Study On The Relationship Between Atherosclerotic Related Gene Polymorphism And Genetic Susceptibility To Lower Extremity Arterial Disease In Type 2 Diabetes Mellitus

China is the country with the largest number of diabetic patients in the world.At present,the prevalence rate of diabetes among people over 18 years old in China has reached 11.2%.The situation is very serious.As one of the most common chronic complications of diabetes,lower extremity arterial disease has a high incidence and insipid progression,which may not only lead to ischemic ulcer,gangrene and amputation of lower limbs,but also significantly increase the risk of cardiovascular events and ischemic stroke in patients.The all-cause mortality can be increased to 3 times.Therefore,it is very important to prevent and cure the occurrence and development of diabetic lower extremity arterial disease.It is suggested that hyperglycemia,smoking,lipid metabolism disorder,elevated blood pressure,old age and gender are all risk factors for lower extremity arterial disease.At the same time,with the further study,it was found that the risk of lower extremity arterial disease in patients with type 2diabetes varies greatly among individuals,races and regions,suggesting that the lower extremity arterial disease in type 2 diabetes is the result of genetic and environmental factors.In recent years,most genetic studies on type 2diabetes and its complications have focused on single nucleotide polymorphism(SNP).SNP refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level,which involves variation of only a single base.SNP occurs frequently in the human genome and it is one of the most common genetic variations inhuman beings.It can affect gene expression and thus be associated with the occurrence of diseases.We know that type 2 diabetes is not a single-gene disease,but rather a genetic predisposition resulting from the combined effects of multiple genetic abnormalities,as well as its complications.In addition,due to ethnic differences and the characteristics of the disease itself,the repeatability of single SNP in the study of disease susceptibility is low.Studies on interaction of SNP-SNP,SNP-Environmental factors and haploid type can better explain the pathogenesis of diseases.At present,studies on the susceptibility genes of lower extremity arterial disease of type 2 diabetes are still dominated by single SNP,and there are few studies on whether such gene-gene and geneenvironmental factors interaction may be involved in its pathogenesis.Therefore,the purpose of this study was to screen out SNPs that may lead to lower extremity arterial disease in type 2 diabetes mellitus from atherosclerotic related genes,so as to analyze the pathogenic factors of lower extremity arterial disease in type 2 diabetes from the genetic level.Analyze the interaction between susceptibility genes and non-genetic factors,providing new ideas for the prevention and treatment of the disease.Part One Analysis of non-hereditary risk factors for lower extremity arterial disease in type 2 diabetes mellitusObjective: To explore non-hereditary risk factors for lower extremity arterial disease in type 2 diabetes mellitus.Methods:1.A total of 300 patients with type 2 diabetes mellitus were included in this study,including 150 patients complicated with lower extremity arterial disease as the case group(T2DM+LEAD group)and 150 patients without lower extremity arterial disease as the control group(T2DM group).2.Basic clinical data and biochemical indicators of all patients were collected,and statistically analyzed whether there were statistical differrences between T2DM+LEAD group and T2 DM group.Binary Logistic regression analysis was used to correct the influence of confounding factors,so as to screen out risk factors of lower extremity arterial disease in type 2 diabetes mellitus.Results:1.There were no significant differences between T2DM+LEAD group and T2 DM group in family history,BMI,DBP,TC,TG,HDL-C,APOB,hypertension,coronary heart disease,cerebral infarction and carotid artery lesions(P>0.05).2.Age,duration of diabetes,male proportion,smoking and drinking ratio,Hb A1 c,WHR,SBP,LDL-C and APOA1 were significantly different between T2DM+LEAD group and T2 DM group(P<0.05).3.Logistic regression analysis showed that gender,age,WHR,Hb A1 c and APOA1 were correlated with the occurrence of lower extremity arterial disease in type 2 diabetes mellitus,and OR(95%CI)were 2.286(1.182-4.421),1.056(1.027-1.085),1.067(1.031-1.104),1.153(1.014-1.312),0.358(0.137-0.940),respectively.Conclusions: Gender,age,WHR and Hb A1 c were independent risk