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. This ing. Multiple logistic regression analysis was used was lower than that reported in a cardiovascular to analyze whether interactions between all of these disease survey preformed by The Ministry of Health, genotypes and the energy and salt intake levels were Labor and Welfare of Japan in 2001, which found associated with hypertension. the prevalence of hypertension in Japanese men A combined analysis of the variants within these aged from 30 to 69 years to be 47.3% (http:// 12 genetic loci was performed by summing up the wwwdbtk.mhlw.go.jp/toukei/kouhyo/data-kou18/ risk alleles (0, 1, 2) of these 12 variants, with each data12/junkan-h12-3.pdf). When compared with individual potentially having from 0 to 36 variants the normotensive group, the mean age, body mass (actual values in the current study ranged from 4 to index, BP and serum lipid profile (except high- 20). Linear regression was used to analyze the density lipid-cholesterol) were significantly higher association between the individual risk allele score in the hypertensive group (Po0.05). There were no and the BP. An association with the prevalence of differences in lifestyles between the two groups for hypertension was expressed as the percentage of items such as daily salt intake, energy intake and hypertension in each risk allele score. For the effect percentage of current smokers or alcohol drinkers. size of the risk allele score on the BP, receiver Table 2 shows the results of the genotyping in the operating characteristic (ROC) analysis calculated 735 subjects for the 16 SNPs of the 12 genes. Success the areas under the ROC curve and used the results rates for genotyping of these SNPs were more than to compare the predictive ability of the risk score for 99.8%. The minor frequencies ranged from 0.13 to all 12 loci and for six of the most significant loci. 0.44. Most of the allele frequencies of these variants All analyses were carried out using Statistical were similar to the Japanese data found in the public Package for Social Science (SPSS) for Windows database from the international HapMap Project version 11.0 (SPSS Inc., Chicago, IL, USA). A P-value (http://www.hapmap.org/). There was no deviation o0.05 was considered statistically significant. from the Hardy–Weinberg equilibrium for any of the alleles (data not shown). Results Among all of the genetic loci, new SNPs on the four genotyped genes (COMT, ATP2B1, CYP17A1, Table 1 presents main characteristics of the total CSK) were successfully replicated. Of these, we subjects, and subjects were classified into normo- strongly confirmed that COMT (rs4680 and rs4633), tensive or hypertensive groups. Mean (±s.d.) age ATP2B1 (rs17249754) and CYP17A1 (rs11191548)

Table 2 Association between eight genetic loci and the prevalence of hypertension or blood pressure

Nearby gene SNPs Allele frequency (risk/non-risk) Hypertension SBP DBP

ORs (95% CI) P-value b-value P-value b-value P-value

CYP17A1 rs11191548 C/T (0.82/0.18) 2.50 (1.08–5.56) 0.032* 3.05 0.026* 2.24 0.013* COMT rs4680 A/G (0.31/0.69) 2.40 (1.41–4.08) 0.001** 4.16 o0.001* 2.38 0.001** rs4633 T/C (0.32/0.68) 2.24 (1.32–3.81) 0.003** 9.15 o0.001* 5.96 o0.001** ATP2B1 rs17249754 G/A (0.65/0.35) 2.12 (1.22–3.70) 0.008* 2.29 0.015* 0.97 0.120 CSK rs1378942 A/C (0.82/0.18) 1.99 (0.92–4.31) 0.083 À4.20 0.009* À2.20 0.055 GREB1 rs3792029 C/G (0.81/0.19) 1.36 (0.95–2.94) 0.099 0.61 0.381 0.50 0.273 CDH13 rs11646213 T/A (0.18/0.82) 1.30 (0.67–2.51) 0.434 1.92 0.457 0.36 0.834 PTK2B rs2241649 G/A (0.39/0.61) 1.18 (0.89–1.69) 0.352 À0.19 0.779 0.25 0.574 rs751019 C/A (0.44/0.56) 1.02 (0.71–1.48) 0.903 À0.002 0.973 À0.33 0.480 HPCAL1 rs3771147 G/A (0.76/0.24) 1.01 (0.72–1.42) 0.956 0.90 0.491 0.71 0.413 rs3771148 G/A (0.78/0.22) 1.08 (0.76–1.53) 0.673 0.54 0.689 0.81 0.361 rs3771149 T/G (0.87/0.13) 1.14 (0.69–1.48) 0.944 0.9 0.538 0.006 0.943 ABCA1 rs1800977 T/C (0.26/0.74) 1.13 (0.57–2.26) 0.722 0.005 0.987 À0.86 0.648 ACADSB rs2277249 G/A (0.29/0.71) 1.05 (0.75–1.47) 0.752 0.21 0.749 À0.11 0.805 CYP11B2 rs1799998 C/T (0.34/0.66) 1.79 (1.01–3.23) 0.045* 0.82 0.417 0.24 0.717 PTGIS rs5629 A/C (0.29/0.71) 1.02 (0.73–1.43) 0.916 À3.02 0.021* 1.87 0.030*

