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Introduction

Hypertension is a complex multifactorial disorder that is believed to result from an interaction between the genetic background of an individual and various environmental factors (1). As hypertension is a significant risk factor for coronary artery disease, stroke and end-stage renal disease, prevention of hypertension in individuals is a crucial aim. One approach to prevention and selection of the most appropriate hypertension treatment is to identify disease susceptibility genes. Previous genome-wide association studies (GWAS) have implicated various loci and genes in predisposition to hypertension in Caucasian and African-American populations (2–7). An adducin 2 gene polymorphism (rs3755351) was shown to be a susceptibility locus for hypertension in Japanese individuals (8), however, the genes that confer susceptibility to this condition in Japanese individuals remains to be definitively identified.

Our previous GWAS showed that the C→T polymorphism (rs6929846) in the 5′ untranslated region of the butyrophilin, subfamily 2, member A1 (BTN2A1) gene was significantly associated with myocardial infarction in Japanese individuals (9). As hypertension is a major risk factor for the development of atherosclerotic disease, including myocardial infarction, it was hypothesized that the rs6929846 polymorphism of BTN2A1 may contribute to the genetic susceptibility to myocardial infarction through affecting the predisposition to hypertension. The aim of the present study was to examine a possible association of rs6929846 with hypertension in community-dwelling Japanese individuals.

Patients and methods

Study population

The study subjects comprised 5,959 community-dwelling Japanese individuals (2,183 subjects with hypertension, 3,776 controls) who were recruited to a population-based cohort study in Inabe (Mie, Japan) between March 2010 and September 2012 (Inabe Health and Longevity Study). The subjects with hypertension had a systolic blood pressure (BP) and/or diastolic BP of ≥140 or ≥90 mmHg, respectively, or were not on antihypertensive medication. Individuals with valvular heart disease, congenital malformations of the heart or vessels, or renal or endocrinological diseases that cause secondary hypertension were excluded from the study. The control individuals had systolic BP and diastolic BP of <140 and <90 mmHg, respectively, and no history of hypertension or of antihypertensive medication. BP was measured at least twice with subjects having rested in the sitting position for >5 min. The measurements were documented by a skilled physician or a nurse according to the guidelines of the American Heart Association (10). The study protocol complied with the Declaration of Helsinki and was approved by the Committees for the Ethics of Human Research of Mie University Graduate School of Medicine and Inabe General Hospital. Written informed consent was obtained from all the subjects.

Polymorphism genotyping

Venous blood (5 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 (SMITEST EX-R&D; Medical and Biological Laboratories, Co., Ltd., Nagoya, Japan). Genotypes of rs6929846 were determined at G&G Science Co., Ltd., (Fukushima, Japan) by a method that combined the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology (Luminex, Austin, TX, USA), as described previously (11,12). Detailed genotyping methodology was also described previously (13).

Statistical analysis

Quantitative data were compared between the subjects with hypertension and the controls by the unpaired Student’s t-test. Categorical data were compared by the χ2 test. The gene counting method estimated the allele frequencies and the χ2 test was used to identify departure from Hardy-Weinberg equilibrium. Multivariable logistic regression analysis was performed with hypertension as a dependent variable and independent variables, including age, gender (0, female; 1, male), body mass index (BMI), smoking status (0, non-smoker; 1, current or former smoker) and rs6929846 genotype; and the P-value, odds ratio (OR) and 95% confidence interval (CI) were calculated. The rs6929846 genotype was assessed according to dominant (0, wild-type homozygotes; 1, the combined group of variant homozygotes and heterozygotes) and recessive (0, the combined group of wild-type homozygotes and heterozygotes; 1, variant homozygotes) genetic models. P<0.05 was considered to indicate a statistically significant difference. Statistical tests were performed with the JMP 5.1 software (SAS Institute, Inc., Cary, NC, USA).

Results

Characteristics

The characteristics of the 5,959 study subjects are shown in Table I. Age, the frequency of males, BMI, the prevalence of smoking, coronary artery disease, stroke, dyslipidemia and diabetes mellitus, as well as serum concentrations of triglycerides and creatinine, fasting plasma glucose level and blood glycosylated hemoglobin (hemoglobin A1c) content, were increased, whereas serum concentrations of high-density lipoprotein (HDL)-cholesterol were lower, in subjects with hypertension compared to the controls.

Table I Characteristics of 5,959 study subjects.

Table I

Characteristics of 5,959 study subjects.

