- Email: [email protected]
Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in Northern Japan
Life Sciences, Vol. 66, No. 26, pp. 2557-2562,200O Copyright 0 2000 Elwicr Science Inc. Printed in the USA. All rights browed 002C3205/00/&w front matter
PIISOO24-3205(00)00589-O
POSITIVE
ASSOCIATIoW POLYXORPEISM
OF mDoT5LIAL NITRIC OXIDs WITS EYPERTl!BlSIOXQ II EiOR-
s -BGEta JAPAN
Masaru Shoji', Shoji Tsutaya', Ran Saito', Hideetsu Takamatu', Minoru Yasujima Department of Laboratory Medicine, Hirosaki University School of Medicine, and Department of Clinical Laboratory*, Hirosaki University Hospital, 5 Zaifu-cho, Hirosaki 036-8562, Japan (Receivedin fmalfonnJanuary 31,200O)
Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.13.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subiects (0.128 vs 0.080, DXo.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (~~0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension. KeyWords:eNOS gene polymorphism,hypertension,Glu298Asp variant Vascular endothelial cells produce nitric oxide (NO), a vascular smooth muscle relaxing factor which plays an important role in the regulation of blood pressure and regional blood flow. NO is produced by NO synthase (NOS) from L-arginine (1). Administration of Larginine decreases blood pressure, while the administration of NOS inhibitor increases blood pressure (2). Endothelium-dependent blood vessel relaxation is impaired in hypertensive patients and in animal models of hypertension induced by hereditary trait, DOCA-salt, or Goldblatt's clamp. The impaired relaxation improves as hypertension 1 Address for correspondence: Masaru Shoji, M.D., Ph.D., Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Tel, 81-172-39-5124; Fax, 81-172-39-5124; E-Mail, [email protected].
2558
eNOS GenePolymorphismsandHypertension
Vol. 66,No.26,2000
normalizes (3). Furthermore, an increase in blood pressure was observed in mice deficient in endothelial NOS (eNO.9) gene (4). In contrast, eNOS gene delivery was reported to reduce systemic blood pressure (5). These findings indicate that the eNOS gene is a putative candidate gene for human hypertension. eNOS is constitutively expressed in vascular endothelial cells (1). The human eNOS gene consists of a total length of 21 kb and 26 exons and has been shown to be polymorphic (6). Recently, these eNOS gene polymorphisms have drawn attention as producing hereditary predisposition to arteriosclerosis-related diseases (7,8). Although there is increased interest in eNOS gene polymorphism as a hereditary factor in hypertension, susceptibility has been the genetic controversial. Recently, hypertension in western Japan was shown to have a linkage with a Glu298Asp eNOS gene polymorphism (9). In northern Japan, there is a high incidence of hypertension and hypertensive cardiovascular diseases. Therefore, to clarify the relationship between hypertension and eNOS gene variants, we examined two polymorphisms of the eNOS gene, 27-bp repeated polymorphism in intron 4 (4b/a) and Glu298Asp polymorphism in exon 7, in hypertensive patients and normal control subjects living in Aomori Prefecture in northern Japan. Methods A total of 376 persons living in Aomori Prefecture in Japan was studied. As shown in Table 1, they consisted of 183 hypertensive patients (83 males and 100 females) and 193 normal control subjects (67 males and 126 females). The diagnostic criteria of 1) patients hypertension used for this study were as follows: in whom hypertension developed under 60 years of age; 2) those under medical treatment; and 3) those with either systolic blood pressure of 160 mmHg or higher, or diastolic blood pressure of 95 mmHg or higher, or both. Normal control subjects were without antihypertensive treatment, and their systolic and diastolic blood pressures were less than 140 and 90 mmHg, respectively. An informed consent was obtained from each subject at the time of recruitment. TABLE
Characteristics
I
of the Study Population. NT
HT
Parameter No. of Age, yrs
subjects
Sex, M/F SBP, mmHg DBP, mmHg BMI, kg/m2 T-Cho, mg/dl HDL-Cho, mq/dl
193 54.4 f 11.9 67/126 125 f 10 76 f 8 23.2 f 3.0 199 f 35 63 f 15
183 56.9 * 9.6 83/100 160 f 20** 95 f 11** 25.4 f 3.5 208 f 37 61 f 17