Abbreviations: ABCA1, ATP binding cassette A1; ACADSB, short/branched chain acyl-CoA dehydrogenase; ATP2B1, plasma membrane calcium- transporting ATPase 1; CI, confidence interval; CDH13, cadherin 13 preprotein; COMT, catecholamine-O-methyltransferase; CSK, c-src tyrosine kinase; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; CYP11B2, cytochrome P450, family 11, subfamily B, polypeptide 2; DBP, diastolic blood pressure; GREB1, gene regulated by estrogen in breast cancer 1; HPCAL1, hippocalcin-like 1 protein; OR, odds ratio; PTGIS, prostaglandin I2 (prostacyclin) synthase; PTK2B, protein kinase 2b; SBP, systolic blood pressure; SNP, single-nucleotide polymorphism. *Po0.05. **Po0.004 (Because 12 genes were tested, the P-value o0.004 was considered to be statistically significant). For the associations with hypertension, the odds ratios were derived from a logistic regression model adjusted for age, body mass index, salt intake and energy intake levels. The non-risk alleles served as the reference groups. For blood pressure associations, the b-coefficients and P-values were obtained from multiple linear regression analyses adjusted for age, body mass index, salt intake and energy intake levels.

Journal of Human Hypertension Combined impact of 12 variants on hypertension K Miyaki et al 433 were significantly associated with the prevalence of hypertension, with odds ratios of 2.40, 95% con- fidene interval (CI) ¼ 1.41–4.08 (P ¼ 0.001), 2.24, 95% CI ¼ 1.32–3.81 (P ¼ 0.003), 2.12, 95% CI ¼ 1.22–3.70 (P ¼ 0.008) and 2.50, 95% CI ¼ 1.08–5.56 (P ¼ 0.032), respectively, (Table 2). In addition, these polymorphisms were also associated with BP traits. When compared with the AG þ GG group, there were higher adjusted systolic ( þ 4.16 mm Hg, Po0.001) and diastolic ( þ 2.38 mm Hg, P ¼ 0.001) pressures found for the AA genotype of rs4680. We also found that there was a significant interaction between the effect of the high-energy intake level and the prevalence of hypertension for the COMT val158met (rs4680) polymorphism. The prevalence of hypertension had a positive correlation with the energy intake level in the AA group (P for inter- Figure 1 Estimation of the increase of hypertension prevalence action ¼ 0.031). When taken together with the pre- in conjunction with the increasing number of hypertension risk valence of hypertension, the COMT gene was alleles from the combination of 12 SNPs. Percentage of the coincidentally associated with hypertension in our hypertensive subjects in each risk score group are plotted, with the vertical bars representing the 95% CIs. The percentage of study. Because of the strong linkage disequilibrium X 2 hypertension in the greatest risk score group ( 18) is 3.5-fold of between rs4633 and rs4680 (r ¼ 0.91), only rs4680 that observed in the fewest risk score group (p8). was examined in the following analysis. SNPs on ATP2B1, CYP17A1 and CSK were also associated with either SBP or DBP, or with both of them. For the GREB1 gene, there was a significant association with hypertension (odds ratio of 2.26 (95% CI ¼ 1.26–4.05, P ¼ 0.006)) for only the Kanagawa cohort. No significance was noted for either the Kyoto cohort or the combined analysis. The other eight SNPs in the PTK2B, HPCAL1, ACADSB, ABCA1 and CDH13 genes did not show any significant association with either the BP or the prevalence of hypertension (Table 2). For the two polymorphisms we previously reported, the current study partially replicated our previous results. The SNP of CYP11B2 appeared to be associated with a risk for hypertension, whereas the SNP of PTGIS appeared to be related to BP (Table 2). Next, we calculated the cumulative effect of multiple risk-associated SNPs on hypertension or BP by using the data set of the 12 SNPs. For COMT, PTK2B and the HPCAL1 gene, only one SNP of each gene (rs4680, rs751019 and rs3771149; with the r2 for the other SNPs on same gene equal to 0.91, 0.53 and 0.97, respectively) was used because all were in linkage disequilibrium with the other polymorph- isms of the gene on which they were located. As a result, there was a significant linear increase in the percentage of hypertension that correlated with the increasing number of risk alleles (Figure 1, P for slope ¼ 0.002). There was a 3.5-fold variation in the percentage of hypertensive subjects between the lowest (p8) and the greatest risk score groups (X18). Moreover, as the number of risk-associated alleles increased, there was a significant increase in the SBP and DBP (1.36 Â and 0.86 Â )ina Figure 2 Estimation of changes in (a) SBP and (b) DBP scores in stepwise manner (P for trend ¼ 7.84 Â 10À6 and conjunction with increasing numbers of hypertension risk alleles À5 when using a combination of 12 SNPs. Black squares represent 1.85 Â 10 , respectively) (Figure 2). In other words, the mean, whereas the bars extending from the squares indicated if an individual carried one additional risk allele, the 95% CI. The shaded columns represent the number of the SBP and DBP increased to 1.36 mm Hg and individuals in each risk score group.

