Journal of Human Hypertension (2012) 26, 430–436 & 2012 Macmillan Publishers Limited All rights reserved 0950-9240/12 www.nature.com/jhh ORIGINAL ARTICLE The combined impact of 12 common variants on hypertension in Japanese men, considering GWAS results K Miyaki1,2, NC Htun2, Y Song1, S Ikeda2, M Muramatsu2 and T Shimbo1 1Division of Clinical Epidemiology, Department of Clinical Research and Informatics, National Center for Global Health and Medicine, Tokyo, Japan and 2Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Genome-wide association studies have identified several results for the CYP11B2 and PTGIS genes. Although there polymorphisms that appear to be on hypertension-sus- were no significant associations found for other variants, ceptible regions. We performed the current replication our results suggested there was a combined impact for study in order to evaluate the association of these loci 12 loci. Individuals carrying more risk alleles had a higher with hypertension in healthy Japanese males and then risk of hypertension (P for the slope ¼ 0.002). Blood examined the combined effect of 12 independent variants. pressures also increased in conjunction with an increas- Overall, 735 Japanese men from two independent co- ing risk allele score (P for trend ¼ 7.84 Â 10À6 and horts were recruited. Association with hypertension was 1.85 Â 10À5 for SBP and DBP, respectively). Our results assessed in 16 polymorphisms on 12 genes and 12 were confirmed the associations between hypertension or chosen to evaluate the combined impact. Polymorphisms blood pressure and four gene variants. We also found a on the COMT, ATP2B1, CYP11A1 and the CSK genes were significant combined effect of the 12 gene loci. confirmed to be associated with hypertension and blood Journal of Human Hypertension (2012) 26, 430–436; pressure (BP). Current findings also replicated previous doi:10.1038/jhh.2011.50; published online 2 June 2011 Keywords: combined impact; polymorphism; blood pressure; genome-wide association study Introduction wide association studies (GWAS) and the candidate gene approach. Hypertension is an important public health problem Recently, results from several GWAS identified found throughout the world. Hypertension preva- several single-nucleotide polymorphisms (SNPs) lence has been increasing, and as of 2006, an esti- that appeared to be related to hypertension in diffe- mated 972 million people in the world currently 1 rent population groups, such as SNP rs17249574, suffer from this problem. In Japan, more than which was near the ATP2B1 gene (encodes PMCA1, 33 million adults, or 45% of the adult population, 2 a plasma membrane calcium/calmodulin-dependent are estimated to have hypertension. Although the ATPase) reported by Korean researchers;3 SNP disease is thought to result from a complex interplay rs11191548, which is near the CYP17A1 (cyto- between genetic predisposition and environmental chrome P450, family 17, subfamily A, polypeptide influences, it is unclear as to what extent the genetic 1) gene and SNP rs1378942, which is near the CSK factors contribute to an individual’s susceptibility (c-src tyrosine kinase) gene identified by a European to hypertension. There are numerous genetic-based GWAS;4 the common variant rs11646213, which studies that have explored the basis of hypertension, was upstream of the CDH13 (cadherin 13 prepro- with the results providing a better understanding of tein) gene found in two European populations.5 The the underlying molecular mechanisms and making candidate gene approach has also been used to it possible to identify the common variants that are investigate associations of diseases with common associated with hypertension or blood pressure variants. Kamide et al. performed a large association (BP). The most used techniques include genome- study and suggested that GREB1 (gene regulated by estrogen in breast cancer 1) and HPCAL1 (hippocal- Correspondence: Dr K Miyaki, Division of Clinical Epidemiology, cin-like 1 protein), which previously revealed by Department of Clinical Research and Informatics, National Center genome-wide scans in Italian6 and Chinese7 were for Global Health and Medicine, Toyama 1-21-1, Shinjuku-ku, candidate hypertension-susceptibility genes in Tokyo 162-8655, Japan. Japanese.8 The polymorphisms of ABCA1 gene E-mail: [email protected] 9 Received 7 December 2010; revised 18 March 2011; accepted 12 (ATP binding cassette A1), ACADSB (short/ April 2011; published online 2 June 2011 branched chain acyl-CoA dehydrogenase),10 COMT Combined impact of 12 variants on hypertension K Miyaki et al 431 (catecholamine-O-methyltransferase)10 and PTK2B diastolic BP (DBP) X90 mm Hg, or the current use (protein kinase 2b)11 were also found to be determi- of an antihypertensive drug. All other subjects nants