| Part Ⅰ: ASSOCIATION BETWEEN THE VEGFA POLYMORPHISMS AND DYSLIPIDEMIA AND ISCHEMIC CARDIO-CEREBROVASCULAR DISEASE IN GUANGXI MAONAN AND HAN CHINESEBackground: In recent years,the incidence and mortality of ischemic cardio-cerebrovascular disease based on atherosclerosis(AS)are still increasing in China.Abnormal lipid metabolism is an independent risk factor for AS.Therefore,active control and intervention of serum lipid levels is of great significance for the prevention and treatment of AS disease.Lipid metabolism is influenced by genetic,environmental factors and genetic-enviromental interaction,and genetic variation usually has race/ethnic specific and multipotent.Previous studies in animal model have confirmed that vascular endothelial growth factor A gene(VEGFA)is closely related to serum lipidlevels and AS development.Large-scale genome-wide association studies(GWASes)have also demonstrated that the VEGFA polymorphisms may be closely related to blood lipid levels and the risk of coronary atherosclerosis heart disease(CHD)and ischemic stroke(IS).However,most of these correlative studies were carried out in European populaitons and the results were inconsistent,to our knowledge,little is known about the association of VEGFA SNPs and serum lipid levels,the risk of CHD and IS in Guangxi populations.Objective: The current study was conducted to explore the association of VEGFA rs3025039,rs10434 and rs3025053 SNPs and serum lipid levels and the risk of CHD and IS in Guangxi Maonan and Han populations.The influences of several environmental factors and gene-enviromental interaction on serum lipid levels were also analyzed.Methods: Genotyping of the VEGFA rs3025039,rs10434 and rs3025053 SNPs was performed by poumerase chain reaction and restriction fragment length polymorphism(PCR-RFLP).Then the genotypes were verified by direct sequencing of PCR products.The epidemiological survey and serum lipid measures were carried out in all of the subjects.Depending on the different research subjects,the study was divided into two stages,the first stage: According to the method of international epidemiological investigation,1210 Maonan subjects and 1240 local Han populations resided in Huanjiang Maonan Autonomous County were randomly selected,the differences in genotypic and allelic frequencies of VEGFA SNPs,and the association of selected SNPs and serum lipid levels in the two populations were determined.The second stage: A case-control study was carried out in Guangxi Han populations,including 644 healthy controls,586 patiens with CHD diagnosed by coronary angiography and 549 patients with IS diagnosed clinically.The differences of genotype and allele frequencies of VEGFA SNPs were determined and then to estimate the association between selected SNPs and the risk of CHD and IS.Results: 1.There were statistically significant differences in serum lipid levels between Guangxi Maonan and Han populations,serum triglyceride(TG)and apolipoprotein(Apo)B levels were higher and high-density lipoprotein cholesterol(HDL-C)and Apo A1 levels were lower in Maonan than in Han populations.2.The distribution of genetype and allele of rs3025039,rs10434 and rs3025053 SNPs was different between the two ethnic groups.3.Several SNPs were associated with total cholesterol(TC,rs3025039 and rs10434),HDL-C(rs10434),LDL-C(rs3025039),Apo A1/Apo B(rs3025039)in Han nationality(P < 0.05/3,adjusted by Bonferroni);with TC(rs10434),Apo B(rs3025053)and Apo A1/Apo B(rs3025039 and rs10434)in Maonan population(P < 0.05/3,adjustd by Boferroni).4.Subgroup analysis according to gender showed that serum LDL-C level and the ratio of Apo A1 to Apo B were different among the genotypes of the VEGFA rs3025039 SNP in Han females,while serum TC and HDL-C levels were different among the genotypes of the VEGFA rs3025039 SNP in Maonan males(P < 0.05/3,adjustd by Boferroni).5.There was a Linkage disequilibrium between rs10434 and rs3025053 SNPs.The most common