NT, normotensives; HT, hypertensives; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index; T-Cho, serum total cholesterol; HDL-Cho, serum HDL cholesterol. Data are expressed by means f S.D.. **p
Vol.66,No.26,2000
eNOSGenePolymorphismsandHypertension
2559
The specific genotype of each individual was determined in the following fashion. DNA was extracted from the peripheral blood of each subject as previously reported (10). The Glu298Asp genotype was identified with restriction fragment length polymorphism as described by Miyamoto et al (9). The restriction enzymes were Ban II for gag (Glu) and Mbo I for gat (Asp). The 4b/a polymorphism was determined as reported by Wang et al (7). The primers used against Glu298Asp polymorphism were 5'-ccccacagctctgcattcag-3' and 5'while those used against 4b/a polymorphism tccatcccacccagtcaatc-31, were 5'.-aggccctatggtagtgcctt-3' and 5'-tctcttagtgctgtggtcat-3'. Data are expressed in terms of mean f SD. The differences in clinical parameters between groups were determined by the unpaired t test. The relationship between gene polymorphism and hypertension was evaluated using Fisher's exact test. The association of 298Asp variant with blood pressure was tested by the unpaired t test in all typed individuals except for those who were under the influence of their antihypertensive treatments. P values less than 0.05 were considered statistically significant. All probability values were based on two-tailed tests.
The systolic and diastolic collection were 125+10/76*8
blood pressures at the time of blood mmHg in normal subjects, and 160+20/95
kll mmHg in hypertensive subjects (Table 1). Thus, both blood pressures were significantly higher in hypertensive patients. However, no differences were observed between the two groups in other factors such as average age, BMI, total cholesterol, and HDLcholesterol. The prevalence of the eNOS alleles in each of the groups satisfied the Hardy-Weinberg equilibrium law. The genotype frequency in codon 298 of the eNOS gene in the normal control group was 85.0% for Glu/Glu and 15.0% for Glu/Asp+Asp/Asp, and allele distributions of Glu and Asp were 92.0% and 8.0%, respectively (Table 2). In hypertensive subjects, the genotype frequency was 76.0% for Glu/Glu
TABLE
Genotypes and Alleles (NT) and Hypertensive
II
of eN0.S Glu298Asp Variant Subjects (HT).
in Normotensive
(nY93) Genotypes Glu/Glu, n (%) Glu/Asp + Asp/Asp, n (%) Significance Odds ratio (95% CI) Alleles Glu, n (%) Asp, n (%I Significance CI,
confidence
interval.
(nf?83)
164 (85.0) 139 (76.0) 29 (15.0) 44 (24.0) p=O.O376 1.8 (1.1-3.0) 355 (92.0) 31 (8.0) p=O.O318
319 (87.2) 47 (12.8)
2560
eNOSGenePolymorphismsandHypertension
TABLE
Vol.66,No. 26,200O
III
Genotypes and Alleles of eNOS 4b/a 27bp-repeats Variant Normotensive (NT) and Hypertensive Subjects (HT).
Genotypes b/b, n (%I b/a + a/a, n (%) Significance Odds ratio (95% CI) Alleles b, n ($1 a, n (%I Significance CI, confidence
in
143 (78.1) 156 (80.8) 40 (21.9) 37 (19.2) p=O.5258 1.2 (0.7-1.9) 347 (89.9) 39 (10.1) p=O.6380
325 (88.8) 41 (11.2)
interval.
and 24.0% for Glu/Asp+Asp/Asp, and allele frequencies of Glu and Asp were 87.2% and 12.8%, respectively. significant There were differences in genotype and allele frequencies between hypertensive patients and normotensive control subjects. The odds ratio was 1.8 and 95% confidence interval was 1.1-3.0. The genotype frequencies of 4b/a polymorphism in normal control subjects were 80.8% for b/b and 19.2% for b/a+a/a, and alleles of b and a in normal control subjects were 89.9% and lO.l%, respectively the (Table 3). On the other hand, in hypertensive subjects, genotype frequency was 78.1% for b/b and 21.9% for b/a+a/a, and frequencies of alleles b and a were 88.8% and 11.2%, respectively. No significant difference in frequency of 4b/a polymorphism was observed between hypertensive and normal control subjects. There were significant differences in diastolic and mean arterial blood pressure measurements between the Glu/Glu and Glu/Asp+Asp/Asp genotypes in hypertensive subjects (Table 4). No differences were found in any blood pressure measurements for the Glu298Asp genotypes in normotensive subjects and for each genotype in 4b/a polymorphism in hypertensives and normotensives (data not shown).
