Positive association of CYP11B2 gene polymorphism with genetic predisposition

(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 Tsukada 1, T Ishimitsu 1, M Teranishi 1, M Saitoh 1, M Yoshii 1, H Inada 1, S Ohta 1, M Akashi 1, J Minami 1, H Ono 1, M Ohrui 2 and H Matsuoka 1 1 Department of Hypertension and Cardiorenal Medicine; 2 Department of Health Care, Dokkyo University School of Medicine, Mibu, Tochigi, Japan Predispositions to essential hypertension and cardiovascular diseases are possibly associated with gene polymorphisms of the renin angiotensin system. Gene polymorphisms of angiotensinogen and angiotensinconverting enzyme genes have been suggested to be risk factors for hypertension and myocardial infarction. Concerning the polymorphism of aldosterone synthase (CYP11B2) gene, earlier studies have shown inconsistent results in terms of its relation to hypertension. In the present case control study, we investigated the association of 344T/C polymorphism in the promoter region of human CYP11B2 gene with genetic predisposition to hypertension. The genotype of 344T/C polymorphism was determined in essential hypertension subjects (n ¼ 250) and normotensive subjects (n ¼ 221). The distributions of three genotypes (TT, TC, and CC) were significantly different between the hypertensive and the normotensive groups (v 2 ¼ 9.61, P ¼ 0.008). Namely, the frequency of C allele was higher in the hypertensive patients than in the normotensive subjects (34.2 vs 26.5%, P ¼ 0.010). Our data suggest that the 344C allele of CYP11B2 gene polymorphism is associated with the genetic predisposition to develop essential hypertension. (2002) 16, 789 793. doi:10.1038/sj.jhh.1001484 Keywords: essential hypertension; gene polymorphism; aldosterone Introduction Aldosterone is the principal mineralocorticoid hormone, which plays a major role in regulating sodium balance and volume homeostasis. 1 Human aldosterone synthase (CYP11B2) is a cytochrome P450 enzyme, which catalyses the terminal steps of aldosterone synthesis in the zona glomerulosa cells of the adrenal. 2,3 Therefore, genetic variations of the CYP11B2 gene that affect its expression may influence the development of salt-sensitive hypertension. Several common polymorphisms have been described in the CYP11B2 gene. 4,5 Among them, the 344T/C polymorphism, which is located at a putative binding site for the steroidogenic transcription factor (SF-1), has been recently attracting interest with regard to its relation to cardiovascular diseases. Several studies have suggested that the C allele of this gene polymorphism is associated with genetic predisposition to cardiovascular diseases Correspondence: Dr T Ishimitsu, Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. E-mail: isimitu@dokkyomed.ac.jp such as hypertension and myocardial infarction. 6 However, the results of earlier studies are not always consistent. Differences in background characteristics of the study subjects such as ethnicity and selection criteria may be responsible for this inconsistency. In the present study, we investigated the 344T/C polymorphism of the CYP11B2 gene in carefully selected normotensive subjects and patients with essential hypertension, and thereby evaluated the association of the genotypes with genetic predisposition to essential hypertension. Subjects and methods Subjects A group of 250 patients with essential hypertension (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 Dokkyo University School of Medicine Hospital. Hypertension was defined as systolic blood pressure exceeding 140 mmhg and/or diastolic blood

790 pressure exceeding 90 mmhg on two or more consecutive visits. 7,8 Individuals diagnosed as having secondary causes of hypertension were excluded. The normotensive control group (NT) included 221 healthy subjects (144 men and 77 women), aged 56 7 5 years, who were older than 50 years and free from a history of any cardiovascular diseases. They were recruited from participants of the health check programme of Dokkyo University School of Medicine Hospital. The subjects were considered to be normotensive when their blood pressure was lower than 140 mmhg in systole and 90 mmhg in diastole on multiple occasions. Blood pressure was measured by a sphygmomanometer, with the subjects in a sitting position after a 10-min rest. All subjects were native Japanese and unrelated to each other. Besides the routine laboratory tests, plasma renin activity, plasma angiotensin II, and aldosterone concentrations were measured by radioimmunoassays in 144 male subjects of the NT group. The study protocol was approved by the institutional review board, and informed consent was obtained from each subject. Genotype determination Genomic DNA was isolated from peripheral leukocytes according to the standard method using Easy DNA kit (Invitrogen, Carlsbad, CA, USA), and the 344T/C polymorphism of the CYP11B2 gene was determined by the analysis of restriction fragment length polymorphism (RFLP). 9 The DNA fragment containing 344T/C of the CYP11B2 gene was amplified by polymerase chain reaction (PCR). The used primers were 5 0 -CAGGGAGGAGACCCCATGT- GAC-3 0 (sense, from 528 to 548) and 5 0 - CCTCCACCCTGTTCAGCCC-3 0 (antisense, from 11 to 29). The PCR profile included 35 cycles of denaturing at 941C for 60 s, annealing at 671C for 60 s, and polymerization at 721C for 120 s. The amplified fragments were digested with HaeIII restriction enzyme (New England BioLabs, MA, USA) and were subjected to electrophoresis on 2.0% agarose gels. HaeIII digestion of the 538 bp PCK product yields 274, 138 and 126 bp fragments. The existence of 344C creates an additional recognition site of HaeIII resulting in digestion of the 274 bp fragment into 203 and 71 bp fragments. Fragments of 274 bp (T allele) and of 203 and 71 bp (C allele) were detected. Statistical analysis Data are presented as means 7 s.d. Clinical characteristics between the two groups were compared by unpaired Student s t-test for parametric data and by w 2 test for categoric data. The allele and genotype frequencies in the two groups were compared by w 2 test. Parametric data of the three genotypes were analysed by ANOVA followed by Dunnett s t-test. A P-value of less than 0.05 was considered to indicate statistical significance. Results Clinical characteristics of study subjects Clinical characteristics of the EH and NT subjects are listed in Table 1. Systolic and diastolic blood pressure was markedly higher in the EH group than in the NT group. Of the 250 EH patients, 229 (91.6%) were taking antihypertensive medication. In addition, as expected naturally, body mass index (BMI), fasting blood glucose, and uric acid were significantly higher in EH than in NT. A total of 68 patients with EH were taking lipid-lowering drugs, and serum total cholesterol was lower in EH than in NT. However, serum triglycerides were higher and serum HDL cholesterol was lower in EH than in NT. 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/m 2 ) 23.9 7 2.4 25.2 7 3.3 o0.001 Habitual smoking, +/ 61/160 127/123 o0.001 Habitual alcohol intake, +/ 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.