haplotype was G-C,the frequencies of G-C and A-T haplotypes were significantly different between the two ethnic groups(P < 0.05).The G-C carriers had higher serum HDL-C levels;A-T carriers had higher serum TC levels than the corresponding haplotype non-carriers in Han populations(P < 0.05/2,adjusted by Bonferroni).The G-C haplotype carriers had lower Apo B levels and higher the Apo A1/Apo B ratio;the A-C haplotype carriers had higher serum TC and Apo B levels than the corresponding haplotype non-carriers in Maonan ethnic group(P < 0.05/2,adjusted by Bonferroni).6.Multivariable linear regression analyses showed that serum lipid levels in both Maonan and Han populations were associated with age,gender,waist circumference,blood pressure,glucose,smoking and alcohol consumption(P < 0.05,respectively).7.In the case-control study,the genotypic and allele frequencies of rs3025039 and rs10434 SNPs;and the allelic frequencies of rs3025053 SNP were significantly different between the CHD patients and controls(P < 0.05).The genotypic and allelic frequencies of rs3025039 SNP;and the allelic frequencies of rs10434 SNP were different between the IS patients and controls(P < 0.05).8.After the potential risk factors were controlled and Bonferroni correction was employed,the rs3025039 and rs10434 SNPs were associated with the risk of CHD and IS in different genetic models.The subjects with TT genotype of rs3025039 SNP was associated with higher risk of CHD(OR = 2.02,95% CI = 1.07-3.84,P = 9E-04)under the comoniant gentic model;the subjects with CT/TT genotypes were associated with higher risk of CHD(OR = 2.02,95% CI = 1.07-3.84,P = 2E-04)and IS(OR = 1.40,95%CI = 1.08-1.80,P = 0.009)under the dominant genetic model;the subjects with CT genotype was associated with higher risk of CHD(OR = 1.53,95%CI = 1.16-2.01,P = 0.0023)under the overdominant genetic model;the rs3025039 T allele carriers was also asscociated with higher susceptibility to CHD(OR = 1.51,95%CI = 1.22-1.90,P = 0.0019)and IS(OR = 1.38,95%CI = 1.11-1.70,P = 0.0003)under the logadditive genetic model.The subjects with AA genotype of rs10434 SNP was associated with higher risk of CHD(OR = 1.65,95% CI = 1.14-2.05,P = 0.0025)under the comoniant gentic model;the subjects with AA/AG genotype were associated with higher risk of CHD(OR = 1.36,95% CI = 1.06-1.74,P = 0.015)and IS(OR = 1.29,95%CI = 1.09-1.77,P = 0.007)under the dominant genetic model;the rs10434 A allele carriers was also asscociated with higher susceptibility to CHD(OR = 1.39,95%CI = 1.13-1.72,P = 0.00197)under the log-additive genetic model.9.The interactions between genetic variations and environment factors were detected,the subjects with rs3025039CT/TT genotype interacted with age > 60 years to increase the risk of CHD and IS,and the subjects with rs3025039CT/TT genotype interacted with BMI ≥ 24 kg/m2 to increase the risk of CHD.The subjects with rs10434AG/AA genotype interacted with hypertension to increase the risk of CHD;the subjects with rs10434AG/AA genotype interacted with smoking to increase the risk of CHD and IS.10.There is a linkage disequilibrium between rs10434 and rs3025053 SNPs in case and control groups.As compared to the C-G haplotype,the A-T haplotype carriers had higher risk of CHD(OR = 1.55,95%CI = 1.14-1.98,P = 0.015)and IS(OR = 1.28,95%CI = 1.03-1.58,P = 0.025).11.Analysis of haplotype interacted with environmental risk factors on the risk of CHD and IS showed that the A-T haplotype interacted with age > 60 years and BMI ≥ 24 kg/m2 to increase the risk of CHD.The A-T haplotype interacted with hypertension and BMI≥24 kg/m2 to increase the risk of IS. 12.Interactions of gene and environmental factors on serum lipid levels were also detected in our study populations.The rs3025039 T allele carriers had higher LDL-C levels and lower Apo A1/Apo B ratio than the T allele non-carriers;the rs10434 A allele carriers had higher serum TC level than the rs10434 A allele non-carriers(P < 