Journal of Human Hypertension (2002) 16, 789–793 & 2002 Nature Publishing Group All rights reserved 0950-9240/02 $25.00 www.nature.com/jhh ORIGINAL ARTICLE Positive association of CYP11B2 gene polymorphism with genetic predisposition to essential hypertension K Tsukada1, T Ishimitsu1, M Teranishi1, M Saitoh1, M Yoshii1, H Inada1, S Ohta1, M Akashi1, J Minami1, H Ono1, M Ohrui2 and H Matsuoka1 1Department of Hypertension and Cardiorenal Medicine; 2Department of Health Care, Dokkyo University School of Medicine, Mibu, Tochigi, Japan Predispositions to essential hypertension and cardio- polymorphism was determined in essential hyperten- vascular diseases are possibly associated with gene sion subjects (n ¼ 250) and normotensive subjects polymorphisms of the renin–angiotensin system. Gene (n ¼ 221). The distributions of three genotypes (TT, TC, polymorphisms of angiotensinogen and angiotensin- and CC) were significantly different between the hyper- converting enzyme genes have been suggested to be tensive and the normotensive groups (v2 ¼ 9.61, risk factors for hypertension and myocardial infarction. P ¼ 0.008). Namely, the frequency of C allele was higher Concerning the polymorphism of aldosterone synthase in the hypertensive patients than in the normotensive (CYP11B2) gene, earlier studies have shown inconsis- subjects (34.2 vs 26.5%, P ¼ 0.010). Our data suggest tent results in terms of its relation to hypertension. In that the À344C allele of CYP11B2 gene polymorphism is the present case–control study, we investigated the associated with the genetic predisposition to develop association of À344T/C polymorphism in the promoter essential hypertension. region of human CYP11B2 gene with genetic predis- Journal of Human Hypertension (2002) 16, 789–793. position to hypertension. The genotype of À344T/C doi:10.1038/sj.jhh.1001484 Keywords: essential hypertension; gene polymorphism; aldosterone Introduction such as hypertension and myocardial infarction.6 However, the results of earlier studies are not always Aldosterone is the principal mineralocorticoid hor- consistent. Differences in background characteris- mone, which plays a major role in regulating sodium 1 tics of the study subjects such as ethnicity and balance and volume homeostasis. Human aldoster- selection criteria may be responsible for this incon- one synthase (CYP11B2) is a cytochrome P450 sistency. enzyme, which catalyses the terminal steps of In the present study, we investigated the À344T/C aldosterone synthesis in the zona glomerulosa cells 2,3 polymorphism of the CYP11B2 gene in carefully of the adrenal. Therefore, genetic variations of the selected normotensive subjects and patients with CYP11B2 gene that affect its expression may essential hypertension, and thereby evaluated the influence the development of salt-sensitive hyper- association of the genotypes with genetic predis- tension. Several common polymorphisms have been position to essential hypertension. described in the CYP11B2 gene.4,5 Among them, the À344T/C polymorphism, which is located at a putative binding site for the steroidogenic transcrip- tion factor (SF-1), has been recently attracting Subjects and methods interest with regard to its relation to cardiovascular Subjects diseases. Several studies have suggested that the C allele of this gene polymorphism is associated with A group of 250 patients with essential hypertension genetic predisposition to cardiovascular diseases (EH) (154 men and 96 women), aged 53 7 11 years, who had developed hypertension before the age of 50 years were selected from the outpatients clinic of Correspondence: Dr T Ishimitsu, Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Dokkyo University School of Medicine Hospital. Mibu, Tochigi 321-0293, Japan. Hypertension was defined as systolic blood pres- E-mail: [email protected] sure exceeding 140 mmHg and/or diastolic blood CYP11B2 gene polymorphism in hypertension K Tsukada et al 790 pressure exceeding 90 mmHg on two or more con- amplified fragments were digested with HaeIII secutive visits.7,8 Individuals diagnosed as having restriction enzyme (New England BioLabs, MA, secondary causes of hypertension were excluded. USA) and were subjected to electrophoresis on The normotensive control group (NT) included 221 2.0% agarose gels. HaeIII digestion of the 538 bp healthy subjects (144 men and 77 women), aged PCK product yields 274, 138 and 126 bp fragments. 