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Introduction

Hypertension remains a global public health problem, as it is an important risk factor for coronary artery disease (CAD), ischemic and hemorrhagic stroke and end-stage renal disease (1). Thus, prevention of hypertension may be an important strategy to reduce the overall burden of cardiovascular, cerebrovascular and renal diseases. Recent meta-analyses of genome-wide association studies (GWASs) for CAD or myocardial infarction in Caucasian populations identified various loci and genes that confer susceptibility to CAD (2,3). As hypertension is a major risk factor of CAD, certain polymorphisms may contribute to the genetic susceptibility to CAD through affecting the predisposition to hypertension. The aim of the present study was to examine a possible association of hypertension in Japanese individuals with 29 single-nucleotide polymorphisms (SNPs) previously identified by the meta-analyses of GWASs as susceptibility loci for CAD or myocardial infarction in Caucasian populations.

Materials and methods

Study population

Study subjects comprised of 5,460 Japanese individuals (3,348 subjects with hypertension and 2,112 controls) that visited outpatient clinics or were admitted to participating hospitals (Inabe General Hospital, Inabe; Gifu Prefectural General Medical Center, Gifu; Gifu Prefectural Tajimi Hospital, Tajimi; Japanese Red Cross Nagoya First Hospital, Nagoya; Hirosaki University Hospital and Hirosaki Stroke Center, Hirosaki, Japan) between 2002 and 2012. The subjects with hypertension had a systolic blood pressure (BP) of ≥140 mmHg or a diastolic BP of ≥90 mmHg (or both) or had taken antihypertensive medication. The control individuals had a systolic BP of <140 mmHg and a diastolic BP of <90 mmHg and no history of hypertension or of taking antihypertensive medication. BP was measured at least twice while subjects were relaxed in a sitting position for >5 min. The measurements were recorded by a skilled physician or a nurse according to the guidelines of the American Heart Association (4). The study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committees in each participating hospital. Written informed consent was obtained from all the subjects.

Selection of polymorphisms

SNPs that were shown to be significantly associated with CAD or myocardial infarction were searched in Caucasian populations by the meta-analyses of GWASs (2,3). These SNPs were examined with the dbSNP database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/SNP/. Accessed March 3, 2013.) to find SNPs with a minor allele frequency of ≥0.015 in a Japanese population. In total, 29 SNPs (data not shown) were finally selected and a possible association with hypertension was examined.

Genotyping of polymorphisms

Venous blood (7 ml) was collected into tubes containing 50 mmol/l ethylenediaminetetraacetic acid (disodium salt), the peripheral blood leukocytes were isolated and genomic DNA was extracted from these cells with a DNA Extraction kit (Genomix; Talent Srl, Trieste, Italy). The SNP genotypes were determined at G&G Science Co., Ltd., (Fukushima, Japan) by a method that combined polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology (Luminex, Austin, TX, USA) as described previously (5–7). Detailed genotyping methodology was also described previously (8).

Statistical analysis

Quantitative data were compared between the subjects with hypertension and controls by the unpaired Student's t-test. Categorical data were compared by the χ2 test. Allele frequencies were estimated by the gene counting method. Departure from the Hardy-Weinberg equilibrium was examined by the χ2 test. Multivariable logistic regression analysis was performed with hypertension as a dependent variable, independent variables, including age, gender (0, female; 1, male), body mass index (BMI), smoking status (0, non-smoker; 1, current or former smoker), and each genotype; and the P-value, odds ratio and 95% confidence interval were calculated. Genotypes of each polymorphism were assessed according to dominant (0, wild-type homozygote; 1, heterozygote and variant homozygote), recessive (0, wild-type homozygote and heterozygote; 1, variant homozygote) and additive genetic models. Additive models comprised additive 1 (heterozygotes vs. wild-type homozygotes) and additive 2 (variant homozygotes vs. wild-type homozygotes) models, which were analyzed simultaneously with a single statistical model. P<0.05 was considered to indicate a statistically significant difference. Statistical test was performed with JMP version 5.1 and JMP Genomics version 6.0 software (SAS Institute, Inc., Cary, NC, USA).

Results

Patient characteristics

Characteristics of the 5,460 subjects are shown in Table I. Age, the frequency of males, BMI, the prevalence of smoking, ischemic stroke, CAD, diabetes mellitus and dyslipidemia, as well as serum concentrations of creatinine and triglycerides and fasting plasma glucose level were greater, whereas estimated glomerular filtration rate and serum concentrations of high-density lipoprotein-cholesterol were lower in the subjects with hypertension than in controls.