0.05/3,adjusted by Bonferroni).The subjects with rs3025039 TT genotype interacted with BMI ≥ 24 kg/m2 to increase the level of serum LDL-C.The subjects with rs10434 AA genotype interacted with smoking to increase the level of serum TC.Conclusions: 1.There was different in serum lipid levels,genotypic and allelic frequencies of VEGFA SNPs between Guangxi Maonan and Han populations.2.There may be a race/ethnic/sex specific association of the VEGFA rs3025039,rs10434 and rs3025053 SNPs and some serum lipid parameters in the two populations.3.There is a linkage disequilibrium between rs10434 and rs3025053 SNPs,the G-C haplotype carriers had lower risk of dyslipidemia,the A-T haplotype carriers had higher risk of dyslipidemia compared to the corresponding haplotype non-carriers.4.The case-control study conducted in Guangxi Han population showed that,the subjects with rs3025039 CT/TT genotypes were associated with increased risk of CHD and IS;the subjects with rs10434 AA/AG genotypes were associated with increased risk of CHD.5.The VEGFA A-T haplotype carriers were associated with increased risk of CHD and IS.The rs3025039,rs10434 SNPs and A-T haplotype interacted with some environmental factors such as age,BMI,hypertension,smoking to influence the risk of CHD and IS.6.The rs3025039 and rs10434 SNPs were associated with the levels of serum lipid in control group,the interaction of SNPs and BMI or smoking on several lipid parameters was detected.These results indicated that the two SNPs may increase the risk of CHD and IS by affecting serum lipid levels.7.These findings suggested that VEGFA rs3025039,rs10434 SNPs and rs10434A-rs3025053 T halotypes interacted with some environmental factors to influence seum lipid levels and the risk of CHD and IS.Part Ⅱ: ASSOCIATION BETWEEN THE SLC22A3-LPAL2-LPA POLYMORPHISMS AND DYSLIPIDEMIA AND ISCHEMIC CARDIOCEREBROVASCULAR DISEASE IN GUANGXI MAONAN AND HAN CHINESEBackground: A large number of studies have indentified that CHD and IS are complex disease affected by both genetic and environmental factors,and genetic factors play a key role in the pathogenesis of the two diseases.The SLC22A3-LPAL2-LPA cluster is located in human chromosome 6q26-27 region.According to different GWAS,several SNPs in the SLC22A33-LPAL2-LPA cluster were confirmed to be associated with serum lipid levels in European and American populations,and this gene cluster also indentified to be significantly associated with the incidence of CHD.The common pathological basis of IS and CHD is AS,and disorders of lipid metabolism is closly related to the incidence of IS,but the studies on the association between the SLC22A3-LPAL2-LPA SNPs and IS are relatively few.However,the relationship between the SLC22A3-LPAL2-LPA polymorphsims and lipid levels and susceptibility to CHD and IS in Guangxi population has not been reported so far.Objective: The current study was conducted to detect the effect of SLC22A3-LPAL2-LPA SNPs(rs394352,rs10455782,rs3106164,rs3088442,rs3127599,rs7767084,rs7765781 and rs783149)and environmental factors on serum lipid levels in Guangxi Maonan and Han populations;and the relationship between SLC22A3-LPAL2-LPA SNPs and the risk of CHD and IS.Methods: Genotyping of the SLC22A3 rs394352,SLC22A3 rs10455782,SLC22A3 rs3106164,SLC22A3 rs3088442,LPAL2 rs3127599,LPA rs7767084,LPA rs776578 and LPA rs783149 SNPs was performed by polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP).Then the genotypes were verified by direct sequencing of PCR products.The epidemiological survey and serum lipid measures were carried out in all of the subjects.Depending on the different research subjects,the study was divided into two stages,the first stage: According to the method of international epidemiological investigation,1234 Maonan subjects and 1255 local Han populations resided in Huanjiang Maonan Autonomous County were randomly selected,the differences in genotypic and allelic frequencies of