TABLE
IV
Blood Pressure (BP) Measurement for Genotypes of eNOS Glu298Asp Variant in Hypertensive Subjects. Genotypes Significance (p value) Glu/Glu Glu/Asp+Asp/Asp (n=113) (n=31) BP, mg Systolic Mean Diastolic Age, yrs
162.5 118.5 96.5 56.0
f 19.6 fi 10.1 f 8.9 f 9.7
166.9 122.6 100.4 56.1
+ 18.1 f 10.2 f 8.8 f 10.2
0.262 0.049 0.032 0.937
Vol. 66,No. 26,200O
eNOS GenePolymorphismsandHypertension
2561
Discussion
The distribution of Glu298Asp polymorphism in the eNOS gene in the normal control group has varied greatly among different reports. In our study the frequency of Glu298 allele was 92%. The rate was the same as that in Tokyo (111, but it was slightly higher in Kyoto and Kumamoto at about 95% (9). In contrast, the analysis in France by Lacolley et al (12) yielded an exceedingly low frequency of 56%. On the other hand, the distribution of 4b/a polymorphism in the normal control group was nearly the same as that found in Nagoya (81, Kyoto and Kumamoto (9). Namely, the 5-time repetition type was dominant, with about 90% being present as allele. However, Wang et al in Australia (7) showed that the occurrence of the 5-time repetition type was slightly less, at 83%. Thus, the eNOS gene 27-bp repeated polymorphism in intron 4 and Glu298Asp polymorphism in the Japanese are more uniform than in other races in terms of genetic background. With respect to Glu298Asp polymorphism, hypertensive patients had a high frequency of 298Asp type in the present study, indicating the presence of a significant relationship. Studies (9) performed in western Japan also revealed a similar relationship with hypertension. However, studies in Tokyo (11) showed no association of Glu298Asp polymorphism with hypertension. These results indicate that environmental and behavioral factors may play a major role in the pathogenesis of hypertension in the larger cities, whereas the eNOS gene might be more important as a hereditary factor in hypertension in the more rural cities of Japan. In France Glu298Asp polymorphism has been reported to be related significantly to hypertension (121, suggesting that Glu298Asp polymorphism in the eNOS gene may represent a genetic marker for hypertension in the French. On the other hand, no significant relationship was observed between 27-bp repeated polymorphism in intron 4 and hypertension. These results conflict with the positive findings by Uwabo et al (131, but are consistent with the report by Miyamoto et al (9). Miyamoto et al found that the 4a polymorphism was strongly linked to the GlOT polymorphism in intron 23. Bonnardeaux et al found (13) that GlOT polymorphism was not associated with hypertension in France. Therefore, neither 4b/a nor GlOT polymorphisms appeared to be genetic markers for hypertension. The eNOS gene has been considered a candidate for an hereditary factor for hypertension. Although earlier studies failed to observe a positive relationship between eNOS gene polymorphism and hypertension (14,151, Arngrimsson et al (16) showed evidence for a familial pregnancy-induced hypertension locus in the eNOS-gene region. Indeed, the recent findings in western Japan and the present study in northern Japan showed that Glu298Asp polymorphism could be a The Glu298Asp site has been genetic susceptibility for hypertension. shown to have neither a functional domain (6) nor a cis element activity on eNOS gene expression. In the present study, however, diastolic and mean arterial blood pressures increased in hypertensive subjects with Glu298Asp variant genotypes. These results may suggest that amino has a the acid substitution at codon 298 pathophysiological significance, at least, in hypertensive subjects. Alternatively, there remains the possibility that the Glu298Asp variant has a link with any other unknown functional locus. In conclusion, a significant relationship has been found between eNOS Glu298Asp polymorphism of the eNOS gene and hypertension, which
eNOSGene PolymorphismsandHypertension
2562
suggests that hypertension.