Table I Characteristics of 5,460 study subjects.

Table I

Characteristics of 5,460 study subjects.

Characteristics	Hypertension (n=3,348)	Controls (n=2,112)	P-values
Age, years	66.2±10.2	61.9±12.1	<0.0001
Gender, men/women, %	63.9/36.1	55.0/45.0	<0.0001
Body mass index, kg/m2	24.0±3.4	23.5±2.9	<0.0001
Current or former smoker, %	25.5	21.7	0.0017
Ischemic stroke, %	20.1	10.5	<0.0001
Coronary artery disease, %	54.2	30.6	<0.0001
Systolic blood pressure, mmHg	151.6±25.5	120.4±12.7	<0.0001
Diastolic blood pressure, mmHg	80.8±16.0	69.1±10.0	<0.0001
Serum creatinine, µmol/l	98.8±126.5	71.0±26.7	<0.0001
Estimated GFR, ml/min/1.73 m2	65.8±24.9	73.7±22.1	<0.0001
Diabetes mellitus, %	43.8	21.4	<0.0001
Fasting plasma glucose, mmol/l	7.41±3.48	6.40±3.17	<0.0001
Dyslipidemia, %	51.3	30.1	<0.0001
Serum triglycerides, mmol/l	1.70±1.13	1.51±1.21	<0.0001
Serum HDL-cholesterol, mmol/l	1.28±0.38	1.39±0.41	<0.0001
Serum LDL-cholesterol, mmol/l	3.12±0.93	3.08±0.90	0.1592

[i] Quantitative data are expressed as mean ± standard deviation. Hypertension, systolic blood pressure of ≥140 mmHg, diastolic blood pressure of ≥90 mmHg, or taking antihypertensive medication; diabetes mellitus, fasting plasma glucose concentration of ≥6.93 mmol/l or taking antidiabetic medication; dyslipidemia, serum concentration of triglycerides of ≥1.65 mmol/l, serum concentration of high-density lipoprotein (HDL)-cholesterol of <1.04 mmol/l, serum concentration of low-density lipoprotein (LDL)-cholesterol of ≥3.64 mmol/l, or taking antidyslipidemic medication; estimated glomerular filtration rate (GFR) (ml/min/1.73 m²) = 194 × [age (years)]^−0.287 × [serum creatinine (mg/dl)]^−1.094 × (0.739 when female).

Comparisons of the genotype distributions or allele frequencies

On the basis of the genotype distribution or allele frequency comparisons by the χ2 test, 4 SNPs (including 2 of borderline significance) were associated with the prevalence of hypertension (Table II). The genotype distributions and allele frequencies for rs12190287 of the transcription factor 21 gene (TCF21) and rs1122608 of the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 gene (SMARCA4) were significantly (P<0.05) associated with the prevalence of hypertension. The allele frequencies for rs9369640 of the phosphatase and actin regulator 1 gene (PHACTR1) and genotype distributions for rs599839 of the proline/serine-rich coiled-coil 1 gene (PSRC1) were also significantly associated with hypertension. These SNPs with a P<0.05 in genotype distributions or allele frequencies by the χ2 test were further examined by multivariable logistic regression analysis with adjustment for covariates.

Table II Comparisons of genotype distributions and allele frequencies of rs12190287, rs1122608, rs9369640 or rs599839 by the χ2 test between the subjects with hypertension and controls.

Table II

Comparisons of genotype distributions and allele frequencies of rs12190287, rs1122608, rs9369640 or rs599839 by the χ2 test between the subjects with hypertension and controls.

Gene	Polymorphism	dbSNP	Hypertension, %	Controls, %	P (genotype)	P (allele)
TCF21	G→C	rs12190287			0.0132	0.0032
	GG		563 (16.8)	402 (19.0)
	GC		1595 (47.6)	1033 (48.9)
	CC		1190 (35.5)	677 (32.1)
	Hardy-Weinberg P		0.4674	0.8215
SMARCA4	G→T	rs1122608			0.0442	0.0143
	GG		2636 (78.8)	1601 (75.9)
	GT		674 (20.1)	481 (22.8)
	TT		37 (1.1)	28 (1.3)
	Hardy-Weinberg P		0.7006	0.2267
PHACTR1	A→C	rs9369640			0.0572	0.0161
	AA		2825 (84.5)	1730 (82.1)
	AC		494 (14.8)	357 (16.9)
	CC		25 (0.7)	21 (1.0)
	Hardy-Weinberg P		0.5057	0.5901
PSRC1	A→G	rs599839			0.0382	0.0681
	AA		2853 (85.8)	1761 (83.6)
	AG		446 (13.4)	332 (15.8)
	GG		26 (0.8)	12 (0.6)
	Hardy-Weinberg P		0.0657	0.3902

[i] dbSNP, the single nucleotide polymorphism database; TCF21, transcription factor 21; Hardy-Weinberg P, a P-value of Hardy-Weinberg equilibrium; SMARCA4, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4; PHACTR1, phosphatase and actin regulator 1; PSRC1, proline/serine-rich coiled-coil 1.