SLC22A3-LPAL2-LPA SNPs,and the association of selected SNPs and serum lipid levels in the two populations were determined.The second stage: A case-control study was carried out in Guangxi Han populations,including 635 healthy controls,579 patiens with CHD diagnosed by coronary angiography and 553 patients with IS diagnosed clinically.The difference in genotype and allele frequencies of SLC22A3-LPAL2-LPA SNPs was determined and then to estimate the association between selected SNPs and the risk of CHD and IS.Results: 1.There were statistically significant differences in serum lipid levels between Guangxi Maonan and Han populations,serum TG and Apo B levels were higher and serum HDL-C levels were lower in Maonan than in Han populations.2.The distribution of genetype and allele of rs10455782,rs3106164 and rs7767084 SNPs was different between the Maonan and Han populations;whereas the allele frequencies of rs394352,rs3088442 and rs783149 SNPs were different between the two ethnic groups.3.Several SNPs were associated with TC(rs10455782 and rs783149),HDL-C(rs7767084),LDL-C(rs3088442),Apo B(rs3106164,rs10455782 and rs7767084)levels and the Apo A1/Apo B ratio(rs3106164)in Han nationality(P < 0.05/8,adjusted by Bonferroni);with TC(rs10455782),HDL-C(rs3088442 and rs7767084),LDL-C(rs10455782,rs3088442 and rs7767084),Apo A1(rs3106164,rs7767084 and LPA rs783149),Apo B(rs3106164)and Apo A1/Apo B ratio(rs783149)in Maonan population(P < 0.05/8,adjustd by Boferroni).4.Subgroup analysis according to gender showed that serum LDL-C levels were different among the rs3088442 genotypes in both Maonan and Han males;serum Apo B levels were different among the rs3106164 genotypes in Han females;serum HDL-C levels were different among the rs3088442 genotypes in Maonan females(P < 0.05/8,adjustd by Boferroni).5.There was a linkage disequilibrium between the SLC22A3 rs394352,SLC22A3 rs10455782,SLC22A3 rs3106164 and SLC22A3 rs3088442 SNPs.The most common haplotype was T-C-C-G,the frequencies of C-T-T-A,C-C-C-A and C-C-T-A haplotypes was significantly different between the two ethnic groups(P < 0.05).The linkage disequilibrium was also observed in LPA rs7767084,rs7765781 and rs783149 SNPs,the most common haplotype was T-G-A,the frequencies of T-G-A and C-C-C haplotypes were significantly different between the two ethnic groups(P < 0.05).6.Several haplotypes were associated with TC(C-T-T-A,T-G-A and C-CC),HDL-C(C-C-C),LDL-C(C-C-C-A and C-C-T-A),Apo B(C-C-C-A)levels and the Apo A1/Apo B ratio(C-C-C-A)in Han nationality(P < 0.05/7,adjusted by Bonferroni);with TC(C-T-T-A and C-C-C),HDL-C(C-C-T-A and T-G-A),LDL-C(C-T-T-A,C-C-CA and T-G-A)and Apo A1(C-C-T-A and C-C-C)in Maonan populatios(P < 0.05/8,adjustd by Boferroni).7.Analysis of gene-gene interaction in Guangxi Maonan and Han populations showed that,the most common haplotype was T-C-C-G-T-G-A,the frequnencies of T-C-C-G-C-C-C,C-C-C-G-T-G-A,C-T-T-A-C-C-C,C-C-C-AT-G-A and C-C-C-A-C-C-C haplotypes were significantly different between the two ethnic groups(P < 0.05).8.Multivariable linear regression analyses showed that serum lipid levels in both Maonan and Han populations were also associated with age,gender,waist circumference,blood pressure,glucose,smoking and alcohol consumption(P < 0.05,respectively).9.The case-control study showed that,the BMI,systolic blood pressure,pulse pressure,serum TC and TG levels were higher;while serum HDL-C levels,the Apo A1/Apo B ratio and alcohol consumption were lower in CHD and IS groups than in control group(P < 0.05).10.The genotypic and allele frequencies of rs10455782,rs3088442,rs7767084 and rs783149 SNPs were different between the CHD patients and controls(P < 0.05).The genotypic and allelic frequencies of rs10455782,rs7767084 and rs783149 SNPs were different between the IS patients and controls(P < 0.05).11.After Bonferroni correction,the subjects with rs10455782CT/TT genotypes were associated with higher risk of CHD(OR = 1.62,95% CI = 1.18-2.23,P = 0.0022)and IS(OR = 1.75,95% CI = 1.26-2.41,P = 0.0007)in the dominant model;and the subjects with CT genotype had higher risk of IS(OR = 1.82,95%CI = 1.29-2.55,P = 0.0005)in the overdominant model.The subjects with rs3088442AG/AA genotypes were associated with lower risk of CHD(OR = 0.77,95% CI = 0.62-0.96,P = 0.0023)and IS(OR = 0.73,95%CI = 0.57-1.03,P = 0.001)in the dominant model;the subjects with rs3088442 AG genotypes had lower risk of CHD(OR = 0.79,95%CI = 0.63-0.99,P = 0.0044)under the overdominant genetic model.The subjects with rs7767084CT/CC genotypes were associated with decreased risk of CHD(OR = 0.83,95%CI = 0.52-1.06,P = 0.0041)in the dominant model.The subjects with rs783149CA/AA genotypes were associated with increased risk of CHD(OR = 2.27,95%CI = 1.05-5.15,P = 0.0035);the rs783149 A allele carriers were associated with higher susceptibility to CHD(OR = 1.87,95%CI = 1.14-3.87,P = 0.0015)under the log-additive genetic model.12.Analysis of the interactions between gene SNPs and environmental factors showed that the subjects with rs10455782CT/TT genotypes interacted with smoking and hypertension to increase the risk of CHD.The subjects with rs3088442AG/AA genotypes interacted with alcohol consumption to decrease the risk of CHD.The subjects with rs783149AC/AA genotypes interacted with BMI ≥ 24 kg/m2 to increase the risk of CHD.The subjects with rs10455782CT/TT genotypes interacted with age>60 years and smoking to increase the risk of IS.The subjects with rs3088442AG/AA genotypes interacted with alcohol consumption to decrease the risk of CHD.13.Linage disequilibrium analysis of SLC22A3 and LPA genetic polymorphisms revealed that the C-T-T-A and T-G-C haplotype carriers had higher risk of CHD and IS;the C-C-C-G and C-C-C haplotype carriers had lower risk of CHD.14.Interactions of the SLC22A3-LPAL2-LPA polymorphsims and environmental factors on serum lipid levels were detected.The rs10455782 T allele carriers had higher levels of TC and Apo B;the rs3106164 T allele carriers had lower Apo A1/Apo B ratio;the rs3088442 A allele carriers had lower level of LDL-C;the rs7767084 C allele carriers had higher level of HDL-C;the rs783149 A allele carriers had higher level of TC(P < 0.05/8,adjusted by Bonferroni)than the corresponding haplotype non-carriers,respectively.The rs10455782 TT genotype interacted with smoking to increase serum Apo B levels;the rs3088442 AA genotype interacted with alcohol consumption to decrease serum LDL-C levels.The rs7767084 CT genotype interacted with males to decrease serum Apo B levels.15.In addition,the rs3088442 AA genotype interacted with alcohol consumption to decrease the incidence of CHD.Conclusions: 1.There was different in serum lipid levels,genotypic and allelic frequencies of SLC22A3-LPAL2-LPA SNPs between Guangxi Maonan and Han populations.2.There may be a race/ethnic/sex specific association of the SLC22A3-LPAL2-LPA SNPs and several serum lipid parameters in the two populations.3.The linkage disequilibrium analysis showed that there was a correlation between haplotypes and serum lipid levels in the two ethnic groups.4.The case-control study conducted in Guangxi Han population revealed that,the rs10455782,rs3088442,rs7767084,rs783149 SNPs and several haplotypes interacted with some environmental factors to change the incidence of CHD and IS.The SLC22A3-LPAL2-LPA rs10455782,rs3088442 and rs7767084 SNPs interacted with some environmental factors to influence serum lipid levels,and the rs3088442 AA genotype interacted with alcohol consumption to influence the risk of CHD.5.These results suggest that the SLC22A3-LPAL2-LPA SNPs were significantly associated with serum lipid paremeters in the Guangxi Maonan and Han populations.6.The interaction between these genetic polymorphsims and their haplotypes and some environmental factors on serum lipid levels may also lead to changes in the risk of CHD and IS. |
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