Characteristic	Hypertension	Control	P-value
Subjects, n	2183	3776
Age, years	60.9±10.6	50.1±12.4	<0.0001
Gender, male/female (%)	62.3/37.7	48.6/51.4	<0.0001
Body mass index, kg/m2	24.1±3.6	22.3±3.1	<0.0001
Current or former smoker, %	47.7	44.5	0.0161
Coronary artery disease, %	4.6	0.8	<0.0001
Stroke, ischemic and hemorrhagic, %	4.1	1.1	<0.0001
Systolic blood pressure, mmHg	133±15	113±11	<0.0001
Diastolic blood pressure, mmHg	83±12	70±10	<0.0001
Mean blood pressure, mmHg	99±12	84±9	<0.0001
Pulse pressure, mmHg	50±13	43±9	<0.0001
Dyslipidemia, %	76.6	54.8	<0.0001
Serum total cholesterol, mmol/l	5.23±0.88	5.24±0.88	0.5910
Serum triglycerides, mmol/l	1.44±0.96	1.21±0.78	<0.0001
Serum HDL-cholesterol, mmol/l	1.60±0.44	1.70±0.45	<0.0001
Serum LDL-cholesterol, mmol/l	3.17±0.79	3.19±0.81	0.2140
Diabetes mellitus, %	20.1	6.7	<0.0001
Fasting plasma glucose, mmol/l	5.83±1.26	5.40±0.96	<0.0001
Blood hemoglobin A1c, %	5.83±0.73	5.59±0.59	<0.0001
Serum creatinine, μmol/l	70.5±20.3	64.7±13.9	<0.0001

[i] Quantitative data are mean ± standard deviation. Dyslipidemia, serum triglyceride concentration ≥1.65 mmol/l, serum high-density lipoprotein (HDL)-cholesterol <1.04 mmol/l, serum low-density lipoprotein (LDL)-cholesterol ≥3.64 mmol/l or undergoing antidyslipidemic medication; diabetes mellitus, fasting plasma glucose concentration ≥6.93 mmol/l, hemoglobin A_1c content ≥6.5% or undergoing antidiabetes medication.

rs6929846 associations

Comparisons of the genotype distributions and allele frequencies by the χ2 test between the subjects with hypertension and the controls revealed that rs6929846 of the BTN2A1 gene was significantly (P<0.05) associated with hypertension (Table II). The genotype distributions of rs6929846 were in Hardy-Weinberg equilibrium among the subjects with hypertension and the controls (Table II).

Table II Comparison of the genotype distributions and allele frequencies of the butyrophilin, subfamily 2, member A1 (BTN2A1) gene polymorphism, rs6929846, by the χ2 test between subjects with hypertension and the controls.

Table II

Comparison of the genotype distributions and allele frequencies of the butyrophilin, subfamily 2, member A1 (BTN2A1) gene polymorphism, rs6929846, by the χ2 test between subjects with hypertension and the controls.

Gene	Polymorphism	Hypertension (%)	Controls (%)	P-value (genotype)	P-value (allele)
BTN2A1	rs6929846			0.0090	0.0051
	CC	1693 (77.6)	3051 (80.8)
	CT	462 (21.2)	677 (17.9)
	TT	28 (1.3)	47 (1.2)
Hardy-Weinberg P-value		0.5765	0.1754

Multivariable logistic regression analysis with adjustment for age, gender, BMI and smoking status revealed that rs6929846 (dominant model) was significantly associated with hypertension, with the minor T allele representing a risk factor (Table III).

Table III Multivariable logistic regression analysis of the BTN2A1 polymorphism, rs6929846, and hypertension.

Table III

Multivariable logistic regression analysis of the BTN2A1 polymorphism, rs6929846, and hypertension.

		Dominant		Recessive
Gene	Polymorphism (C→T)	P-value	OR (95% CI)	P-value	OR (95% CI)
BTN2A1	rs6929846	0.0008	1.29 (1.11–1.50)	0.8582	1.05 (0.60–1.79)

[i] Multivariable logistic regression analysis was performed with adjustment for age, gender, body mass index and smoking status. BTN2A1, butyrophilin, subfamily 2, member A1 gene. OR, odds ratio; CI, confidence interval.

The association of rs6929846 with systolic, diastolic or mean BP, or pulse pressure was also examined among all the individuals or individuals not on antihypertensive medication. Among all the individuals, systolic, diastolic and mean BP was significantly higher in the combined group of individuals with the CT or TT genotype compared to those with the CC genotype. Among the individuals not on antihypertensive medication, diastolic and mean, but not systolic BP, were significantly higher in the combined group of individuals with the CT or TT compared to those with the CC genotype. There was no significant difference in pulse pressure between the rs6929846 genotypes (Table IV).

Table IV Association of the BTN2A1 polymorphism, rs6929846, with systolic, diastolic or mean blood pressure, or pulse pressure.

