Hypertension Research (2011) 34, 722–727 & 2011 The Japanese Society of Hypertension All rights reserved 0916-9636/11 $32.00 www.nature.com/hr ORIGINAL ARTICLE CYP7A1 genotypes and haplotypes associated with hypertension in an obese Han Chinese population Lingyu Fu1,3, Yanyan Zhao2, Xiaomei Wu1, Hong Liu2, Jingpu Shi1,3, Jingyu Lu2 and Bo Zhou1 This study investigated the association between single-nucleotide polymorphisms (SNPs; rs3808607 and rs1125226) within the CYP7A1 promoter and hypertension susceptibility in a Han Chinese population. From 2003 through 2006, a population- based case–control study was performed in a cohort of 1187 randomly selected Han Chinese subjects. A sib-pair study for a transmission disequilibrium test analysis was carried out in 76 hypertensive (HT) families (n¼312) from northeastern Liaoning province. SNPs were detected using real-time PCR. No significant differences were found in the genotype or allele frequencies of either SNP (P40.05), with no excessive allele sharing. For rs3808607, the frequency of the AA genotype in obese hypertensive patients was 31.91%, significantly higher than in normotensive (NT) subjects (12.73%; odds ratio (OR)¼3.21, 95% confidence interval (CI)¼1.35–7.66). For rs3808607, the AA genotype frequency was significantly higher in obese male HT subjects (27.87%) than in matched NTs (7.41%; OR¼4.83, 95% CI¼1.03–22.65). After adjustment for environmental risk factors in obese participants, the AA genotype was associated with hypertension (OR¼3.395, 95% CI¼1.412–8.162). Among subjects with body mass index X28 kg mÀ2, the HT and NT groups had significantly different frequencies of Hap I (C/C) and Hap IV (A/A). The frequencies of rs3808607 alleles in the CYP7A1 gene differed significantly between obese HT and NT men. Haplotypes I and IV were associated with hypertension in obese participants. Hypertension Research (2011) 34, 722–727; doi:10.1038/hr.2011.18; published online 24 February 2011 Keywords: association; body mass index; cholesterol 7-hydroxylase promoter; haplotype; single-nucleotide polymorphism. INTRODUCTION which is a cytochrome P-450 enzyme. In the study by Toshio et al.,5 The population of northeastern China has an age-standardized the expression of CYP7A1 mRNA was decreased in spontaneously HT prevalence of hypertension of almost 35%, which has made it an hyperlipidemic rats. Furthermore, in our study population, intake of ideal region for the study of this disease.1 Several genetic cohort animal oil and blood lipid metabolism disorders were independent studies have been performed in this population, and associations have risk factors for hypertension, according to the preliminary results of been reported between the likelihood of developing hypertension and this study. Therefore, we selected CYP7A1 as a candidate gene for single-nucleotide polymorphisms (SNPs) within CYP4F2 (À421G/C)2 hypertension in our study population. and NOS2 (À1026C/A).3 Hypertension mostly arises as a complex The CYP7A1 gene spans approximately 10 kb and contains six quantitative trait that is affected by varying combinations of genetic exons, five introns, one 5¢-untranslated region and one 3¢-untranslated and environmental factors; the role of each gene is relatively minor, region, and maps onto chromosome 8q11.6 Several studies have so many additional studies on susceptibility genes are required. been performed to assess the association of CYP7A1 with diseases Hypertension rarely occurs in the absence of other metabolic related to lipid metabolism. Most studies concerning genetic varia- disturbances. Dyslipidemia is one of the earliest and most frequent tions in the CYP7A1 gene have focused on an A-C SNP in the metabolic disturbances in hypertensive (HT) individuals, occurring promoter region, which is at position À278 from the translation concomitantly in over one-third of patients with hypertension. The initiation codon or position À204 from the transcriptional start site. liver has a central role in the regulation and maintenance of whole- The results appear to be conflicting as to whether this polymorphism body sterol balance. Conversion of cholesterol into bile acids in the is significantly associated with, for example, plasma lipid levels,7–9 risk liver, together with the secretion of cholesterol into bile, is the major of arteriosclerosis10 and gallstone disease 11–13 in adults and children quantitative pathway for eliminating cholesterol from the body.4 The from Caucasian and Asian populations. Some intervention trials have initial stage of this process is catalyzed by the rate-limiting enzyme in suggested that the polymorphism has an important role in the the catabolism of cholesterol, cholesterol 7-hydroxylase (CYP7A1),4 metabolism of lipids.14,15 1Department of