factors for lower extremity artery disease in type 2 diabetes mellitus,and APOA1 was protective factor for lower extremity artery disease in type 2diabetes mellitus.Part Two Study on the relationship between atherosclerotic related gene polymorphism and genetic susceptibility to lower extremity arterial disease in type 2 diabetes mellitusObjective: To screen arteriosclerotic related genes and explore the correlation between their polymorphism and lower extremity artery disease in type 2 diabetes mellitus.Methods:1.In this study,150 patients with type 2 diabetes mellitus complicated with lower extremity arterial disease were included as the case group(T2DM+LEAD group),and 150 patients with type 2 diabetes mellitus without lower extremity arterial disease were included as the control group(T2DM group).2.A total of 75 SNPs related to type 2 diabetic lower extremity arterial disease,type 2 diabetic atherosclerosis and type 2 diabetic macrovascular disease were searched from CNKI and Pub Med in recent 10 years,and they were located on 56 genes.STRING database and Cytoscape software were used to screen the top 20 key genes through gene interaction,and a total of 26 SNPs were obtained.Genotyping was performed by Mass ARRAY time-flight mass spectrometry,Taqman fluorescence quantitative PCR and firstgeneration sequencing.3.Hardy-weinberg equilibrium test was used to analyze genotype distribution.Chi-square test was used to compare the SNPs genotype and allele frequency distribution between the two groups.Haplotypes were analyzed using Haploview software.The influence of environmental confounding factors was corrected by binary Logistic regression analysis.The odds ratio(OR value)and its 95% confidence interval(95% CI)were used to represent the relative risk.The ultimate goal is to screen out SNPs associated with lower extremity arterial disease in type 2 diabetes mellitus.Results:1.A total of 24 SNPs were included in the analysis.2.There were three SNPs that were different in genotype or allele distribution between the two groups.The distribution of genotype and allele frequentcy of rs7412 on APOE gene were significantly different between the two groups(P=0.015 and 0.013,respectively).The genotype distribution of rs35767 on IGF1 gene was significantly different between the two groups(P=0.022),but there was no difference in allele frequency distribution(P>0.05).There was a significant difference in genotype distribution of rs699947 on VEGFA gene between the two groups(P=0.036),but no significant difference in allele frequency distribution(P>0.05).3.Rs35767 on IGF1 gene: in codominant model,the risk of lower extremity arterial disease in T2 DM patients with GA genotype was 0.434 times that of GG genotype carriers(95%CI: 0.228-0.823,P=0.011);In dominant model,the risk of AA+GA carriers was 0.489 times that of GG carriers(95%CI: 0.288-0.829,P=0.008);In overdominant model,the risk of GA genotype carriers was 0.430 times that of GG+AA genotype carriers(95%CI:0.260-0.730,P=0.002).Other genetic patterns and allele distribution of rs35767 were not associated with disease(P>0.05).Rs699947 on VEGFA gene: the risk of AA and CA genotype carriers were 0.420 and 3.069 times that of CC genotype carriers under codominant model,and OR(95%CI)values were 0.420(0.180-0.979)and 3.069(1.189-7.919),respectively.The risk of AA genotype carriers was 0.159 times that of CC+CA genotype carriers under recessive model(95%CI: 0.030-0.850,P=0.031).Other genetic patterns and allele distribution of rs35767 showed no correlation with disease(P>0.05).Rs1024611 on MCP-1 gene: The risk of AA genotype carriers was1.512 times that of GG genotype carriers under codominant model(P<0.05),OR(95%CI)was 1.512(1.004-2.279).The risk of AA genotype carriers was2.382 times that of GG+GA genotype carriers under codominant model(95%CI: 1.103-5.141).Other genetic patterns and allele distribution of this SNP also showed no correlation with disease(P>0.05).Rs1862513 on RETN gene: the risk of GG+CG genotype carriers was 0.569 times that of CC genotype carriers in the dominant model(95%CI: 0.329-0.984,P=0.044).Other genetic patterns and allele distribution of this SNP showed no correlation with disease(P>0.05).Rs7412 on APOE gene: the risk of TT+TC genotype carriers was 0.384 times that of CC genotype carriers in the dominant model(95%CI: 0.167-0.884,P=0.024).In the overdominant model,the risk of TC genotype carriers was 0.390 times that of CC+TT genotype carriers(95%CI: 0.170-0.890,P=0.022).T allele carriers had 0.384 times risk than C allele carriers(95%CI: 0.173-0.856,P=0.019).There was no correlation between