56 7 5 years, who were older than 50 years and free The existence of À344C creates an additional from a history of any cardiovascular diseases. They recognition site of HaeIII resulting in digestion of were recruited from participants of the health check the 274 bp fragment into 203 and 71 bp fragments. programme of Dokkyo University School of Medi- Fragments of 274 bp (T allele) and of 203 and 71 bp cine Hospital. The subjects were considered to be (C allele) were detected. normotensive when their blood pressure was lower than 140 mmHg in systole and 90 mmHg in diastole on multiple occasions. Blood pressure was mea- Statistical analysis sured by a sphygmomanometer, with the subjects in 7 a sitting position after a 10-min rest. All subjects Data are presented as means s.d. Clinical char- were native Japanese and unrelated to each other. acteristics between the two groups were compared Besides the routine laboratory tests, plasma renin by unpaired Student’s t-test for parametric data and by w2 test for categoric data. The allele and genotype activity, plasma angiotensin II, and aldosterone 2 concentrations were measured by radioimmunoas- frequencies in the two groups were compared by w says in 144 male subjects of the NT group. test. Parametric data of the three genotypes were The study protocol was approved by the institu- analysed by ANOVA followed by Dunnett’s t-test. A tional review board, and informed consent was P-value of less than 0.05 was considered to indicate obtained from each subject. statistical significance. Results Genotype determination Clinical characteristics of study subjects Genomic DNA was isolated from peripheral leuko- cytes according to the standard method using Easy Clinical characteristics of the EH and NT subjects DNA kit (Invitrogen, Carlsbad, CA, USA), and the are listed in Table 1. Systolic and diastolic blood À344T/C polymorphism of the CYP11B2 gene was pressure was markedly higher in the EH group than determined by the analysis of restriction fragment in the NT group. Of the 250 EH patients, 229 length polymorphism (RFLP).9 The DNA fragment (91.6%) were taking antihypertensive medication. In containing À344T/C of the CYP11B2 gene was addition, as expected naturally, body mass index amplified by polymerase chain reaction (PCR). The (BMI), fasting blood glucose, and uric acid were used primers were 50-CAGGGAGGAGACCCCATGT- significantly higher in EH than in NT. A total of 68 GAC-30 (sense, from À528 to À548) and 50- patients with EH were taking lipid-lowering drugs, CCTCCACCCTGTTCAGCCC-30 (antisense, from and serum total cholesterol was lower in EH than in À11 to À29). The PCR profile included 35 cycles NT. However, serum triglycerides were higher and of denaturing at 941C for 60 s, annealing at 671C for serum HDL cholesterol was lower in EH than in NT. 60 s, and polymerization at 721C for 120 s. The The serum creatinine concentration was not sig- Table 1 Clinical characteristics of the normotensive subjects and hypertensive patients Parameter Normotensive (n=221) Hypertensive (n=250) P-value Age (year) 56.1 7 5.4 52.7 7 11.0 o0.001 Sex, male/female 144/77 154/96 Body mass index (kg/m2) 23.9 7 2.4 25.2 7 3.3 o0.001 Habitual smoking, +/À 61/160 127/123 o0.001 Habitual alcohol intake, +/À 101/120 110/140 Systolic blood pressure (mmHg) 117 7 11 171 7 22 o0.001 Diastolic blood pressure (mmHg) 72 7 8 104 7 12 o0.001 Fasting blood glucose (mg/dl) 95 7 9 103 7 16 o0.001 Serum total cholesterol (mg/dl) 208 7 37 197 7 32 o0.001 Serum HDL cholesterol (mg/dl) 54 7 13 51 7 12 0.026 Serum triglycerides (mg/dl) 128 7 75 154 7 88 o0.001 Serum creatinine (mg/dl) 0.9 7 0.5 1.0 7 0.5 Serum uric acid (mg/dl) 5.3 7 1.3 5.8 7 1.2 o0.001 Data are means 7 s.d. HDL, high-density lipoprotein. In the hypertensive group, 229, 68, and 23 patients were on medications for hypertension, hyperlipidaemia and hyperuricaemia, respectively. Blood pressure of the hypertensive group is the value when antihypertensive drugs were not given. Journal of Human Hypertension CYP11B2 gene polymorphism in hypertension K Tsukada et al 791 Table 2 Genotype and allele frequencies of the À344T/C polymorphism of the CYP11B2 gene in Normotensive subjects and hypertensive patients Normotensive Hypertensive (n=221) (n=250) Genotype frequency T/T, n (%) 124 (56.1) 105 (42.0) T/C, n (%) 77 (34.8) 119 (47.6) C/C, n (%) 20 (9.1) 26 (10.4) Figure 1 Plasma renin activity (left panel) and plasma concentra- Allele frequency tions of angiotensin II (middle panel) and aldosterone (right T (%) 73.5 65.8 panel) in 144 male subjects with TT (n ¼ 81), TC (n ¼ 50), and CC C (%) 26.5 34.2 (n ¼ 13) genotypes. *Po0.05, **Po0.01. Genotype distribution in hypertensive vs normotensive: w2=9.61, P=0.008. Figure 1 depicts the indices of the circulating Allele distribution in hypertensive vs normotensive: w2=6.60, P=0.010. renin–angiotensin–aldosterone system in male NT subjects with TT (n ¼ 81), TC (n ¼ 50), and CC Table 3 Numbers of smokers and nonsmokers in normotensive (n ¼ 13) genotypes. The CC group had lower plasma subjects and hypertensive patients grouped by the CYP11B2 renin activity and lower plasma concentrations of genotype angiotensin II and aldosterone than the TT and TC groups. Genotype TT TC CC Discussion Normotensive (n=221) Nonsmoker 92 52 16 In the present study, we investigated the association Smoker 32 25 4 between the À344T/C polymorphism in the promo- Hypertensive (n=250) ter region of the human CYP11B2 gene and the Nonsmoker 55 61 13 development of hypertension.