| Part Ⅰ Study on the association between MC4 R SNPs and obesityBackground: Obesity is affected by the interaction of environment and heredity,and it is a complex multi-factor disease,which is essentially caused by chronic positive energy balance,that is,dietary energy intake exceeds energy consumption.In recent years,a number of obesity susceptibility genes have been screened by genome-wide association studies,and MC4 R gene is one of them.Subsequently,studies were carried out to repeatedly verify the association between the MC4 R gene and obesity among different populations around the world,but the results were different.The research groups are mainly European and American people,and the Chinese Han population is also involved,but other ethnic minorities in China have not yet been involved.The previous investigations of our research group found that the average body mass index of Maonan population was higher than that of local Han population.Purpose: Three polymorphic loci(rs17782313,rs476828 and rs12970134)of MC4 R obesity susceptibility genes screened by genome-wide association analysis(GWAS)were selected for genotyping to analyze the association between these SNP and their haplotypes,gene-gene(G × G)and gene-environment(G × E)interaction with obesity and its possible mechanism,so as to provide theoretical basis for the prevention and treatment of obesity.Methods: A cross-sectional study comprised of 1,836 participants(obesity group,858;and control group,978)was conducted in the Maonan ethnic group,an isolated minority in China.The general information and clinical materials of the participants were collected in the form of questionnaire,and genomic DNA was extracted from leucocytes of venous blood.Genotypes of the three SNPs(rs17782313,rs476828 and rs12970134)were determined by the next-generation sequencing(NGS)technology.Allele and genotype frequency distribution,the Hardy-Weinberg equilibrium(HWE),Pair-wise linkage disequilibrium(LD)and frequencies of haplotype were calculated by the SHEsis Main software.Unconditional logic regression was used to test the correlation of genotypes with the risk hazard of obesity,but also the SNP-or haplotype-environment interactions on the risk hazard of obesity after gender,age,Waist circumference,smoking,alcohol consumption,hypertension and hyperlipidemia were adjusted.The best interactive combination between the SNPs,haplotypes and environmental factors(Waist circumference,age,smoking,drinking and sex)was screened by generalized multifactor dimensionality reduction(GMDR).In addition,different types of interaction dendrogram and circle graph for SNP-environment and haplotype-environment interactions on the risk of obesity were generated by MDR,and the form and size of the interaction were evaluated by Logistic regression analysis.Results: 1.The mean values of BMI,WC,systolic blood pressure(SBP),diastolic blood pressure(DBP),TC,TG and LDL-C levels and the percentages of subjects who smoked cigarettes were higher while HDL-C value was lower in obese patients compared to the control subjects(P < 0.05).However,no discrepancies were noted in terms of age structure,glucose levels,sex ratio,and drinking between the two groups(P > 0.05 for all).2.Chi-square(χ~2)test revealed that the frequency distribution of MC4 R rs17782313 T and C alleles was significantly different between the case group and control group(P < 0.001).Logistic regression analysis implied that the CT/CC genotype was related to a raised risk of obesity in the dominant model(P = 0.019),after adjusting for confounding factors.There was also significant difference in the frequency distribution of T and C alleles at MC4 R rs476828locus(P < 0.001).Logistic regression analysis showed that the CT/CC genotype was associated with an increased risk of obesity in the dominant model(P =0.003),after adjustment for confounding factors.The frequency distribution of MC4 R rs12970134 G and A alleles was significantly different(P < 0.001).Logistic regression analysis showed that the AG /AA genotype had no statistically significant effect on obesity in the dominant model(P = 0.15),after adjusting for confounding factors.3.SHEsis software analysis showed that MC4 R rs17782313-rs476828-rs12970134 in the study population showed strong LD between control and obesity groups(D’ = 0.77–0.99).And the results for haplotypes showed that MC4 R T-T-G,MC4 R C-C-A,MC4 R T-C-G and MC4 R T-T-A were the most common haplotypes(haplotype frequency > 0.03).MC4 R C-C-A haplotype increased the risk of obesity with statistical significance(P <0.001).However,both MC4 R T-T-A(P = 0.02)and MC4 R T-T-G(P < 0.001),as protective haplotype,were associated with obesity.4.GMDR software screened out two best three-locus models.A significant three-locus model(P < 0.001)involving rs12970134 SNP,drinking and WC was found,with a cross-validation consistency(10/10)and a testing accuracy of 83%,indicating a potential interaction between SNPs and these environmental factors.Moreover,the three-locus model also tested haplotype-environment interactions(Waist circumference,drinking and T-T-A,P < 0.001),with a cross-validation consistency(10/10)and a testing accuracy of 82%,revealing