Study On The Correlation Between Loci Polymorphisms On Chromosome 1p13 And Peripheral Arterial Disease In Diabetes And Their Mechanism

Diabetes with lower-extremity arterial disease(LEAD) is also called diabetes with peripheral arterial disease(PAD). At present, it is generally believed that the main pathogenesis of PAD in diabetes is associated with atherosclerosis.Atherosclerosis is the common pathological basis of lower extremity vascular disease and coronary artery disease(CAD). Similarly to CAD, dyslipidemia, smoking, hypertension, and diabetes are also risk factors for PAD. In addition, genetic factors play an important role in the occurrence and development of PAD. Genome-wide Association Study(GWAS) have discovered multiple loci associated with the risk of disease. Some of them will enhance the risk of disease, while some of them will reduce the risk of disease. GWAS have been used to predict(CAD) and PAD. While rs1333049 on chromosome 9p21 was not only associated with CAD, but also associated with PAD at the same time. It shows that there may be a common genetic basis between coronary artery disease and peripheral arterial disease.Several studies have found that there is a significant correlation between rs599839 single nucleotide polymorphism(SNP) and coronary heart disease(CHD), and genotyping on 1p13 region confirmed rs599839 and rs646776 SNP and acute myocardial infarction(MI) risk also have obvious relevance. Meanwhile, GWAS found that rs646776, rs599839 and rs12740374 on human chromosome 1p13 were strongly correlated with serum total cholesterol(TC) and or low density lipoprotein cholesterol(LDL-C). Rs599839 was related to the risk of myocardial infarction and LDL-C, and the mechanism was associated with the rs12740374 SNP. Sortilin 1 is the intracellular protein located in the golgi apparatus, which mediates the transport of particles containing apolipoprotein B. Rs12740374 T locus increased SORT1 m RNA and sortilin 1 protein expression, reduced the VLDL and LDL levels in the plasma, and then may decrease the risk of coronary disease. While the effect of rs12740374 G locus is on the opposite.Blood glucose and hemoglobin A1C(Hb A1C) level are important index reflecting the status of glucose metabolism. Impaired regulation of glucose can increase the risk of type 2 diabetes and vascular disease. GWAS have identified 32 locus associated with blood glucose, several of which was also associated with the risk of type 2 diabetes. DK Sanghera etc. found that rs599839 on chromosome 1p13 was associated with fasting glucose and type 2 diabetes in sikhs, but it has not yet been reported in China.In summary, genetic loci on 1p13 have a certain correlation with risk of coronary artery disease, plasma glucose and dyslipidemia, and these sites are also related to CAD and MI. Therefore, this paper will discuss the correlation between rs599839, rs646776 and rs12740374 single nucleotide polymorphism and diabetic lower extremity atherosclerotic vascular disease, and to explore its possible mechanism, In order to understand the pathophysiology of disease mechanism better and find new therapeutic target for disease. Part one Study on the correlation between rs599839, rs646776 and rs12740374 polymorphism on 1p13 and peripheral arterialdisease in diabetesObjective:To investigate the correlation between rs599839, rs646776 and rs12740374 polymorphism on chromosome 1p13 and peripheral arterial disease.Methods:1 DM group involved 442 cases of patients with type 2 diabetes and DMPAD group involved 440 cases of PAD with diabetes, all of which were Chinese Han population and unrelated. Hyperthyroidism, hypothyroidism, severe liver and kidney dysfunction and genotyping losers were excluded.2 The height, weight, blood pressure, ABI, Hb A1 C and lipids of all subjects were detected. Genomic DNA was extracted from peripheral white blood cells, and 3 candidate SNPs were genotyped by Taqman probe technique.3 Statistical analysis was performed by SPSS 16.0 software, and chi-square test was to assess