- Email: [email protected]
Association analyses of endothelial nitric oxide synthase gene polymorphisms in essential hypertension
AJH
2000;13:994 –998
Association Analyses of Endothelial Nitric Oxide Synthase Gene Polymorphisms in Essential Hypertension Adam V. Benjafield and Brian J. Morris
Endothelial nitric oxide synthase (eNOS), encoded by NOS3, is a potent regulator of vasomotor tone and peripheral resistance. Congenic experiments indicate that a chromosomal segment containing the rat eNOS gene contributes to rat spontaneous hypertension (HT). A role for NOS3 in onset of essential hypertension (HT) is, however, controversial. We therefore decided to test NOS3 polymorphisms in a set of patients who have an accentuated ability to show an existing genetic association. The 112 HT subjects had two HT parents and the normotensive (NT) subjects had two NT parents. All were Anglo-Celtic whites. The two most promising polymorphisms, viz, a biallelic variable number of tandem repeats (VNTR) in intron 4 and an exon 7 variant that leads to an amino acid change (Glu298Asp), were genotyped by PCR (and BanII digestion in the case of the latter). Frequency of the minor allele of the VNTR was 0.11 in the NT and 0.10 in the HT subjects (P
ⴝ .9). For the exon 7 variant, Asp298 frequency was 0.30 and 0.32 in each respective group (P ⴝ .6). Tracking was seen for the Asp298 allele with elevation in body mass index (P ⴝ .034), and the minor allele of the VNTR with elevation in LDL (P ⴝ .007) and reduction in HDL (P ⴝ .048). In conclusion, we saw no association of NOS3 markers with HT in the population studied. However, possible genotypic effects on plasma lipids and body mass index might warrant further studies, especially in view of possible associations with heart disease. Am J Hypertens 2000; 13:994 –998 © 2000 American Journal of Hypertension, Ltd.
E
sure (BP),5 endothelium-dependent vasodilation is blunted,6 NO deficiency induces vascular and cardiac hypertrophy,7 and inhibition of eNOS reduces coronary flow and raises BP.8 Deletion of the eNOS gene (NOS3) leads to hypertension9 –11 and BP variability,12 and transfer from a normotensive (NT) strain to a spontaneously hypertensive rat (SHR) background reduced BP 15–20%,13 supporting a role of reduced expression in SHR HT.14 NOS3, on chromosome 7q35– q36,15 is a 21-kb, 26exon gene16 with three single nucleotide polymorphisms (SNP) in 5⬘-flanking DNA (at ⫺788, ⫺924, and ⫺1474 bp),17,18 two SNP in coding DNA (C774T: silent; G894T: Glu298Asp19), and in introns, six SNP,20,18 a variable number of tandem (27-bp) repeats (VNTR)
ndothelial nitric oxide (NO) synthase (eNOS; NOS3), via generation of NO, is the major endothelial-derived arteriolar vasodilator.1 In essential hypertension (HT), NO is reduced,2– 4 NO correlates negatively with blood pres-
Received September 21, 1999. Accepted February 8, 2000. From the Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, The University of Sydney, Sydney, New South Wales, Australia. This study was funded by the National Health and Medical Research Council of Australia. Address correspondence and reprint requests to B. J. Morris, DSc, Hypertension Gene Laboratory, Department of Physiology and Institute for Biomedical Research, Building F13, The University of Sydney, NSW 2006, Australia; e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Association study, blood pressure, body mass index, chromosome 7, hypertension, lipids, molecular genetics, nitric oxide, endothelial nitric oxide synthase, single nucleotide polymorphism, variable number of tandem repeats polymorphism.
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TABLE 1. GENOTYPE AND ALLELE FREQUENCIES FOR NOS3 VNTR VARIANT AND RESTRICTION FRAGMENT-LENGTH POLYMORPHISM IN HYPERTENSIVE AND NORMOTENSIVE GROUPS Genotype Frequencies VNTR Group NT
n 184
HT
112
4b/4b 147 (0.80)
4b/4a 35 (0.19)
4a/4a 2 (0.01)
89 (0.79) G/T (Glu298Asp) variant Group n GG NT 149 70 (0.47)
23 (0.21)
2
4a 39 (0.11)
0 (0)
201 (0.90)
23 (0.10)
GT 68 (0.46)
TT 11 (0.07)
G 208 (0.70)
T 90 (0.30)
43 (0.47)
8 (0.09)
123 (0.68)
59 (0.32)
0.29 91
40 (0.44)
Allele Frequencies 4b 329 (0.89)
1.3
HT
P
0.52
0.87
2
P
0.02
0.90
0.26
0.61
VNTR ⫽ variable number of tandem repeats; NT ⫽ normotensive subjects; HT ⫽ hypertensive subjects. Values in parentheses are fractions.