Table 2 Genotype and allele frequencies of the 344T/C polymorphism of the CYP11B2 gene in Normotensive subjects and hypertensive patients 791 Normotensive (n=221) Hypertensive (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) Allele frequency T (%) 73.5 65.8 C (%) 26.5 34.2 Genotype distribution in hypertensive vs normotensive: w 2 =9.61, P=0.008. Allele distribution in hypertensive vs normotensive: w 2 =6.60, P=0.010. Table 3 Numbers of smokers and nonsmokers in normotensive subjects and hypertensive patients grouped by the CYP11B2 genotype nificantly different between the two groups. Although the ratios of habitual drinkers were not different between EH and NT, smokers were more frequent in EH than in NT. In the EH group, 41 subjects had complications because of other cardiovascular diseases. Namely, 37 subjects had present or past history of coronary artery disease, five subjects had past history of cerebral stroke, and one subject had both. Genotype and allele frequencies Genotype TT TC CC Normotensive (n=221) Nonsmoker 92 52 16 Smoker 32 25 4 Hypertensive (n=250) Nonsmoker 55 61 13 Smoker 50 58 13 Table 2 shows the frequencies of each genotype and allele. The distributions of three genotypes were in accordance with the Hardy Weinberg equilibrium both in the NT and EH groups. However, the genotype distribution was significantly different between the two groups (w 2 ¼ 9.61, P ¼ 0.008). Namely, the frequency of the C allele was higher and the frequency of the T allele was lower in EH than in NT (w 2 ¼ 6.60, P ¼ 0.010). In the EH group, there was no significant difference in the C allele frequency between the 41 subjects with cardiovascular diseases and the 209 subjects without them (0.34 vs 0.34). As shown in Table 3, the ratios of habitual smokers were not different among the three genotypes either in the NT or the EH group. Figure 1 Plasma renin activity (left panel) and plasma concentrations of angiotensin II (middle panel) and aldosterone (right panel) in 144 male subjects with TT (n ¼ 81), TC (n ¼ 50), and CC (n ¼ 13) genotypes. *Po0.05, **Po0.01. Figure 1 depicts the indices of the circulating renin angiotensin aldosterone system in male NT subjects with TT (n ¼ 81), TC (n ¼ 50), and CC (n ¼ 13) genotypes. The CC group had lower plasma renin activity and lower plasma concentrations of angiotensin II and aldosterone than the TT and TC groups. Discussion In the present study, we investigated the association between the 344T/C polymorphism in the promoter region of the human CYP11B2 gene and the development of hypertension. Our results indicate that the genotype distribution differs between hypertensive and normotensive groups, and that the 344C allele is associated with genetic predisposition to develop EH. A number of studies have suggested the implication of CYP11B2 gene polymorphism in the pathophysiology of cardiovascular diseases. It was first reported that the 344C allele of CYP11B2 gene polymorphism was associated with left ventricular mass in Finnish young adults without clinical heart diseases. 10 The same authors have also reported that the 344C allele was associated with higher systolic blood pressure levels in Finnish men. 11 With regard to the mechanism of developing hypertension, it has been reported that baroreflex sensitivity is decreased in subjects carrying the C allele. 12 However, in the French population, the frequency of the C allele was rather lower in hypertensive patients than in normotensive subjects. 13 It was also reported that 344T/C polymorphism is associated with the risk of myocardial infarction in Finland. 14 However, such association was not observed in later studies undertaken in England and Germany. 15,16 In the Finnish study, the risk of myocardial infarction was increased especially when the C allele coexisted with habitual smoking; 14 however, such interaction between CYP1B2 gene polymorphism and smoking was not observed in our study as to the development of essential hypertension. The relationship between gene polymorphism and predisposition to cardiovascular diseases may