Hardy-Weinberg equilibrium of rs599839, rs646776 and rs12740374 genotype in both groups. Independent samples t test and rank sum test were used to compare the measurement data between groups. Gene counting methods were used to calculate the gene frequency and allele frequency. Measure data and the frequencies of genotypes and alleles in each group were compared by chi square test. The Correlation between variables and indicators was determined by using spearman related analysis. Univariate and multivariate analysis use Logistic regression analysis.Results:1 Compared with DM group, differences of age, course of disease, smoking, hypertension, Hb A1 C, TC, high density lipoprotein cholesterol(HDL-C), LDL-C, body mass index(BMI), CAD, MI in DMPAD group were considered to be statistically significant(P<0.05), and there was no significant difference in gender and triglycerides(TG) between the two groups;2 Compared with DM group, frequencies of rs599839 A and G, rs646776 A and G, rs12740374 C and T genotypes were statistically significantly different in DMPAD group;frequencies of rs599839 AA and AG+GG, rs646776 AA and AG+GG, rs12740374 CC and CT+TT genotypes were statistically significantly different(P<0.05) in dominant model in DMPAD group,but frequencies of rs599839 AA+ AG and GG, rs646776 AA +AG and GG, rs12740374 CC+CT and TT genotypes were not statistically significantly different(P>0.05) in recessive model in DMPAD group;3 In the DMPAD and DM group, the result of Hardy Weinberg equilibrium test for rs599839 was that c2 values were 0.069 and 0.439, P values were 0.792 and 0.507, respectively; For rs646776, c2 values were 0.881 and 1.393, P values were 0.347 and 0.237, respectively; For rs12740374, c2 values were 0.376 and 0.340, P values were 0.539 and 0.559, respectively. The genotype frequencies of these loci were in Hardy-Weinberg genetic equilibrium(P > 0.05);4 Univariate logistic regression analysis showed that age, duration of disease, smoking, hypertension, Hb A1 C, TC, HDL-C, LDL-C, BMI, CAD, MI, rs599839, rs646776 and rs12740374 genotype and PAD with diabetes were correlated(P<0.05). Multivariate logistic regression analysis indicated that age, Hb A1 c, TG, LDL-C, smoking, hypertension, duration of diabetes were the independent risk factors for PAD with diabetes(P<0.05) and HDL-C, rs599839, rs646776, rs12740374 genotype were protective factors for PAD with diabetes(P<0.05). Part two Correlation of rs599839, rs646776 and rs12740374 polymorphis-m on 1p13 and glucose and lipid metabolismObjective:To investigate the correlation of rs599839, rs646776 and rs12740374 on chromosome 1p13 with plasma glucose and lipids.Methods:1 The study population is the same as those in part one. Height, weight, blood pressure, ABI, fasting plasma glucose(FPG), post-prandial glucosese(PPG), Hb A1 C, TC, TG, HDL-C and LDL-C of all subjects were detected.2 Genomic DNA was extracted from peripheral blood mononuclear cells, and 3 candidate SNPs were genotyped by Taqman probe technique.3 Statistical analysis was performed by SPSS 16.0 software. Normal distribution measurement data was expressed with the mean and standard deviation, and the non-normal distribution data was applied to analyze the data after logarithm transition.Count data expressed with the percentage.The Correlation between variables and indicators was determined by covariance analysis and spearman related analysis.Results:1 Adjusting for age, sex, smoking, hypertension, lipid-lowering therapy, in total study population, there were statistically significant distinctions in LDL-C between rs599839 AA and AG+GG, rs646776 AA and AG+GG, rs12740374 GG and GT+TT genotype in dominant model, and they were linearly correlated(P<0.05). The spearman rank correlation coefficient were-0.166,-0.131 and-0.146 respectively; 2 Adjusting for age, sex, smoking,hypertension, lipid-lowering therapy, in total study population, there were not statistically significant distinctions in FPG, PPG, Hb A1 c, TC, TG, HDL-C and BMI between rs599839 AA and