Journal of Human Hypertension Combined impact of 12 variants on hypertension K Miyaki et al 434 0.86 mm Hg, respectively. This significant combined (c-src tyrosine kinase), LMAN1L (lectin, mannose- impact of the 12 genetic polymorphisms on the binding, 1 like) and ARID3B (encoding AT-rich effect of BP suggested that an accumulation of these interacting domain protein). However, at the present polygenic polymorphisms increased the BP and the time it has yet to be agreed upon as to which of these risk of hypertension in the Japanese population. genes actually contributes to the association with To evaluate the discriminatory power of a genetic BP. Our results are notable in that the association test based on these variants, we calculated the was with SBP, but not with DBP. area under the ROC curve. The area under the curve Although numerous common variants have been for all 12 SNPs studied was 0.571 (95% CI ¼ 0.527– suggested to be disease-associated, these only account 0.616, P ¼ 0.002) for both the SBP and DBP, con- for a small fraction of the genetic variation of the firming the accuracy of this assay. complex traits found in human populations,17 and most of the individual causal risk alleles have genotype relative risks in the range of 1.1 to 2.0.18 Discussion This may be due to the incomplete linkage disequili- brium between the causal variants and the genotyped Our replication study in a general Japanese popula- SNPs, with the exacerbation by the causal variants tion-based cohort confirmed that the COMT, ATP2B1, having a lower minor allele frequency than the SNPs CYP17A1 and CSK genes significantly predicted the already examined.19 However, a recent study that risk of hypertension and were related to BP traits. examined the heritability for human height developed These findings were independent of confounding a linear model analysis that could be used to estimate factors such as age, body mass index, salt intake and the proportion of variance for height related to 294 831 energy intake levels. SNPs. The analyses showed that 45% of the variance COMT is a ubiquitous enzyme that is crucial to the could be explained using the model, which was a metabolism of carcinogenic catechols and catecho- nearly 10-fold increase relative to the published and lamines, and regulation of the human COMT gene validated individual SNPs.19 It has been assumed expression may be important in the pathophysiology that the combined effect of risk variants might of various human disorders, including estrogen- make it possible for us to identify individuals with induced cancers, Parkinson’s disease, depression an accumulated genetic risk.20 In the current study, and hypertension.13 Previous studies have revealed we evaluated not only the individual variants that an association with hypertension10,14,15 and the may be associated with hypertension, but also the current study replicated these findings. ATP2B1 combined impact that they may have on hypertension encodes PMCA1, which is a plasma membrane or BP. The other six SNPs (ACADSB, GREB1, ABCA1, calcium/calmodulin-dependent ATPase that is ex- PTK2B, HPCAL1 and CDH13) were not successfully pressed in vascular endothelium. PMCA1 functions replicated in our study, which may have been due to as a modifier locus for phasic vascular contraction, the relatively small sample size. Even so, we were able and has a critical role in intracellular Ca þþ to identify the significant combined impact of these homeostasis.16 SNP rs11191548 is located on a large 12 genetic polymorphisms on the effect of BP cluster of associated SNPs that span a B430-kb and hypertension, suggesting