Association Of SLC30A8 Gene Polymorphism And Promoter Region Methylation With The Incidence Of T2DM And Conversion In Glycemic Status In Uygur

Objective:（1）To understand the association between SLC30A8 gene polymorphism and T2DM and insulin resistance in uygur,and to explore the risk effect of this gene on the future incidence of T2DM and Pre-DM in NGT population.（2）Analysis the association of serum zinc,copper,calcium,magnesium level with T2DM risk in uygur and investigate the factors affecting the zinc ion level.（3）To explore the correlation between the methylation in the promoter region of SLC30A8 gene and the incidence of type 2diabetes and investigate the factors that affect the level of methylation.Analysis the effect of methylation in the promoter region of SLC30A8 gene on the naturnal conversion of glucose metabolism.Methods:（1）The subjects were from a previous study on genetic differences in the metabolic syndrome of uyghurs,who were admitted to the first affiliated hospital of xinjiang medical university or the first hospital in kashgar from March 2012 to June 2013,.The 474 study subjects were divided into NGT group,Pre-DM group and T2DM group.The serum zinc,copper,calcium and magnesium was determined by flame atomic absorption spectrometry.Compare the level differences among three groups and analyse the factors affecting the level of serum zinc ions.logistic regression analyzed the correlation between the four ionic levels and the risk of T2DM.Logistic regression was also used to analyze the risk factors which affect the naturnal conversion of glucose metabolism.（2）A total of 547 patients were followed up for 54 months（4.5 years）of baseline population from the previous study,who were included in the study of the second part.The subject was divided into three groups:191cases in NGT group,62cases in pre-diabetes and 221cases in T2DM.The differences between the three groups were compared include general demographic characteristics,biochemical index and the SNP of SLC30A8 gene,comparing the FPG and OGTT 2h PG in NGT and Pre-DM,The effects of rs13266634 SNP on the naturnal conversion of glucose metabolism were compared with chi-square test.（3）Pyrosequencing is used to detect SLC30A8 gene promoter region 6 CpGs site methylation rate in target populations（include 90cases in the NGT group,45 in the pre-diabetes group and 80 in the T2DM group）.Comparing the differences of methylation of 6 CpG sites between the three groups by.variance analysis.Furthermore,the correlation between the methylation and risk factors（blood lipid,smoking,exercise,sleep and stress）was further analyzed by bivariate correlation.A total of 36 new T2DM and pre-DM were found during the 4.5years of follow-up.To analyze the risk factors that affect the naturnal conversion of glucose metabolism by logistic regression.Results:（1）Part 1:In 474 cases the proportion of rural origin in the pre-diabetes group and the diabetes group（44.0%and44.7%,respectively）were significantly higher than that in the control group（11.4%）.In terms of cultural level,the composition ratio of primary and lower education groups in pre-diabetes group and diabetes group（22.9%and 17.5%,respectively）was higher than that in the control group（3.0%）.The incidence of obesity increased from NGT group to pre-DM and T2DM group（19.4%,40.3%and 49.3%respectively）,The incidence of abdominal obesity also increased from NGT group to pre-DM and T2DM group（78.3%,85.0%and 91.8%respectively）（P<0.05）;Triglycerides also showed a rising tendency from the NGT group to the pre-DM group and the T2DM group,while the HDL showed the opposite trend.and the difference was statistically significant（P<0.05）.There was no difference in serum zinc ion concentration in the three groups.After adjusting the confounding factors,the concentration of serum Zn²⁺was not correlated with T2DM or IGR.The adjusted OR value of copper ion for T2DM is 1.350（95%CI:1.087-1.677,P<0.05）,and OR for T2DM+Pre-diabetes is 1.23（95%CI:1.01-1.51,P<0.05）.Serum Mg²⁺levels were significantly correlated with pre-diabetes group and diabetes group（P<0.05）.Logistic regression showed that HDL decreased（OR:7.57,95%CI:1.615-35.489）,smoking（OR:8.27,95%CI:1.453-47.101）are the risk factors for glucose metabolism disorder in men.And low density lipoprotein（OR:11.61,95%CI:2.464-54.720）,family history（OR:6.26,95%CI:1.050-37.291）and low magnesium（OR:0.435,95%CI:0.218-0.867）were risk factors for glucose metabolism disorder in women.