rs7412 and disease in other genetic patterns(P>0.05).4.There were linkage disequilibrium among three groups of SNPs:rs1800872,rs1800871 and rs1800896 on IL10 gene of chromosome1(Chr1),rs699947 and rs2010963 on VEGFA gene of chromosome 6(Chr6),rs1800470 and rs1800469 on TGFB1 gene of chromosome 19(Chr19).However,there was no statistical difference in the frequency of haplotypes distribution between the two groups,and Logistic regression analysis did not show that haplotypes were associated with the risk of lower extremity arterial disease in type 2 diabetes mellitus(P>0.05).Conclusions:1.GA genotype of rs35767 on IGF1 gene,GG+CG genotype of rs1862513 on RETN gene,TC genotype and T allele of rs7412 on APOE gene were protective factors of lower extremity arterial disease in type 2 diabetes mellitus.AA genotype of rs699947 on VEGFA gene was a protective factor of lower extremity arterial disease in type 2 diabetes mellitus,while CA genotype may increase the risk of disease.AA genotype of rs1024611 on MCP-1 gene was a risk factor of disease.2.The haploid types formed by rs1800872,rs1800871 and rs180089 on IL10 gene,rs699947 and rs2010963 on VEGFA gene and rs1800470 and rs1800469 on TGFB1 gene were all not associated with the risk of lower extremity arterial disease in type 2 diabetes mellitus.Part Three Effects of interaction between atherosclerotic related gene polymorphism and non-genetic factors on lower extremity arterial disease in type 2 diabetes mellitusObjective: To investigate whether the interaction of gene-gene and gene-non-genetic factors related to atherosclerosis affects the risk of lower extremity arterial disease in type 2 diabetes mellitus.Methods:1.In this study,150 patients with type 2 diabetes mellitus complicated with lower extremity arterial disease were included as the case group(T2DM+LEAD group),and 150 patients with type 2 diabetes mellitus without lower extremity arterial disease were included as the control group(T2DM group).2.Atherosclerotic related gene polymorphism was retrieved from CNKI and Pub Med.Key genes were screened by STRING database and Cytoscape software.Genotyping was performed by Mass ARRAY time-flight mass spectrometry,Taqman fluorescence quantitative PCR and first-generation sequencing.3.Hardy-weinberg equilibrium test was used to analyze genotype distribution.Generalized multifactor dimensionality reduction software(GMDR)was used to construct gene-gene and gene-non-genetic interaction models.Logistic regression model was used to evaluate the multiplicative interaction,and cross-generation analysis was used to evaluate the additive interaction of two factors.Results:1.GMDR analysis of 24 SNPs gene-gene interactions suggested that the optimal model of three-factor gene-gene interaction composed of rs35767,rs688 and rs1800470 was not associated with the occurrence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was0.5407,cross-validation consistency was 6/10,but P value was 0.1719>0.05).Other SNPs combination models also showed no statistical significance(P>0.05).2.GMDR analysis of SNPs and non-genetic factors suggested that the optimal interaction model between age and SNPs(age,rs7412)had no influence on the occurrence of lower extremity arterial disease in type 2diabetes mellitus(accuracy of test set was 0.5607,cross-validation consistency was 4/10,P=0.3770>0.05).The optimal interaction model between gender and SNPs(gender,rs1024611)may influence the incidence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was 0.6267,cross-validation consistency was 10/10,P=0.0010).The risk of the high-risk groups was 3.1105 times that of the low-risk groups(OR=3.1105,95%CI1.5912-6.0805).The three-order interaction model between gender and SNPs(gender,rs699947,rs3918242)also showed statistical significance(test set accuracy was 0.6210,cross-validation consistency was 6/10,P=0.0010),and the risk of the high-risk groups was 4.7939 times that of the low-risk groups(OR=4.7939,95%CI: 2.3660-9.7131).The optimal interaction model between disease course and SNPs(disease course,rs1799750,rs1800871,rs688,rs1800470,rs7799039)had no correlation with the incidence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was0.5771,cross-validation consistency was only 2/10,P=0.0107<0.05).The optimal interaction model between smoking history and SNPs(smoking history,rs429358)had no effect on the occurrence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was 0.5057,cross-validation consistency was only 3/10,P=0.1719>0.05).The optimal interaction model between BMI and SNPs(BMI,rs35767)was not associated with the