a potential haplotype-environment interaction.5.Entropy-based interaction dendrograms and circle graph obtained from MDR analysis revealed the strongest redundancy effect in the SNP-environment interaction(rs12970134 and waist circumference)with interaction entropy of-0.55 %,and haplotype-environment interaction(MC4R T-T-A and waist circumference)with interaction entropy of-0.11%,respectively.6.In order to acquire the OR and 95%CI for the combined effects,we performed an interaction study using logistic regression analyses.When the SNP-environment interaction was analyzed,compared to the participants with rs12970134 GG and WC(male<90 cm,female<80 cm),we revealed that not only the participants with rs12970134 GA/AA genotypes and WC(male ≥ 90 cm or female ≥ 80 cm)had higher risk of obesity(P < 0.001),but also rs12970134 GG genotypes and WC(male ≥ 90 cm,female ≥ 80 cm)could raise the risk of obesity(P < 0.001).In addition,when the haplotype-environment interactions were studied,compared to the non-carriers and WC(male<90cm or female < 80),non-carriers and WC(male ≥ 90 cm,female ≥ 80 cm)raise the risk of obesity(P < 0.001),the carriers of T-T-A haplotype and WC(male ≥ 90 cm,female ≥ 80 cm)also had higher obesity risk(P < 0.001).Conclusions: 1.Waist circumference,total cholesterol,low density lipoprotein,triglyceride and smoking may be the risk factors for obesity,while HDL-C may reduce the risk of obesity.2.MC4 R rs17782313(C)allele,rs476828(C)allele,rs12970134(A)allele may be risk factors for obesity.For haplotype analysis,MC4 R C-C-A haplotype increased the risk of obesity;however,MC4 R T-T-A and MC4 R T-T-G haplotypes could reduce the risk of obesity.In a word,MC4 R SNPs,their haplotypes might be involved in the susceptibility to obesity.3.Our outcomes show that the interaction of MC4 R rs1297013 and waist circumference was noted on the risk of obesity,as well as between haplotype T-T-A and waist circumference.MC4 R rs12970134 gene carriers(GA+AA)and WC(male ≥ 90 cm,female ≥ 80 cm)were the risk factors for hypertension,as well as the haplotype T-T-A carrier and waist circumference(male ≥ 90 cm,female ≥ 80 cm).In a word,our results implied that MC4 R and environmental factors(G × E)interaction might be involved in the susceptibility of obesity.Part Ⅱ Study on the association between CYP17A1-ATP2B1 SNPs and hypertensionBackground: Hypertension is a complex disease affected by both environment and heredity.With the emergence of genome-wide association analysis,more and more hypertension susceptibility genes have been unveiled,including CYP17A1 gene and ATP2B1 gene.Studies on the association between CYP17A1 gene or ATP2B1 gene and hypertension have been repeatedly verified in different populations around the world,but the results are different.And there are few studies on the effects of gene-gene and environment-gene interaction on hypertension.Purpose: Four polymorphic loci(CYP17A1 rs1004467,CYP17A1rs11191548,ATP2B1 rs1401982 and ATP2B1 rs17249754)of MC4 R hypertension susceptibility genes screened by genome-wide association analysis(GWAS)were selected for genotyping to analyze the association between these SNP and their haplotypes,gene-gene(G × G)and gene-environment(G × E)interaction with hypertension and its possible mechanism,so as to provide theoretical basis for the prevention and treatment of hypertension.Methods: A cross-sectional study comprised of 1652 participants(hypertension group,816;and control group,836)was conducted in the Maonan ethnic group,an isolated minority in China.The general information and clinical materials of the participants were collected in the form of questionnaire,and genomic DNA was extracted from leucocytes of venous blood.Genotypes of the four SNPs(CYP17A1 rs10044,CYP17A1 rs11191548,ATP2B1rs1401982 and ATP2B1 rs17249754)were determined by the next-generation sequencing(NGS)technology.Allele and genotype frequency distribution,the Hardy-Weinberg equilibrium(HWE),Pair-wise linkage disequilibrium(LD)and frequencies of haplotype were calculated by the SHEsis Main software.Unconditional logic regression was used to test the correlation of genotypes with the risk hazard of hypertension,but also the SNP-or haplotypeenvironment interactions on the risk hazard of hypertension after gender,age,WC,smoking,alcohol consumption,hypertension and hyperlipidemia were adjusted.The best interactive combination between the SNPs,haplotypes and environmental factors(WC,age,smoking,drinking and sex)was screened by generalized multifactor dimensionality reduction(GMDR).In addition,different types of interaction dendrogram for SNP-environment and haplotype-environment interactions on the risk of hypertension were generated by MDR,and the form and size of the interaction were evaluated by Logistic regression analysis.Results: 1.The mean values of BMI,SBP,DBP,TC,TG and LDL-C levels were higher while HDL-C value was lower in hypertensive patients compared to the control subjects(P < 0.05).However,no discrepancies were noted in terms of age structure,glucose levels,sex ratio,and the percentages of subjects who smoked cigarettes or drinking between the two groups(P > 0.05 for all).2.Chi-square(χ~2)test showed that the