Table IV

Association of the BTN2A1 polymorphism, rs6929846, with systolic, diastolic or mean blood pressure, or pulse pressure.

rs6929846 individuals	Genotype			P-value (CC vs. CT+TT)	P-value (CC+CT vs. TT)
	CC	CT	TT
All
Subjects, n	4721	1134	75
Systolic blood pressure, mmHg	120±16	121±16	123±16	0.0390a	0.1100
Diastolic blood pressure, mmHg	74±12	75±12	76±12	0.0045a	0.4342
Mean blood pressure, mmHg	90±12	91±13	92±12	0.0069a	0.2389
Pulse pressure, mmHg	46±11	46±11	48±11	0.8956	0.1430
Not on antihypertensive medication
Subjects, n	3709	850	59
Systolic blood pressure, mmHg	117±15	118±16	120±16	0.0657	0.1577
Diastolic blood pressure, mmHg	73±12	74±12	75±11	0.0067a	0.3204
Mean blood pressure, mmHg	88±12	89±13	90±12	0.0116a	0.2220
Pulse pressure, mmHg	45±10	44±10	46±10	0.6717	0.3445

a P<0.05. Data for blood pressure and pulse pressure are mean ± standard deviation.

{ label (or @symbol) needed for fn[@id='tfn4-br-02-06-0818'] } BTN2A1, butyrophilin, subfamily 2, member A1 gene.

Discussion

The present study has shown that the rs6929846 polymorphism of the BTN2A1 gene was significantly associated with the prevalence of hypertension in community-dwelling Japanese individuals, with the minor T allele representing a risk factor for this condition. Our previous study showed that rs6929846 was significantly associated with hypertension in a different hospital-based study population (14). The results of the present population-based study are consistent with the previous observations in the hospital-based study (14) and validated the association of rs6929846 in BTN2A1 with hypertension.

BTN2A1 is a cell surface transmembrane glycoprotein that is a member of the butyrophilin superfamily. The butyrophilin family was originally identified due to its ability to induce the production of milk fat globules (15), however, a number of proteins belonging to the butyrophilin and butyrophilin-like families were shown to regulate immune function and polymorphisms in the protein coding sequences were associated with the predisposition to inflammatory diseases (16). Our previous study showed that the T allele of the rs6929846 polymorphism of the BTN2A1 gene was associated with an increased risk of myocardial infarction and with an increased transcription activity of BTN2A1 (9). The serum high sensitivity C-reactive protein concentrations were significantly increased in the individuals in the combined group of CT or TT compared to those with the CC genotype among healthy individuals without neoplastic, infectious or inflammatory disease (9,17). Therefore, the T allele of rs6929846 may accelerate the inflammatory processes.

Previous studies have indicated that chronic vascular inflammation influences BP and vascular remodeling (18–21). Systolic and diastolic BP and pulse pressure have been shown to be positively associated with interleukin-6 plasma concentrations in healthy males (18). Plasma concentrations of high sensitivity C-reactive protein were greater in individuals with hypertension compared to those with normal BP, and were positively correlated with systolic BP and pulse pressure (19). Oxidative stress and vascular inflammation have been shown to affect BP, and chronic inflammation may play a critical role in the pathogenesis of hypertension (20,21). The present study showed that rs6929846 of BTN2A1 was significantly associated with hypertension, with the minor T allele representing a risk factor for this condition. The enhancement of chronic inflammation by the T allele of rs6929846 may account for its association with hypertension, although the molecular mechanism of the affect of rs6929846 on the development of hypertension remains to be elucidated.

In the results of the present study, systolic, diastolic or mean BP were increased by 2–3 mmHg in individuals with the TT genotype compared to those with the CC genotype. Such a difference is small at the individual level and may not have practical clinical implications. However, this increase in BP is important at the population level, due to the high incidence of coronary artery disease and stroke. The reduction in mortality estimated for each 2-mmHg decrease in systolic BP is 4% for coronary artery disease and 6% for stroke (22). Small differences in the average BP indicate significant differences in population mortality (22). In Japan, the reduction of morbidity or mortality estimated for each 2-mmHg decrease in systolic BP for coronary artery disease is 5.4% (5,367 individuals) or 4.0% (3,944 individuals), respectively, and for stroke it is 6.4% (19,757 individuals) or 3.0% (9,127 individuals), respectively (23). Therefore, identification of genetic variants that contribute to the increased risk of hypertension is clinically important.

There were limitations in the present study: i) As the study subjects comprised of only Japanese individuals, further study is required in other ethnic groups. ii) rs6929846 of BTN2A1 is possibly in linkage disequilibrium with other polymorphisms in BTN2A1 or in other nearby genes, which are responsible for hypertension development. iii) The functional relevance of rs6929846 of BTN2A1 to pathogenesis of hypertension remains unclear.

In conclusion, the results of the present study indicate that BTN2A1 may be a susceptibility gene for hypertension in Japanese individuals. Determining the rs6929846 genotype may provide genetic risk assessment informative for hypertension. As multiple variants, which each have a small effect, are believed to be responsible for a large fraction of the genetic component of essential hypertension, further identification of hypertension susceptibility genes will allow more accurate assessment of the genetic component of this condition.

Acknowledgements

The present study was supported by research grants from the Japan Health Foundation and Okasan Kato Culture Promotion Foundation (to Y.Y.).

References