that accumulation of region at 10q24 and which shows an association in a the polygenic polymorphisms reliably increased the meta-analysis.4 The locus includes six genes, with BP and the risk of hypertension in the Japanese the mutation near to the CYP17A1, AS3MT, CNNM2 population. and NT5C2 gene. The most reasonable candidate is It has been demonstrated that the utility of a CYP17A1, which encodes the cytochrome P450 genetic test may be better validated by the method of enzyme CYP17A1 (also known as P450c17) that is ROC curves, as this methodology provides the responsible for mediating steroid 17a-hydroxylase ability to discriminate between individuals who and 17, 20-lyase activity. The first enzymatic action may or may not be prone to developing the disease.20 is a key step in the biosynthesis of mineralocorti- In the current study, we used the ROC curve to coids and glucocorticoids that affect sodium hand- compare the six SNPs that were confirmed to be ling in the kidney and the second action involves associated with the BP and for the 12 SNPs that were the sex-steroid biosynthesis. In the current analysis, found to have a higher area under the curve (0.571 this SNP appeared to be associated with both vs 0.558 for SBP, 0.571 vs 0.557 for DBP). These the prevalence of hypertension and BP. In fact, our results indicate there is an elevated accuracy of results suggest a much broader mechanism of action discrimination when using this method, although than that reported by Newton-Cheh et al.,4 who only the overall change was quite small in the current found an association with SBP. These researchers case. Regardless, our results showed there was an also reported finding a polymorphism, rs1378942, inadequate discriminatory ability when there was which was in the intron of the CSK gene at 15q24. an area under the curve of 0.571. This suggests This polymorphism was one of a cluster of asso- limited predictive value of these genetic tests for ciated SNPs that spanned B72 kb and that were complex diseases such as hypertension when there strongly associated with DBP.4 Genes in the region is only information on a few variants available. include CYP1A2 (cytochrome P450 enzyme), CSK Although investigations of more common variants

Journal of Human Hypertension Combined impact of 12 variants on hypertension K Miyaki et al 435 may improve the overall accuracy, the potential Conflict of interest limitations of this approach need to be considered. For example, in the well-known large-scale GWAS The authors declare no conflict of interest. that was reported by the Wellcome Trust Case Control Consortium,21 none of the variants that were pre- viously associated with hypertension showed any evidence for association. Thus, when dealing with Acknowledgements complicated diseases, in addition to genetic factors This study was supported by Grant-in-Aid for that may contribute to the pathogenesis and develop- Scientific research (B) (19390173) from the Ministry ment of the disease, environmental factors, such as of Education, Culture, Sports, Science and Technol- dietary energy and salt intake levels, as well as the ogy (to Dr Miyaki) and Grant-in-Aid for Scientific interaction of genetic and environmental factors, also research (C) (22590547) from Ministry of Education, need to be considered when evaluating the suscept- Culture, Sports, Science and Technology (to Dr ibility. In the present study, SNP rs4680 in the COMT Muramatsu). gene showed an interaction between the genotype and dietary energy intake that appeared to be associated with hypertension. 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