this polymorphism
may represent
Vol. 66,No.26,2000
a genetic
marker
of
Acknowledgment The authors are grateful to Dr. Kei Sato for valuable comments on our manuscript and to Miss Akiko Inaba for her expert secretarial assistance. The current study was supported in part by Grant-in-Aid for Scientific Research (No.09672347) from the Culture, Ministry, Japan, the Karoji Medical Fund for Medical Research in Hirosaki University, Kurozumi Medical Foundation, and Charitable Trust Clinical Pathology Research Foundation of Japan. References 1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
C.NATHAN, FASEB J. 6 3051-3064 (1992). T.YAMAMOTO, T.KIMURA, K.OTA, M.SHOJI, M.INOUE, M.OHTA, K.SATO, T.FUNYU, and K.ABE, Tohoku J. Exp. Med. 174 59-69 (1994). C.CARDILLO and J.A.PANZA, Vast. Med. 3 138-44 (1998). P.L.HUANG, Z.HUANG, H.MASHIMO, K.D.BLOCH, M.A.MOSKOWITZ, J.A.BEVAN, and M.C.FISHMAN, Nature 377 239-242 (1995). K.F.LIN, L.CHAO, and J.CHAO, Hypertension 30 307-13 (1997). K.MIYAHARA, T.KAWAMOTO, K.SASE, Y.YUI, K.TODA, L.X.YANG, R.HATTORI, T.AOYAMA, Y.YAMAMOTO, Y.DOI, S.OGOSHI, K.HASHIMOTO, C.KAWAI, S.SASAYAMA, and Y.SHIZUTA, Eur. J. Biochem. 223 719726 (1994). X.L.WANG, A.S.SIM, R.F.BADENHOP, R.M.MCCREDIE, and D.E.WILCKEN Nat. Med. 2 41-45 (1996). S.ICHIHARA, Y.YAMADA, T.FUJIMURA, N.NAKASHIMA, and M.YOKOTA, Am. J. Cardiol. 81 83-86 (1998). Y.MIYAMOTO, Y.SAITO, N.KAJIYAMA, M.YOSHIMURA, Y.SHIMASAKI, M.NAKAYAMA, S.KAMITANI, M.HARADA, M.ISHIKAWA, K.KUWAHARA, E.OGAWA, I.HAMANAKA, N.TAKAHASHI, T.KANESHIGE, H.TERAOKA, T.AKAMIZU, N.AZUMA, Y.YOSHIMASA, T.YOSHIMASA, H.ITOH, I.MASUDA, H.YASUE, and K.NAKAO, Hypertension 32 3-8 (1998). S.TSUTAYA, H.KITAYA, Y.SAITO, S.NAKATA, H.TAKAMATSU, and M.Yasujima, Tohoku J. Exp. Med. 182 151-155 (1997). N.KATO, T.SUGIYAMA, H.MORITA, T.NABIKA, H.KURIHARA, Y.YAMORI, and Y.YAZAKI, Hypertension 33 933-936 (1999). P.LACOLLEY, S.GAUTIER, O.POIRIER, B.PANNIER, F.CAMBIEN, and A.BENETOS, J. Hypertens. 16 31-35 (1998). J.UWABO, M.SOMA, T.NAKAYAMA, and K.KANMATSUSE, Am. J. Hypertens. 11 125-128 (1998). A.BONNARDEAUX, S.NADAUD, A.CHARRU, X.JEUNEMAITRE, P.CORVOL, and F.SOUBRIER, Circulation 91 96-102 (1995). S.C.HUNT, C.S.WILLIAMS, A.M.SHARMA, I.INOUE, R.R.WILLIAMS, and J.M.LALOUEL, J. Hum. Hypertens. 10 27-30 (1996). R.ARNGRIMSSON, C.HAYWARD, S.NADAUD, A.BALDURSDOTTIR, J.J.WALKER, W.A.LISTON, R.I.BJARNARNADOTTIR, D.J.BROCK, R.T.GEIRSSON, J.M.CONNOR, and F.SOUBRIER, Am. J. Hum. Genet. 61 354-362 (1997).
