, alteration of life style and aging. DM has become a major chronic disease with the current number of diabetic patients reaching 130 millions, in which type 2 diabetes mellitus (T2DM) accounted for about 90%. In China the number has surpassed 40 millions, and the cost of treatments for DM has become a heavy economic burden to our country, which is effecting the development of economy seriously.The etiological factor and pathogenesis of T2DM remain unclear for complication. Family, twin and race studies have precisely indicated a genetic component might be involved in T2DM. Most T2DM are not simple Mendelian disease but complex disease with polygenic or multifactorial genetic disease. Besides genetic predisposition, environmental agents affect T2DM too. Obesity, high calorie diet and underactivity are the major environmental agents. And T2DM has the genetic and phenotype heterogeneity.It is helpful to detect patients early and adopt intervention study by elucidating the genetic factor of T2DM. The above mentioned is the reason that the world concerns molecular genetics of T2DM.Mass genome scanning study has suggested a number of chromosomal regions possibly containing T2DM susceptibility gene with homo-genome information and bioinformatics. These chromosomal regions include 1q,2p,8p,9q,12q,20q and so on, but there is less study on chromosome 16. In addition, linkage study has found that ras-related associated with diabetes (RRAD) loci was located on chromosome 16q22, while metallothionein (MT) gene family members were on 16q13 within 10cM distance from RRAD loci. Therefore it is possible to find susceptibility genes for T2DM on this region by screening SNP.To investigate a genetic association between the polymorphism of MT gene and T2DM, the present study tried to reveal the predisposing genes and molecule genetic mechanism for T2DM with case-control association study. The present study also investigated the relationship of the SNPs on MT gene and the indexes of blood plasma and T2DM involving interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), superoxide dismutase (SOD) and hexokinase (HK).The present study selected the T2DM patients and unrelated healthy control individuals as objects from Han Chinese in North China, and all the objects were above 40 years old. We collected 851 samples, including 397 T2DM patients and 457 control individuals. The PCR-RFLP methods were adopted to detect 7 SNPs on MT gene, including rs8052394 and rs11076161 on MT1A gene, rs8052334, rs964372 and rs7191779 on MT1B gene, rs708274 on MT1E gene, rs10636 on MT2A gene. The goodness-of-fitnessχ2 test was used to detect whether the genotype frequency distribution fit the law of Hardy-Weinberg genetic equilibrium. The differences of allelic gene and genotype distribution between T2DM and control group were detected byχ2 test. The conditional test was used to test the combined effect of distinct loci on the disease by conditioning on allele (COA) or by conditioning on genotype (COG). Theχ2 test was used to test the association between the SNPs and clinical situations of T2DM. The ANOVA test was used to test the association among the indexes, T2DM and SNPs. All the tests were analyzed by SPSS.The following are the major results obtained in this study.1. The H-W equilibriumTheχ2 goodness-of-fit test showed that the genotypic distributions of all 7 SNPs were not deviated from the H-W equilibrium, and thus this sample pool was suitable for the genetic analysis (P>0.05).2. Comparison of SNPs genotype between case and controlThere were no significant differences between the T2DM group and control group (P>0.05) of the frequency distributions of the genotype for MT1A gene rs8052394, but when we put GG and GA together (for GG was only 2.3% in all objects), there was significant differences between the T2DM group and control group (P<0.05). The other six SNPs failed to show remarkable differences between the T2DM group and control group (P>0.05).3. Comparison of SNPs allele between case and controlThere was significant differences between the T2DM group and control group (P<0.05) of the frequency distributions of the allelic gene for MT1A gene rs8052394. The point mutation of G allele increased the morbid risk of T2DM. The other six SNPs failed to show remarkable differences between the T2DM group and control group (P>0.05). 4. Analysis of the combined effect of distinct locusThe conditional test was used to test the combined effect of distinct locus on the disease by COA or by COG. The test showed a disease association for rs8052394,rs11076161,rs8052334,rs964372,rs7191779,rs708274 and rs10636 combined with other different SNPs.5. Association among obesity, SNPs and T2DMThere were more obesity objects in the T2DM group than control group (P<0.01). The allele gene for rs8052334 were found significant differences with obesity (P<0.05), the point mutation of C allele increased the risk of obesity. There were no differences in other SNPs (P>0.05).6. Association between the genotype, allele of SNPs and clinical situationsIn 256 patients, we investigated the relationships between the allele and clinical situations or genotype of SNPs and clinical situations. We found rs708274 was associated with polyuria (P<0.05), rs8052394 and rs708274 were associated with polyphagia (P<0.05), rs964372 was associated with polydipsia and emaciation (P<0.05), rs11076161 was associated with polydipsia, polyphagia, polyuria and emaciation (PPHE) (P<0.05), rs8052334 and rs10636 were associated with hyperlipemia (P<0.05), rs964372 was associated with the increase of TC (P<0.05), rs11076161 and rs10636 were associated with diabetic neuropathy (P<0.05). There were no remarkable differences between other SNPs and other clinical situations (P>0.05).7. Association among blood plasma indexes, SNPs and T2DMWe detected IL-6, TNF-α, SOD and HK in blood plasma in 43 patients and 41 control objects. There were significant differences of IL-6 (P<0.05), TNF-α(P<0.05), and SOD (P<0.01) between the T2DM group and control group (P<0.05). But we did not find significant differences of HK between two groups. On rs8052394 locus, the level of SOD was lower of GG (P<0.01), GA (P<0.05) carrier when compared with AA carrier in patient group and three genotypes in control group (P<0.01). There were significant interactions between disease status and genotypes for SOD. No significant interactions were found between disease status and genotypes for all other parameters considered.In a word, these findings thoroughly suggest that MT gene associate with T2DM, MT1A rs8052394 may be one of the predisposition gene locus, and its genotypes may associate with disease status and the level of SOD. The allele gene of rs8052334 may associate with obesity. There were some SNPs associated with different clinical situations of DM.This study is one of the important contents of disease genomics in post-genomic era. These findings are very important for elucidating the genetic mechanisms of T2DM at the molecular level, and also for the development of prediction of T2DM risk, genetic diagnosis and new drugs for the treatment of the illness.