Clinical Epidemiology, The First Affiliated Hospital, China Medical University, Shenyang, China and 2Department of Medical Genetics, China Medical University, Shenyang, China 3These authors contributed equally to this work Correspondence: Professor J Shi and Professor L Fu, Department of Clinical Epidemiology, The First Affiliated Hospital, China Medical University, No. 155, Nan Jing Bei Street, Shenyang, Liaoning Province 110001, China. E-mail: [email protected] or [email protected] Received 11 October 2010; revised 26 December 2010; accepted 27 December 2010; published online 24 February 2011 CYP7A1 is associated with hypertension in obesity LFuet al 723 The HapMap linkage disequilibrium patterns and haplotype are listed in Table 1. Subgroups were divided according to their body structure can serve as a reference to select haplotype tagging mass index (BMI):17 low body weight, BMI o18.5 kg mÀ2;normal SNPs (htSNPs) for association studies. Various degrees of linkage body weight, BMI 18.5–23.9 kg mÀ2; overweight, BMI 24.0– disequilibrium exist across different regions in different populations. 27.9 kg mÀ2;obese,BMIX28 kg mÀ2. Ethical approval and informed As inferred from public HapMap data and analyzed by Nakamoto consent were obtained from the local ethics committee and from all et al.,16 rs3808607 (CYP7A1 À204C/A) and rs1125226 (CYP7A1 subjects prior to their inclusion into the study. À6791C/A) are likely to be the relevant htSNPs in the Han Chinese population in Beijing, China (CHB). Genotyping of CYP7A1 Therefore, rs3808607 and rs1125226 were selected in this study as DNA was isolated from whole blood with the use of FlexiGene DNA Kits tag variants to assess the genotypes of an extensive population in our (QIAgen, Hilden, Germany). The patients and the controls were genotyped for study. To the best of our knowledge, no studies, including the genome- the rs3808607 and rs1125226 variants by a TaqMan Assay with a 7500 HT Real- wide association studies, have investigated whether these two poly- Time PCR System (Applied Biosystems, Foster City, CA, USA); all of the morphisms are associated with the risk of hypertension. We aimed to reagents and synthesized probes were purchased from Applied Biosystems identify hypertension risk haplotypes of the CYP7A1 regulatory region (C_8893122_10 and C_27492121_20). Real-time PCR was performed using and estimate their impact on gene transcription, as well as their 12.5 mlTaqMan2Â universal master mix, 0.625 ml primer probe, 10.875 ml interaction with environmental factors. DNase-free water and 1 ml DNA sample, for a total volume of 25 ml per single reaction in a 96-well plate. In each assay run, DNase-free water was used as a non-template control, and DNA of a known genotype was used as a positive METHODS control. Assay conditions were 2 min at 501C, 10 min at 951C, 40 cycles of 921C Subjects for 15 s and 601C for 1 min. The post-assay analysis was performed using SDS From 2003 through 2006, a population-based case–control study was software (Applied Biosystems). The samples of genotyped CC, AA and CA were performed with 1187 subjects of Han Chinese origin, who were selected randomly and confirmed by sequencing (3100 Genetic Analyzer, selected by random sampling in the northeastern region of Liaoning Applied Biosystems). province, China. Additionally, a family-based study was conducted, consisting of 76 families with 312 members; each family had at least Statistical analyses two offspring and one parent with hypertension. For all the indivi- Data were analyzed by SPSS, version 13.0, for Windows (SPSS, Chicago, IL, duals, blood pressure (BP) was measured three separate times on the USA). The Hardy–Weinberg equilibrium was assessed with the use of w2-tests. right arm of seated subjects, with at least a 5-min rest between The differences in clinical characteristics between HTs and normotensives (NTs) measurements; the individual average was calculated from these were assessed by a two-sample t-test for quantitative variables and a w2-test for three measurements. Hypertension was defined as an average systolic categorical variables. Allele and genotype frequencies between the HT and BP X140 mm Hg and/or an average diastolic BP X90 mm Hg; sub- normotensive (NT) groups were compared using the w2-test. Multivariate jects with diabetes mellitus, renal disease or secondary hypertension, logistic regression analyses were used to assess the relationship between each as diagnosed by medical specialists, were excluded from the study. polymorphism and hypertension while considering the effects of other predictor Normotension was defined as BPo140/90 mm Hg with no history of variables. The probability for stepwise entry was 0.05 and the probability for stepwise removal was 0.1. ORs and 95% confidence