PIISOO24-3205(00)00589-O
POSITIVE
ASSOCIATIoW POLYXORPEISM
OF mDoT5LIAL NITRIC OXIDs WITS EYPERTl!BlSIOXQ II EiOR-
s -BGEta JAPAN
Masaru Shoji', Shoji Tsutaya', Ran Saito', Hideetsu Takamatu', Minoru Yasujima Department of Laboratory Medicine, Hirosaki University School of Medicine, and Department of Clinical Laboratory*, Hirosaki University Hospital, 5 Zaifu-cho, Hirosaki 036-8562, Japan (Receivedin fmalfonnJanuary 31,200O)
Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.13.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subiects (0.128 vs 0.080, DXo.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (~~0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension. KeyWords:eNOS gene polymorphism,hypertension,Glu298Asp variant Vascular endothelial cells produce nitric oxide (NO), a vascular smooth muscle relaxing factor which plays an important role in the regulation of blood pressure and regional blood flow. NO is produced by NO synthase (NOS) from L-arginine (1). Administration of Larginine decreases blood pressure, while the administration of NOS inhibitor increases blood pressure (2). Endothelium-dependent blood vessel relaxation is impaired in hypertensive patients and in animal models of hypertension induced by hereditary trait, DOCA-salt, or Goldblatt's clamp. The impaired relaxation improves as hypertension 1 Address for correspondence: Masaru Shoji, M.D., Ph.D., Department of Laboratory Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Tel, 81-172-39-5124; Fax, 81-172-39-5124; E-Mail, [email protected].
2558
eNOS GenePolymorphismsandHypertension
Vol. 66,No.26,2000
normalizes (3). Furthermore, an increase in blood pressure was observed in mice deficient in endothelial NOS (eNO.9) gene (4). In contrast, eNOS gene delivery was reported to reduce systemic blood pressure (5). These findings indicate that the eNOS gene is a putative candidate gene for human hypertension. eNOS is constitutively expressed in vascular endothelial cells (1). The human eNOS gene consists of a total length of 21 kb and 26 exons and has been shown to be polymorphic (6). Recently, these eNOS gene polymorphisms have drawn attention as producing hereditary predisposition to arteriosclerosis-related diseases (7,8). Although there is increased interest in eNOS gene polymorphism as a hereditary factor in hypertension, susceptibility has been the genetic controversial. Recently, hypertension in western Japan was shown to have a linkage with a Glu298Asp eNOS gene polymorphism (9). In northern Japan, there is a high incidence of hypertension and hypertensive cardiovascular diseases. Therefore, to clarify the relationship between hypertension and eNOS gene variants, we examined two polymorphisms of the eNOS gene, 27-bp repeated polymorphism in intron 4 (4b/a) and Glu298Asp polymorphism in exon 7, in hypertensive patients and normal control subjects living in Aomori Prefecture in northern Japan. Methods A total of 376 persons living in Aomori Prefecture in Japan was studied. As shown in Table 1, they consisted of 183 hypertensive patients (83 males and 100 females) and 193 normal control subjects (67 males and 126 females). The diagnostic criteria of 1) patients hypertension used for this study were as follows: in whom hypertension developed under 60 years of age; 2) those under medical treatment; and 3) those with either systolic blood pressure of 160 mmHg or higher, or diastolic blood pressure of 95 mmHg or higher, or both. Normal control subjects were without antihypertensive treatment, and their systolic and diastolic blood pressures were less than 140 and 90 mmHg, respectively. An informed consent was obtained from each subject at the time of recruitment. TABLE
Characteristics
I
of the Study Population. NT
HT
Parameter No. of Age, yrs
subjects
Sex, M/F SBP, mmHg DBP, mmHg BMI, kg/m2 T-Cho, mg/dl HDL-Cho, mq/dl
193 54.4 f 11.9 67/126 125 f 10 76 f 8 23.2 f 3.0 199 f 35 63 f 15
183 56.9 * 9.6 83/100 160 f 20** 95 f 11** 25.4 f 3.5 208 f 37 61 f 17
NT, normotensives; HT, hypertensives; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index; T-Cho, serum total cholesterol; HDL-Cho, serum HDL cholesterol. Data are expressed by means f S.D.. **p
Vol.66,No.26,2000
eNOSGenePolymorphismsandHypertension
2559
The specific genotype of each individual was determined in the following fashion. DNA was extracted from the peripheral blood of each subject as previously reported (10). The Glu298Asp genotype was identified with restriction fragment length polymorphism as described by Miyamoto et al (9). The restriction enzymes were Ban II for gag (Glu) and Mbo I for gat (Asp). The 4b/a polymorphism was determined as reported by Wang et al (7). The primers used against Glu298Asp polymorphism were 5'-ccccacagctctgcattcag-3' and 5'while those used against 4b/a polymorphism tccatcccacccagtcaatc-31, were 5'.-aggccctatggtagtgcctt-3' and 5'-tctcttagtgctgtggtcat-3'. Data are expressed in terms of mean f SD. The differences in clinical parameters between groups were determined by the unpaired t test. The relationship between gene polymorphism and hypertension was evaluated using Fisher's exact test. The association of 298Asp variant with blood pressure was tested by the unpaired t test in all typed individuals except for those who were under the influence of their antihypertensive treatments. P values less than 0.05 were considered statistically significant. All probability values were based on two-tailed tests.