Multivariable logistic regression analysis with adjustment for age, gender, BMI and smoking status revealed that rs12190287 of TCF21 (dominant, recessive and additive 2 models) was significantly (P<0.05) associated with hypertension, and the C allele represented a risk factor for this condition (Table III). Similar analyses revealed that rs1122608 of SMARCA4, rs9369640 of PHACTR1 and rs599839 of PSRC1 (dominant and additive 1 models) were also associated with hypertension, with the minor T, C and G alleles, respectively, protecting against this condition.

Table III Multivariable logistic regression analysis for the association of rs12190287, rs1122608, rs9369640 or rs599839 with hypertension with adjustment for age, gender, body mass index and smoking status.

Table III

Multivariable logistic regression analysis for the association of rs12190287, rs1122608, rs9369640 or rs599839 with hypertension with adjustment for age, gender, body mass index and smoking status.

		Dominant		Recessive		Additive 1		Additive 2
Gene	Polymorphism	P-value	OR (95% CI)	P-value	OR (95% CI)	P-value	OR (95% CI)	P-value	OR (95% CI)
TCF21	rs12190287 (G→C)	0.0168a	1.20 (1.03–1.38)	0.0014a	1.21 (1.08–1.37)	0.1620		0.0010a	1.32 (1.12–1.55)
SMARCA4	rs1122608 (G→T)	0.0305a	0.86 (0.75–0.99)	0.7103		0.0330a	0.86 (0.75–0.99)	0.6202
PHACTR1	rs9369640 (A→C)	0.0119a	0.82 (0.71–0.96)	0.2908		0.0190a	0.83 (0.71–0.97)	0.2497
PSRC1	rs599839 (A→G)	0.0248a	0.84 (0.72–0.98)	0.2232		0.0110a	0.81 (0.69–0.95)	0.2567

a P<0.05. OR, odds ratio; CI, confidence interval; TCF21, transcription factor 21; SMARCA4, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4; PHACTR1, phosphatase and actin regulator 1; PSRC1, proline/serine-rich coiled-coil 1.

Finally, the association of rs12190287, rs1122608, rs9369640 or rs599839 with systolic or diastolic BP was examined among all the individuals (Table IV). There were no significant differences in systolic or diastolic BP among the genotypes of these SNPs.

Table IV Association of systolic or diastolic blood pressure (BP) with the TCF21, SMARCA4, PHACTR1 or PSRC1 genotypes in all the subjects.

Table IV

Association of systolic or diastolic blood pressure (BP) with the TCF21, SMARCA4, PHACTR1 or PSRC1 genotypes in all the subjects.

Variables		Genotype		P (ANOVA)	P (dominant)	P (recessive)
TCF21 (rs12190287)	GG	GC	CC
No. of subjects	687	1916	1344
Systolic BP, mmHg	143±25	143±27	144±27	0.8477	0.8928	0.5673
Diastolic BP, mmHg	77±16	78±15	78±16	0.5614	0.4006	0.3632
SMARCA4 (rs1122608)	GG	GT	TT
No. of subjects	3056	840	43
Systolic BP, mmHg	144±27	143±27	141±24	0.8213	0.6111	0.6456
Diastolic BP, mmHg	78±15	78±16	77±15	0.9333	0.8666	0.7701
PHACTR1 (rs9369640)	AA	AC	CC
No. of subjects	3340	569	26
Systolic BP, mmHg	143±27	144±26	143±30	0.9155	0.6785	0.9906
Diastolic BP, mmHg	78±16	77±14	76±12	0.3185	0.1406	0.5346
PSRC1 (rs599839)	AA	AG	GG
No. of subjects	3363	523	29
Systolic BP, mmHg	143±26	144±28	147±24	0.8109	0.7541	0.5351
Diastolic BP, mmHg	78±15	78±16	76±11	0.7536	0.8679	0.4957

[i] TCF21, transcription factor 21; SMARCA4, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4; PHACTR1, phosphatase and actin regulator 1; PSRC1, proline/serine-rich coiled-coil 1; ANOVA, analysis of variance.