frequency distribution of CYP17A1rs1004467 A and G alleles was significantly different between the hypertension group and the control group(P < 0.001).After adjustment for confounding factors,logistic regression analysis implied that the AG/GG genotype was related with a declined risk of hypertension in the dominant model(P < 0.001).There was also significant difference in the frequency distribution of CYP17A1rs11191548 T and C alleles(P < 0.001).After adjustment for confounding factors,logistic regression analysis revealed that the TC/CC genotype reduced the susceptibility of hypertension in the dominant model(P < 0.001).The frequency distribution of ATP2B1 rs1401982 G and A alleles was also significantly different(P < 0.001).After adjustment for confounding factors,logistic regression analysis implied that GA/AA genotype was related with an increased susceptibility of hypertension in the dominant model(P = 0.002).The frequency distribution of ATP2B1 rs17249754 G and A alleles was also significantly different(P < 0.001).Logistic regression analysis after showed that the AG/AA genotype was associated with a reduced risk of hypertension in the dominant model(P < 0.001),adjusting for confounding factors.3.SHEsis software analysis showed that CYP17A1 rs1004467-rs11191548(D’ = 0.95)and ATP2B1 rs1401982-rs17249754(D’ = 0.95)showed strong linkage in two groups,respectively.And the results of haplotypes showed that CYP17A1 A-T,CYP17A1 G-C,CYP17A1 G-T,ATP2B1 A-G,ATP2B1 G-A and ATP2B1 G-G were the most common haplotypes(haplotype frequency > 0.03).Both of CYP17A1 A-T and ATP2B1 A-G haplotypes increased the risk of hypertension with statistical significance(P < 0.001 for all).However,both CYP17A1 G-C and ATP2B1 G-A,as protective haplotypes,were associated with hypertension,which were statistically significant(P < 0.001 for all).The correlation analysis of CYP17A1 G-T and ATP2B1 G-G haplotypes with hypertension showed no statistical significance(P > 0.05).4.GMDR software analysis showed that there were no two or three-locus models with statistical significance for four SNPs(CYP17A1 rs1004467,CYP17A1 rs11191548,ATP2B1 rs1401982 and ATP2B1 rs17249754),suggesting that there might be no interaction effect between CYP17A1 and ATP2B1 on hypertension.A significant two-locus model(rs11191548 and BMI≥ 24 kg/m~2,P < 0.05)revealed a potential SNP-environment interaction between rs11191548 and BMI ≥ 24 kg/m~2,with a cross-validation consistency(7/10)and a testing accuracy of 63%.Another significant two-locus model(CYP17A1 G-C and BMI ≥ 24 kg/m~2)(P < 0.05)indicated a potential haplotype-environment interaction,with a cross-validation consistency(9/10)and the testing accuracy of 63%.5.Entropy-based interaction dendrograms and circle graph obtained from MDR analysis revealed the strongest redundancy effect in the SNP-SNP interaction(rs1401982 and rs17249754)with interaction entropy of-0.98%,SNP-environment interaction(rs11191548 and BMI ≥ 24 kg/m~2)with interaction entropy of-0.70%,haplotype-haplotype interaction(CYP17A1 A-T and CYP17A1 G-C)with interaction entropy of-0.53%,and haplotype-environment interaction(CYP17A1 G-C and age)with interaction entropy of-0.14%,respectively.6.In order to acquire the OR and 95%CI for the combined effects,we performed an interaction study using logistic regression analyses.The interaction between CYP17A1 and ATP2B1 polymorphisms was not statistically significant(P > 0.016).When analyzed with the SNP-environment interaction,compared to the individuals with rs11191548 TT and BMI<24 kg/m~2,we found that not only the individuals with rs11191548 TT genotypes and BMI ≥ 24kg/m~2 could raise the risk of hypertension(P < 0.001),but also rs11191548TC/CC genotypes and BMI ≥ 24 kg/m~2 could raise the risk of hypertension(P=0.014).When haplotypes-environment interactions were studied,compared to non-carriers and age(age < 60),non-carriers and age(age ≥ 60)raise the risk of hypertension(P < 0.001),however,carriers and age(age < 60)was not statistically significant,and neither was carriers and age(age ≥ 60)(P > 0.016).Conclusions: 1.Body mass index,waist circumference,total cholesterol,triglyceride,blood glucose and low density lipoprotein may be the risk factors for hypertension,while HDL-C may reduce the risk of hypertension.2.CYP17A1 rs1004467(G)allele and ATP2B1 rs1401982(A)allele may be risk factors for hypertension,while CYP17A1 rs11191548(C)allele and ATP2B1 rs17249754(A)allele can reduce the risk of hypertension.For haplotype analysis,CYP17A1 A-T and ATP2B1 A-G haplotypes increased the risk of hypertension;however,CYP17A1 G-C and ATP2B1 G-A haplotypes reduced the risk of hypertension.In a word,the CYP17A1-ATP2B1 SNPs,their haplotypes might be involved in the susceptibility to hypertension.3.Our outcomes show that the interaction of rs11191548 and body mass index was observed on the risk of hypertension,which might be the risk factors for hypertension.In a word,our results implied that CYP17A1 and ATP2B1(G× G)interaction may have no association with the hypertension in Maonan population,however,CYP17A1 G × E interaction might be involved in the risk of hypertension. |
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