The systolic and diastolic collection were 125+10/76*8
blood pressures at the time of blood mmHg in normal subjects, and 160+20/95
kll mmHg in hypertensive subjects (Table 1). Thus, both blood pressures were significantly higher in hypertensive patients. However, no differences were observed between the two groups in other factors such as average age, BMI, total cholesterol, and HDLcholesterol. The prevalence of the eNOS alleles in each of the groups satisfied the Hardy-Weinberg equilibrium law. The genotype frequency in codon 298 of the eNOS gene in the normal control group was 85.0% for Glu/Glu and 15.0% for Glu/Asp+Asp/Asp, and allele distributions of Glu and Asp were 92.0% and 8.0%, respectively (Table 2). In hypertensive subjects, the genotype frequency was 76.0% for Glu/Glu
TABLE
Genotypes and Alleles (NT) and Hypertensive
II
of eN0.S Glu298Asp Variant Subjects (HT).
in Normotensive
(nY93) Genotypes Glu/Glu, n (%) Glu/Asp + Asp/Asp, n (%) Significance Odds ratio (95% CI) Alleles Glu, n (%) Asp, n (%I Significance CI,
confidence
interval.
(nf?83)
164 (85.0) 139 (76.0) 29 (15.0) 44 (24.0) p=O.O376 1.8 (1.1-3.0) 355 (92.0) 31 (8.0) p=O.O318
319 (87.2) 47 (12.8)
2560
eNOSGenePolymorphismsandHypertension
TABLE
Vol.66,No. 26,200O
III
Genotypes and Alleles of eNOS 4b/a 27bp-repeats Variant Normotensive (NT) and Hypertensive Subjects (HT).
Genotypes b/b, n (%I b/a + a/a, n (%) Significance Odds ratio (95% CI) Alleles b, n ($1 a, n (%I Significance CI, confidence
in
143 (78.1) 156 (80.8) 40 (21.9) 37 (19.2) p=O.5258 1.2 (0.7-1.9) 347 (89.9) 39 (10.1) p=O.6380
325 (88.8) 41 (11.2)
interval.
and 24.0% for Glu/Asp+Asp/Asp, and allele frequencies of Glu and Asp were 87.2% and 12.8%, respectively. significant There were differences in genotype and allele frequencies between hypertensive patients and normotensive control subjects. The odds ratio was 1.8 and 95% confidence interval was 1.1-3.0. The genotype frequencies of 4b/a polymorphism in normal control subjects were 80.8% for b/b and 19.2% for b/a+a/a, and alleles of b and a in normal control subjects were 89.9% and lO.l%, respectively the (Table 3). On the other hand, in hypertensive subjects, genotype frequency was 78.1% for b/b and 21.9% for b/a+a/a, and frequencies of alleles b and a were 88.8% and 11.2%, respectively. No significant difference in frequency of 4b/a polymorphism was observed between hypertensive and normal control subjects. There were significant differences in diastolic and mean arterial blood pressure measurements between the Glu/Glu and Glu/Asp+Asp/Asp genotypes in hypertensive subjects (Table 4). No differences were found in any blood pressure measurements for the Glu298Asp genotypes in normotensive subjects and for each genotype in 4b/a polymorphism in hypertensives and normotensives (data not shown).