（2）Part 2:The incidence of hypertension in diabetes group increased from the control group to pre-diabetes group and diabetes group（17.4%,37.8%and 57.4%respectively）（P<0.05）.The incidence of coronary heart disease and lipid metabolism was no difference between the pre-DM and T2DM group,but both were higher than the control group（P<0.05）.The genotype frequency of the SLC30A8 gene rs13266634 was different in three groups and the CC genotype is risk gene.Considering that blood glucose in patients with diabetes affected by drugs,so the combination of NGT group and T2DM to compare FPG and 2h PG group in different genotypes,showing that FPG decreased from CC type,CT and TT（respectively 5.57,5.21 and 5.07mmol/L,P<0.05）,2h PG shows a downward trend,but the difference is not statistically significant（P>0.05）.Homa-beta in 44 newly diagnosed diabetic patients.shows a downward trend from CC to TT（68.50,69.88 and 82.17 respectively,P>0.05）.During follow-up,11cases in NGT group have been newly diagnosed pre-diabetes（the incidence of Pre-DM was4.54%）,15 cases were newly diagnosed T2DM（incidence 6.19%）,26 cases in Pre-DM group are newly diagnosed T2DM（incidence 57.77%）.Comparing the distribution of rs13266634 genotypes between progress groups and no progress groups,it was found that the frequency of CC type in the progressive group was higher than that in the non-progress group.（60.4%and 44.4%,respectively）and the OR value of the development of glucose metabolism in CC genotype was 1.905（95%CI:1.035-3.506,P<0.05）.（3）Part three:The 6 Cp G sites in the promoter region of SLC30A8 were highly methylated（average methylation rate 89.79%）.The methylation rate of CpG3 is lower than that of pre-diabetes group and diabetes group（91.28%,92.20%and 92.04%respectively）in object with reduced HDL,But there is no such trend in normal HDL objects.The ratio of waist-to-hip ratio was correlated with CpG2 methylation rate（r=-0.278,P<0.001）,there is a correlation between HDL and CpG2.Methylation rate of CpG1,CpG3,Cp G4,Cp G5 and CpG6 in non-OSAHS are lower than those of OSAHS,and CpG3 loci difference was statistically significant（P<0.05）.The methylation rate of CpG4,Cp G5 and CpG6 was positively correlated with the number of cigarettes smoked per week（r=0.321、0.266 and 0.401,P<0.05）,And we can see that the methylation rate was different in different psychological pressure groups between CpG1、CpG2、CpG5and CpG6.In general,the methylation rate increased with pressure（P<0.05）,but the highest methylation rate was in the pressure medium group,not the maximum group.According to the conversion of glucose metabolism status during the follow-up period,multivariate Logistic regression was performed and results showed that Cp G2 and Cp G3were risk factors for disease progressionOR value respectively（CpG2:OR=3.42,95%CI:1.15-10.176,CpG3:OR=4.67,95%CI:1.066-20.487）.Conclusion:（1）We did not find the association between Serum Zn²⁺level and T2DM/Pre-DM,which may be related to the overall Serum Zn²⁺level in uygurs of Xinjiang is high.Serum Zn²⁺level was associated with gender and age,and decreased significantly in patients with diabetes mellitus combined with nephropathy.Increased serum copper ions and reduced magnesium ions were associated with increased risk of T2DM,suggesting that the association between ions and diabetes may vary with regions and populations.（2）The rs13266634 of SLC30A8 C allele is the risk gene of T2DM in xinjiang uygur,the risk genotype of the site can improve insulin resistance in non-diabetic patients,and promote the development of glucose metabolism disorders in non diabetes population.（3）It is suggested that impaired glucose regulation is the most important risk factor for the onset of diabetes,while elevated TG and lower HDL are important indicators for predicting the occurrence of diabetes.（4）Methylation in promoter region of SLC30A8 gene are related with the risk factor of T2DM,Lower HDL may be the important factors that affect the level of methylation rate.Environmental factors,such as obesity,smoking,psychological pressure and nocturnal oxygen desaturation caused by OSAHS affect the gene promoter region methylation,Which may increase the risk of T2DM by regulating the methylation of the gene.