AG+GG, rs646776 AA and AG+GG, rs12740374 GG and GT+TT genotype in dominant model(P>0.05);3 Adjusting for age, sex, smoking, hypertension, lipid-lowering therapy, in DMPAD group,there were statistically significant distinctions in LDL-C between rs599839 AA and AG+GG, rs646776 AA and AG+GG, rs12740374 GG and GT+TT genotype in dominant model, and they were linearly correlated(P<0.05). The spearman rank correlation coefficient were-0.172,-0.129 and-0.150 respectively; 4 Adjusting for age, sex, smoking, hypertension, lipid-lowering therapy, in DMPAD group, there were not statistically significant distinctions in FPG, PPG, Hb A1 c, TC, TG, HDL-C and BMI between rs599839 AA and AG+GG, rs646776 AA and AG+GG, rs12740374 GG and GT+TT genotype in dominant model(P>0.05). Part three Study on the mechanism of the correlation between rs599839,rs646776, rs12740374 polymorphisms on 1p13 and peripheral arterial disease in diabetesObjective:To investigate the mechanism about rs599839, rs646776 and rs12740374 polymorphisms on chromosome 1p13 on the occurrence and development of diabetic lower extremity vascular disease.Methods:1 Genotyping data in DM and DMPAD group of part 1 were analyzed by linkage disequilibrium methods and haplotype detection respectively.2 Considering the frequency of rs599839, rs646776 and rs12740374 genotype, our study randomly selected subjects with rs599839 AA, rs646776 AA and rs12740374 GG genotype, each genotype of which included 25 cases. And then we selected subjects with rs599839 AG+GG, rs646776 AG+GG, rs12740374 GT+TT genotype, each genotype of which included 25 cases in DMPAD group of part 1, and the ratio of the two genotype frequencies of each locus was consistent with that of the total DMPAD population. Detect sort1 m RNA expression of the selected samples.3 She SIS software was applicated for rs599839, rs646776 and rs12740374 linkage disequilibrium and haplotype analysis. SPSS 16.0 software was used for statistical analysis. The expression of sort1 m RNA in different genotypes was compared with two independent samples t test, P values less than 0.05 were considered to be significant.Results:1 Linkage disequilibrium coefficients D’ and r2 of rs12740374 and rs599839, rs12740374 and rs646776, rs599839 and rs646776 were 0.933, 0.977, 0.930 and 0.689, 0.914, 0.655 respectively in DM group. Linkage disequilibrium between each 2 of the 3 loci was observed, with the strongest linkage disequilibrium between rs12740374 and rs646776;2 In DMPAD group, Linkage disequilibrium coefficients D’ and r2 of rs12740374 and rs599839, rs12740374 and rs646776, rs599839 and rs646776 were 0.915, 0.959, 0.922 and 0.558, 0.849, 0.613 respectively. Each 2 of the 3 loci were also in Linkage disequilibrium, and linkage disequilibrium between rs12740374 and rs646776 was the strongest as well; 3 Frequencies of haplotype TGG for rs12740374, rs599839, and rs646776 between DM and DMPAD group were statistically significantly different(P<0.01), odds ratio and 95% CI were 0.502(0.297-0.846);4 Compared with GG, AA, AA genotype, rs12740374 GT+ TT,rs646776 AG+GG, and rs599839 AG+GG genotype were statistically significantly related with increased SORT1 m RNA(P<0.01). 2-△△Ct value was 5.81(3.07, 11.00), 3.94(1.64, 944), 2.36(0.73, 7.62) separately.Conclusion:1 Age, Hb A1 C, TG, LDL-C, smoking, hypertension, diabetes duration, CAD, MI were independent risk factors for PAD with diabetes, and HDL-C was protective factor for PAD in diabetes.2 Rs599839, rs646776, rs12740374 genotype were protective factor for PAD in diabetes.3 In dominant model, LDL-C levels between different genotypes of rs599839, rs646776, rs12740374 were statistically significantly different, and they showed a linear correlation in DMPAD group and total study population.4 Haplotype TGG for rs12740374, rs599839, and rs646776 may be a protective factor for PAD in diabetes.5 The difference of SORT1 m RNA expression of different genotypes reveals the probable mechanism between rs599839, rs646776 and rs12740374 gene polymorphism and PAD in diabetes.