Discussion

Although GWASs have implicated various loci and genes in predisposition to hypertension in Caucasian (9–13) or African-Americans populations (14), the genes that confer susceptibility to this condition in the Japanese population remain to be definitively identified. The associations of the 29 SNPs identified by the meta-analyses of GWASs for CAD in Caucasian populations (2,3) to hypertension were examined in 5,460 Japanese individuals. The large-scale association study revealed that rs12190287 (G→C) of TCF21 was significantly associated with the prevalence of hypertension in the Japanese population with the C allele representing a risk factor for this condition. The rs1122608 of SMARCA4, rs9369640 of PHACTR1 and rs599839 of PSRC1 were also associated with hypertension, and the minor T, C and G alleles, respectively, were protective against this condition.

TCF21 is a class II basic helix-loop-helix transcription factor, which is expressed in the mesenchyme of developing organs, including lung, kidney and epicardium (15). TCF21 binds specific DNA sequences and negatively regulates various cell differentiation through modulating cell cycle arrest and tissue-specific gene expression (15), indicating that TCF21 has a role in the development of malignant tumors (16). Expression quantitative trait locus (eQTL) data and a reporter gene assay showed that the C allele of rs12190287 increased the expression of TCF21 in vascular smooth muscle cells (VSMCs) (2,17). An in vitro study of cultured MG63 cells showed that expression of the cyclin-dependent kinase inhibitor, P21, was reduced when TCF21 was introduced (15), indicating that TCF21 functions as a negative regulator of P21, which is crucial in VSMC proliferation (18). As microvascular structural narrowing caused by aberrant proliferation of VSMCs may play an important role in the development of hypertension (19), altered TCF21 expression may affect BP trait through P21-dependent microvascular remodeling.

SMRACA4 is located adjacent to the low-density lipoprotein receptor gene and regulates the transcription of various genes through disrupting chromatin structure using the chemical energy of adenosine triphosphate hydrolysis (20). Previous eQTL data indicated that the expression of SMARCA4 is likely modulated by rs1122608 (2). A possible regulating role of SMARCA4 in P21 expression and in VSMCs differentiation through myocardin-mediated pathway has also been reported (21,22).

PHACTR1 is expressed predominantly in neuronal tissues in brain (23). Although the precise function of PHACTR1 remains unclear, associations with cell migration, motility and invasiveness in tumor tissues have been reported (24). PHACTR1 may affect the activity of phosphatase 1, which is indicated to be involved in the regulation of nitric oxide synthesis in endothelial cells (25), through modulating G-actin binding affinity to its RPEL motif (23).

PSRC1 is a microtubules-associated protein that directly binds microtubules and controls the density, assembly and dynamics of microtubules, indicating that it plays a key role in the regulation of chromosome congression and segregation during mitosis (26). PSRC1 was also shown to be a susceptibility locus for a plasma concentration of low-density lipoprotein-cholesterol in a previous GWAS (27).

Although 4 SNPs were significantly associated with the prevalence of hypertension, genotypes of these polymorphisms were not associated with systolic or diastolic BP among all the subjects. These results did not change when untreated hypertensive subjects or control individuals were examined separately (data not shown). Although the reason for this discrepancy remains unclear, there were certain possibilities: i) The number of hypertensive subjects not taking antihypertensive medication was small; ii) information for drug compliance obtained by a questionnaire was incomplete; and iii) a substantial proportion of the subjects had white-coat hypertension.

The present study had several limitations: i) As the results of the present study were not replicated, validation of the findings is required in other independent subject panels or in other ethnic groups; ii) it is possible that 4 SNPs identified in the present study are in linkage disequilibrium with other polymorphisms in the same gene or in other nearby genes that are actually responsible for the development of hypertension; and iii) the functional relevance of rs12190287, rs1122608, rs9369640 or rs599839 to pathogenesis of hypertension remains to be elucidated.

In conclusion, rs12190287 (G→C) of TCF21 may be a susceptibility locus for hypertension in Japanese individuals. The rs1122608 of SMARCA4, rs9369640 of PHACTR1 and rs599839 of PSRC1 were also associated with hypertension. Determination of genotypes of these SNPs may prove informative for assessment of the genetic risk for hypertension in Japanese individuals.

Acknowledgements

The present study was supported by a Collaborative Research Grant from the Gifu Prefectural General Medical Center (no. H24-26 to Y.Y.) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 24590746 to Y.Y.).

References