Association Study Of Gene Polymorphisms In TGF-β Signal Pathway With Essential Hypertension

Background and objectivesCardiovascular disease is the leading cause of death for adults in developed or developing countries, including China. Hypertension has been proved the primary and independent modifiable risk factor for stroke and coronary heart disease (CHD) so that controlling hypertension is one of the most effective ways to prevent cardiovascular disease.With changes in lifestyle (such as smoking, overdrinking and unhealthy diet, etc.) and the increase in life expectancy, the prevalence of hypertension has been increasing significantly in China in the past decades. Particularly, the prevalence of hypertension has also increased rapidly in younger people and the rural areas. These results underscore the urgent need to carry out active researches on the etiology and develop effective strategies to improve prevention, diagnosis, and treatment of hypertension.Hypertension among adults with no identifiable cause is essential hypertension (EH). EH arises as a complex quantitative trait that is affected by varying combinations of genetic and environmental factors. Probably, genetic factor influences the blood pressure through numerous minor-effect genes, and biological process of hypertension involves multiple physiological pathways with gene and environment factors. Recently, two-stage association study based community population is a cost effective approach for identifying complex disease genes in genetic studies and it has received much attention.As the major basis in pathologic process of EH, large artery stiffening, peripheral resistance increasing caused by endothelial dysfunction and small artery remodeling have been proved the primary agent for blood pressure increase or even organ impairment. Researchers have focused on the molecular genetics of the pathology of artery stiffening and remodeling. However, the mechanism of human hypertension is still not fully understood. In animal study, proof indicated that Emilinl modulates TGF-βavailability in the pathogenesis of hypertension and links TGF-βmaturation to blood pressure homeostasis. This led us to further research the development and remodeling of artery in EH.With the purpose of exploring the relationship between development and remodeling of artery and EH, we collected and analyzed some biological data about the development and remodeling of blood vessel and selected several candidate genes, including FBN1, EMLIN1, FURIN, THSD1, TGFB1 and TGFBR1 gene, which proved being involved in the TGF-βsignal pathway. We applied a two-stage case-control study. We selected tagSNPs with consideration of predicted SNP (Single nucleotide polymorphisms) functions and obtained twenty three representative SNPs covered the candidate genes. Furthermore, we examined the relationship between the twenty three polymorphisms and EH in Chinese.Methods and SubjectsSubjectsAll the studied subjects recruited from the International Collaborative Study of Cardiovascular Disease in Asia (InterASIA in China), from which all the DNA samples and clinical data for participants were obtained. The local bioethical committee approved the protocol, and informed consent was obtained from each participant. InterASIA used a four-stage stratified sampling method to select a representative sample of the general population aged 35 to 74 years in China. We enrolled 1,317 unrelated hypertensive patients and 1,269 age and gender-matched unrelated normotensive from four northern field centers of InterASIA, namely Beijing, Jilin, Shandong, and Shanxi province. In this study, hypertensive were selected with systolic BP (SBP)≥150 mmHg, and/or diastolic BP (DBP)≥95 mmHg, or current use of antihypertensive medication, whereas subjects with a clinical history of secondary hypertension, coronary heart disease and diabetes were excluded. At the same time, healthy normotensives with SBP<140 mmHg and DBP<90 mmHg were selected from the same target study population as controls.MethodsPart 1We conducted a two-stage association study and took total 2,586 unrelated subjects as the main study population in this study. 