TABLE
IV
Blood Pressure (BP) Measurement for Genotypes of eNOS Glu298Asp Variant in Hypertensive Subjects. Genotypes Significance (p value) Glu/Glu Glu/Asp+Asp/Asp (n=113) (n=31) BP, mg Systolic Mean Diastolic Age, yrs
162.5 118.5 96.5 56.0
f 19.6 fi 10.1 f 8.9 f 9.7
166.9 122.6 100.4 56.1
+ 18.1 f 10.2 f 8.8 f 10.2
0.262 0.049 0.032 0.937
Vol. 66,No. 26,200O
eNOS GenePolymorphismsandHypertension
2561
Discussion
The distribution of Glu298Asp polymorphism in the eNOS gene in the normal control group has varied greatly among different reports. In our study the frequency of Glu298 allele was 92%. The rate was the same as that in Tokyo (111, but it was slightly higher in Kyoto and Kumamoto at about 95% (9). In contrast, the analysis in France by Lacolley et al (12) yielded an exceedingly low frequency of 56%. On the other hand, the distribution of 4b/a polymorphism in the normal control group was nearly the same as that found in Nagoya (81, Kyoto and Kumamoto (9). Namely, the 5-time repetition type was dominant, with about 90% being present as allele. However, Wang et al in Australia (7) showed that the occurrence of the 5-time repetition type was slightly less, at 83%. Thus, the eNOS gene 27-bp repeated polymorphism in intron 4 and Glu298Asp polymorphism in the Japanese are more uniform than in other races in terms of genetic background. With respect to Glu298Asp polymorphism, hypertensive patients had a high frequency of 298Asp type in the present study, indicating the presence of a significant relationship. Studies (9) performed in western Japan also revealed a similar relationship with hypertension. However, studies in Tokyo (11) showed no association of Glu298Asp polymorphism with hypertension. These results indicate that environmental and behavioral factors may play a major role in the pathogenesis of hypertension in the larger cities, whereas the eNOS gene might be more important as a hereditary factor in hypertension in the more rural cities of Japan. In France Glu298Asp polymorphism has been reported to be related significantly to hypertension (121, suggesting that Glu298Asp polymorphism in the eNOS gene may represent a genetic marker for hypertension in the French. On the other hand, no significant relationship was observed between 27-bp repeated polymorphism in intron 4 and hypertension. These results conflict with the positive findings by Uwabo et al (131, but are consistent with the report by Miyamoto et al (9). Miyamoto et al found that the 4a polymorphism was strongly linked to the GlOT polymorphism in intron 23. Bonnardeaux et al found (13) that GlOT polymorphism was not associated with hypertension in France. Therefore, neither 4b/a nor GlOT polymorphisms appeared to be genetic markers for hypertension. The eNOS gene has been considered a candidate for an hereditary factor for hypertension. Although earlier studies failed to observe a positive relationship between eNOS gene polymorphism and hypertension (14,151, Arngrimsson et al (16) showed evidence for a familial pregnancy-induced hypertension locus in the eNOS-gene region. Indeed, the recent findings in western Japan and the present study in northern Japan showed that Glu298Asp polymorphism could be a The Glu298Asp site has been genetic susceptibility for hypertension. shown to have neither a functional domain (6) nor a cis element activity on eNOS gene expression. In the present study, however, diastolic and mean arterial blood pressures increased in hypertensive subjects with Glu298Asp variant genotypes. These results may suggest that amino has a the acid substitution at codon 298 pathophysiological significance, at least, in hypertensive subjects. Alternatively, there remains the possibility that the Glu298Asp variant has a link with any other unknown functional locus. In conclusion, a significant relationship has been found between eNOS Glu298Asp polymorphism of the eNOS gene and hypertension, which
eNOSGene PolymorphismsandHypertension
2562
suggests that hypertension.
this polymorphism
may represent
Vol. 66,No.26,2000
a genetic
marker
of
Acknowledgment The authors are grateful to Dr. Kei Sato for valuable comments on our manuscript and to Miss Akiko Inaba for her expert secretarial assistance. The current study was supported in part by Grant-in-Aid for Scientific Research (No.09672347) from the Culture, Ministry, Japan, the Karoji Medical Fund for Medical Research in Hirosaki University, Kurozumi Medical Foundation, and Charitable Trust Clinical Pathology Research Foundation of Japan. References 1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. 12. 13. 14. 15. 16.
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