of BP and responsible for the development of were considered to be normotensive. The study hypertension in Japanese individuals. was approved by the ethics review committee of the In the current study, we chose 14 SNPs in 10 genes Medical Research Institute of Tokyo Medical and that had been previously reported by GWAS or Dental University. All participants provided written were from candidate gene studies. After genotyping informed consent. them in two unrelated Japanese cohorts, we were able to replicate associations in the general Japanese population. In addition, our previous results have Genotyping discovered several SNPs that were associated with Genomic DNA was extracted from the peripheral hypertension in Japanese men, including C-344 blood of each subject by conventional methodology. T SNP of the CYP11B2 gene12 and C1117A SNP of Genotyping for all polymorphisms was performed the PTGIS gene (unpublished). In the current study, by PCR using a TaqMan genotyping assay (ABI) we also included these polymorphisms and per- followed by allelic discrimination analysis using formed a multiple regression analysis in order to SDS software (Applied Biosystems, Carlsbad, CA, evaluate the combined risk for BP from these 12 loci USA). For each sample, 10 ng of purified DNA was in our samples. mixed with 0.25 mlof20Â SNP genotyping assay mix that contained forward and reverse primers, VIC and FAM probes, 2.5 ul of TaqMan universal PCR Subjects and methods master mix and 2.25 ml of Dnase-free water. PCR amplification was performed by using the Applied Subjects Biosystems 9700 Thermal Cycler, with holding A total of 735 Japanese men from two separate temperatures of 90 1C for 10 min, 40 cycles of the occupational cohorts participated in the current denaturation temperature at 92 1C for 15 s and an study (Table 1). The first cohort, which has been annealing temperature of 60 1C for 1 min, with a 12 described elsewhere, comprised of 311 popula- final cooling step at 4 1C. tion-based Japanese men who worked for a company Following PCR, allelic discrimination was per- in Kanagawa Prefecture. The second cohort com- formed by using the Sequence Detection System prised 424 population-based Japanese men in Kyoto (ABI PRISM 7900HT, USA, SDS software package Prefecture who worked for a subsidiary of the version 2.2.1). We obtained successful genotyping first company. Information on age, current smoking call rates of 499.98% for the whole characterized status, drinking and the energy intake of all subjects sample. was obtained by means of a self-reported question- naire. A food frequency questionnaire was used for this purpose. Medical history was acquired Statistical analysis by interview. Height and weight were measured, The allele frequencies were determined by direct with the body mass index calculated as weight in counting. Deviation of the genotype distribution kilograms divided by height in meters squared. After from the Hardy–Weinberg disequilibrium was con- subjects rested quietly for at least 10 min in a supine firmed by a w2-test. Difference in the mean values of position, BP was measured using a posterior wall age, clinical characteristics, current smoking status, velocity/ankle-brachial index device (Nippon Colin, drinking and energy intake levels between the Aichi, Japan). Pressure was measured twice, with genotype groups were compared by an analysis of the average mean value then recorded. Hypertension variance or w2-test. Logistic regression analyses were was defined as a systolic BP (SBP) X140 mm Hg or a performed in order to examine the relationship Table 1 Baseline characteristics of the subjects Characteristics Total subjects (n ¼ 735) Normotensive (n ¼ 474) Hypertensive (n ¼ 261) Age (years) 47.0±8.9 45.3±9.2 50.1±7.4 BMI (kg mÀ2) 23.4±3.4 22.8±3.0 24.6±3.8 Systolic blood pressure (mm Hg) 129.9±17.5 120.1±10.3 147.8±13.2 Diastolic blood pressure (mm Hg) 79.8±11.8 73.5±8.0 91.1±8.6 Total serum cholesterol (mg dlÀ1) 203.0±34.5 199.0±33.4 210.3±35.4 Serum triglycerides (mg dlÀ1) 151.7±118.1 144.1±113.2 165.7±125.7 Serum HDL-cholesterol (mg dlÀ1) 56.7±14.4 57.0±13.9 56.3±15.4 Salt intake (g per day) 9.5±3.4 9.5±3.4 9.5±3.5 Energy intake (kJ per day) 7675.1±2190.3 7664.3±2179.4 7694.4±2214.6 Current smoking (%) 69.9 71.6 66.9 Alcohol use (%) 73.0 71.1 76.3 Abbreviations: BMI, body mass index; HDL, high-density lipoprotein. Data are presented as mean±s.d. or percentage. Journal of Human Hypertension Combined impact of 12 variants on hypertension K Miyaki et al 432 between the genotype and hypertension, as adjusted and body mass index were 47.0±8.9 years and for potential confounders, which might correlate to 23.4±3.4 kg mÀ2, respectively. The total prevalence BP, such as age, current smoking status and drink- of hypertension was 35.5% in our subjects.