792 not be always universal among different ethnic groups. In the Japanese, unlike the results of our study, two earlier studies have failed to find a significant relationship between CYP11B2 gene polymorphism and hypertension. 17,18 Tsujita et al 17 have enrolled a large number of subjects randomly selected from the municipal population and could not find significant differences in blood pressure between TT, TC, and CC genotypes. This result is not necessarily in conflict with our findings. Young subjects of the study by Tsujita et al may possibly develop hypertension later in their life and systolic hypertension caused by arteriosclerosis in the elderly may not reflect genetic predisposition to EH. In the present study, the subject selection was performed so that such ambiguity was excluded, because all subjects in the EH group had developed hypertension before they were 50 years old and all subjects in the NT group were older than 50 years. On the other hand, Kato et al 18 have performed a case control study in which they failed to find any relation between the gene polymorphisms of the renin angiotensin aldosterone system and EH. In the subanalysis of their study, similar criteria to our study were applied to select the normotensive and hypertensive subjects. Namely, even when the hypertensive patients who had developed hypertension before 45 years of age and the normotensive subjects more than 60 years of age were compared, a significant difference in genotype frequencies was not detected. Discordance of the results of the study by Kato et al with our results may be attributed to the influence of environmental factors. Most of our study subjects lived in Tochigi, a rural to suburban district, while the participants of the study by Kato et al had been recruited in Tokyo, a big city. The quality and quantity of stress, which contribute to the development of hypertension, are supposed to be different between urban and rural lives. In addition, people in Tochigi are known to ingest much salt as compared to urban people. According to the national nutritional survey performed in 1999, the average salt intake was 13.9 g/day in Tochigi and 12.5 g/day in Tokyo. Therefore, salt-induced hypertension is more likely to occur in our study subjects than in the subjects living in Tokyo. In this aspect, the observed relationship between the C allele of the CYP11B2 gene and the development of EH may only be applicable to rural Japanese subjects consuming more salt. Indices of salt intake such as urinary Na excretion were not evaluated in our study, and the salt-loading study in Spanish subjects did not demonstrate any relationship between salt sensitivity and CYP11B2 gene polymorphism. 19 Therefore, further salt balance studies are needed to elucidate the implication of the CYP11B2 genotype in salt sensitivity in Japanese subjects. Matsubara et al 20 have examined the association of CYP11B2 gene polymorphism with circadian blood pressure rhythm. They observed no significant difference in 24-h ambulatory blood levels among the genotypes; however, the nocturnal decline in blood pressure was greater in CC homozygotes. Moreover, the prevalence of cardiovascular disease was less in CC than in other genotypes. In the EH group of our study, the presence or absence of cardiovascular complications did not affect the genotype distribution of CYP11B2 gene polymorphism, although the number of patients with cardiovascular diseases is limited. Further large-scale study is needed to elucidate the relation of CYP11B2 gene polymorphism to the incidence of cardiovascular diseases. With regard to the plasma aldosterone levels, Pojoga et al 21 have shown that the CC genotype was associated with increased plasma aldosterone in French patients with EH, while Brand et al 22 have reported that plasma aldosterone was not affected by the genotypes of 344T/C polymorphism in German normotensive subjects. In the present study, most patients with EH who were already treated pharmacologically and plasma aldosterone levels were not evaluated. In male NT subjects of this study, the circulating renin angiotensin system was suppressed and the plasma aldosterone level was lower in the CC group than in the TC or TT groups. In accordance with our results, Davies et al 23 have reported that urinary excretion of aldosterone is less in the CC genotype than in the TC and TT genotypes. Therefore, it is speculated that the CC genotype may be related to the development of lowrenin hypertension; however, the mechanism is not supposed to be brought about by the increased transcription of the aldosterone synthetase gene. The results of this study revealed the association of CYP11B2 gene polymorphism with genetic predisposition to hypertension. The frequency of the 344C allele was higher in patients with EH than in normotensive subjects. However, the cross-sectional studies, including our current one, cannot be completely free from some bias in selecting the study subjects. The NT subjects of this study were recruited from the participants of a health check programme and the EH patients were the outpatients of our university hospital. Besides the genetic predisposition to hypertension, it may be possible that unexpected difference in genetic background had existed between the case and control groups of this study. Therefore, the results of this study should be confirmed by further prospective studies and linkage analyses in sib-pairs. Acknowledgements We thank Ms Yasuko Kawamura, Ms Mika Nomura, Ms Jun Chou, Ms. Masako Minato, Ms Machiko Sakata and Ms Yasuko Mamada for technological assistance in executing the study. This study was supported in part by a grant-in-aid for scientific

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