incidence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was 0.5279,cross-validation consistency was5/10,P=0.3770>0.05).The optimal interaction model between Hb A1 c and SNPs(Hb A1 c,rs7799039)may affect the incidence of lower extremity arterial disease in type 2 diabetes mellitus(accuracy of test set was 0.6323,cross-validation consistency was 10/10,P=0.0010<0.05).The risk of high-risk group was 3.3220 times that of low-risk group(OR=3.3220,95%CI1.6413-6.7237).Other non-genetic factors,such as drinking history,dyslipidemia,hypertension and other macrovascular diseases,WHR,etc.,had no interaction model with SNPs under GMDR operation.3.Correlation analysis between gender and rs1024611 suggested that compared with female-GG type(rs1024611 wild homozygous type),male-GG type and male-(GA+AA)type were correlated with disease occurrence(P<0.05)after adjusted age,smoking history,Hb A1 c and WHR,and OR(95%CI)values were 4.839(1.911-12.254)and 3.332(1.431-7.759),respectively.But the results of the additive model showed that there was no interaction between them based on the additive model(the confidence intervals of RERI,AP and S did not meet the requirements of non-0,non-0and non-1 at the same time).Gender,rs1024611 and their interaction terms(rs1024611×gender)were introduced into the equation by binary Logistic regression analysis Enter method,after adjusted for the covariables such as age,smoking history,Hb A1 c and WHR,and the results showed that rs1024611 had no multiplicative interaction with gender(P=0.129).Correlation analysis between rs7799039 and Hb A1 c suggested that Hb A1c<9%-(AG+GG),Hb A1c≥9%-AA,Hb A1c≥9%-(AG+GG)were all correlated with disease occurrence compared with Hb A1c<9%-AA(P<0.05),and OR(95%CI)values were 2.673(1.242-5.751),3.743(1.793-7.813)and2.741(1.282-5.860),respectively.However,the results of the additive model indicate that there was no interaction between them based on the additive model(the confidence intervals of RERI,AP and S do not meet the requirements of non-0,non-0 and non-1 at the same time).Hb A1 c,rs7799039and their interaction term(rs7799039 ×Hb A1c)were introduced into the equation using binary Logistic regression analysis Enter method.The results showed that(AG+GG)genotype of rs7799039 and Hb A1c≥9% were risk factors for lower extremity arterial disease in type 2 diabetes mellitus,and the OR(95%CI)values were 2.785(1.304-5.946)and 3.668(1.768-7.610),respectively.The interaction of the two factors was also associated with disease(OR=0.244,95%CI: 0.080-0.746,P=0.013).OR<1,indicating antagonism between rs7799039 and Hb A1 c.Correlation analysis of rs699947 and rs3918242 with gender suggested that compared with female-CC-CC(rs699947 and rs3918242 were all wild homozygous types),the male-CC-CC type and the male-(CA+AA)-(CT+TT)type had statistical significance for the occurrence of disease alone(P<0.05).Adjusted for age,smoking history,Hb A1 c and WHR,OR(95%CI)values were 3.864(1.518-9.833),5.525(1.283-23.800),respectively.Gender,rs699947,rs3918242 and their interaction terms(rs699947×rs3918343×gender)were introduced into the equation by binary Logistic regression analysis Enter method,after adjusted for the covariables such as age,smoking history,Hb A1 c and WHR,and the results showed that rs3918242(CT+TT type)and gender(male)had statistical significance(P<0.05),while rs699947 showed no statistical difference(P>0.05).Model fitting suggested interaction among the three factors(OR=5.359,95%CI: 1.172-24.498,P=0.030).Conclusions:1.Gene-gene interaction of candidate SNPs may not play a role in the pathogenesis of lower extremity arterial disease in type 2 diabetes mellitus.2.The interaction between gender and rs1024611 increased the risk of lower extremity arterial disease in type 2 diabetes mellitus,but there was neither additive model interaction nor multiplicative interaction between the two factors.3.The interaction between gender and rs699947 and rs3918242 can increase the risk of lower extremity arterial disease in type 2 diabetes mellitus.4.The interaction between Hb A1 c and rs7799039 can increase the risk of lower extremity arterial disease in type 2 diabetes mellitus,but there was no interaction based on additive model,there was a multiplication interaction,and the multiplication interaction was antagonistic.5.The interaction between SNPs and age,course of disease,smoking history,BMI may have no correlation with the incidence of lower extremity arterial disease in type 2 diabetes mellitus.GMDR analysis showed no interaction between SNPs and drinking history,dyslipidemia,hypertension and other macrovascular diseases,WHR,etc.