993 subjects were selected as stage 1 subsample containing 503 hypertensive patients (SBP≥160 mmHg and/or DBP≥100 mmHg) and 490 age- and gender-matched normotensive controls (SBP<140 mmHg and DBP<90 mmHg). The criterion for case selection of the subsample based on the hypothesis that individuals with higher BP were likely to be enriched for genetic susceptibility, which might increase the difference in frequency of susceptibility alleles between cases and controls to improve power. At stage 2, subsample included 814 cases with hypertension and 779 age- and gender-matched normotensives.Combinating SNP functional prediction analysis, We selected twenty three representative tagSNP from HapMap database of Han Chinese in Beijing, China (CHB) according to the linkage disequilibrium (LD) analyses of all SNPs, which covered the candidate gene, reasonable length upstream and downstream. All the SNPs were examined the association with EH.Part 2We applied three methods to explore gene-gene and gene-environment interactions for EH. First, we utilized multivariate adaptive regression splines (MARS) and generalized multifactor-dimensionality reduction (GMDR) to identify latent interactions involved in the TGF-βpathway gene polymorphisms associated with EH. Furthermore, we employed Logistic regression to test the interactions identified by MARS or GMDR in three different models.ResultsPart 1 By single locus analysis, rs2011616 and rs3754734 of EMILIN1, rs4932178 of FURIN, rs12980942 of TGFB1, rs12346650 and rs1888223 of TGFBR1, and rs140598 and rs6493333 of FBN1 showed significant associations with EH (P<0.05) in stage 1 and we further genotyped the eight SNPs among remained sample. Joint analysis indicated that three SNPs presented significant association with EH. TT (vs. CC+CT) genotype of rs4932178 and allele A (vs.G) of rs12346650 increased the risk of EH significantly, odds ratios (ORs) were 2.543 (95% CI 1.324-4.886) and 1.473 (95% CI 1.315-1.650) respectively. G (vs.C) of rs140598 showed a protective effect on EH (OR=0.827 95% CI 0.703-0.973) after adjustment for conventional risk factors.Haplotypes of TGFBR1 gene were composed of rs12346650 and rs1888223. With the haplotype G-W used as reference, the adjusted ORs of haplotype A-G and haplotype A-W were 1.269 (95% CI: 1.095 to 1.470), 1.550 (1.299 to 1.856) respectively, and the OR of haplotype G-G was 0.112 (0.094 to 0.134). With the diplotype Hap1 (G-W)-Hap1 (G-W) used as reference, diplotype Dip2 (Hap1-Hap2), Dip3 (Hap1-Hap4), Dip4 (Hap2-Hap2), Dip5 (Hap3-Hap3) and Dip6 (Hap2-Hap3) were at higher risk for EH (after adjusted by Bonferroni correction, P<0.003). The haplotypes of FBN1 were composed of rs140598 and rs6493333. Haplotype C-C of FBN1 gene was used as reference and the adjusted OR of haplotype G-C was 0.823 (95% CI, 0.715 to 0.948). With diplotype Hap (C-C)-Hap (C-C) used as reference, the adjusted OR of diplotype Hap (G-C)-Hap (G-C) was 0.619 (95% CI, 0.436 to 0.880). Part 2First, MARS identified a significant interaction among smoking, rs140598 of FBN1 gene and rs12346650 of TGFBR1 gene, and rs12346650 presented a main effect for EH in model 1. In model 2, rs12346650 main effect showed in six out of seven significant interactions and rs140598 only appeared in five interactions whereas rs4932178 only presented an independent main effect. In model 3, three interactions were detected among drinking and four SNPs with latent minor effect.Subsequently, GMDR verified rs12346650 main effect in four interactions among rs4932178, rs140598, smoking and drinking in model 1. In model 2, Analogously, GMDR verified rs12346650 main effect in four interactions of rs4932178, rs140598, rs12980942, smoking and driking. However, there were no interactions identified by GMDR in model 3.Furthermore, Logistic regression tested three major 3-way interactions of rs12346650-rs140598-smoking, rs12346650-rs140598-rs3754734 and rs1888223 -rs12980942-drinking, which had been identified by MARS in the three models respectively, and achieved an explainable concordance with MARS.ConclusionsOur findings indicated TGF-βpathway genes might harbor some susceptible variations to essential hypertension in north Han Chinese population. Three methods, GMDR, MARS and Logistic regression analyses identified consistently significant gene by gene and gene by environmental interactions that associated with hypertension and the findings provide further support for the possible contributions of TGF-βpathway genes to essential hypertension.