in intron 4,15 and a (CA)-repeat in intron 13.15 In whites, linkage20,21 and association22 involving the latter have proved negative for HT, as have intronic SNP.20,23 In Japanese an association was seen in HT without left ventricular hypertrophy.24 Although the exon 7 (Glu298Asp) variant, detected as a BanII restriction fragment-length polymorphism (RFLP), has shown an association with HT in two25,26 of three27 Japanese studies and in one study of whites,23 this concerned contrasting alleles. The intron 4 VNTR was associated with plasma nitrite and nitrate,28 and with HT in one Japanese study,29 but not in two others,25,26 nor with HT in whites with coronary artery disease (CAD).30 The present study tested the two most promising polymorphisms in a well-studied cohort having two HT parents and early-onset, moderate to severe disease.31,32
0.1 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT), 63 mmol/L KCl, 13 mmol/L Tris-HCl, pH 8.3, 2 mmol/Lp MgCl2). PCR products of 393 bp (4a allele) and 420 bp (4b allele) were seen on 3% agarose gel electrophoresis. Genotypes for the G/T (Glu298Asp) variant were determined by PCR using primers (sense) 5⬘-TCC CTG AGG AGG GCA TGA GGC T-3⬘, (antisense) 5⬘-TGA GGG TCA CAC AGG TTC CT-3⬘, synthesized by GeneWorks. After 94°C for 5 min, 30 cycles of 94°C, 61°C, and 72°C for 1 min each were performed in 20 L (100 ng genomic DNA, 13.5 pmol each primer, 3 mmol/L each dNTP, 0.1 U AmpliTaq DNA polymerase, 63 mmol/L KCl, 13 mmol/L Tris-HCl, pH 8.3, 2 mmol/L MgCl2). PCR products (20 L) were digested with 2 U BanII (Promega, Madison, WI) at 37°C for 8 h. With T (Asp298) present the 457-bp PCR product was not cut, but was for the G (Glu298) allele, to fragments of 137 and 320 bp.
MATERIALS AND METHODS
Statistical Analyses 2 and ANOVA was performed with StatView (Abacus Concepts, Berkeley, CA). Linkage disequilibrium was tested by the method of Hill.33
Subjects These, their characteristics, and methods for determination of various parameters were as described previously.31,32 Genotyping DNA was isolated from whole blood using a QIAamp DNA Mini Kit (Qiagen). Genotypes for the VNTR ‘4a/b’ polymorphism were determined by polymerase chain reaction (PCR) using the primers (sense) 5⬘-AGG CCC TAT GGT AGT GCC TTT-3⬘, (antisense) 5⬘-TCT CTT AGT GCT GTG GTC AC-3⬘, synthesized by GeneWorks (Adelaide). Steps were: 94°C for 5 min, then 35 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min in 20 L (100 ng genomic DNA, 13.5 pmol each primer, 3 mmol/L each dNTP,
RESULTS Genotype data for each variant are shown in Table 1. Genotype frequencies did not deviate from HardyWeinberg expectations. No significant association with HT was seen. For a relative risk of 1.6, our study had 97% power to detect significant association of the Glu298Asp variant with HT, given the P value we obtained, and for the VNTR marker was 96%. For the VNTR, no HT subjects were homozygous for the 4a allele, which is rare: frequency ⫽ 3% in whites.30 Examination of haplotypes of the VNTR and G/T
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TABLE 2. GENOTYPIC ASSOCIATIONS WITH BODY MASS INDEX AND PLASMA LIPIDS Parameter 2
BMI (kg/m ) Triglycerides HDL cholesterol LDL cholesterol
Variant
MM
Mm
mm
F
P
RFLP RFLP VNTR VNTR
25 ⫾ 4 2.2 ⫾ 0.2 1.2 ⫾ 0.1 3.4 ⫾ 0.1
27 ⫾ 4 2.8 ⫾ 0.4 0.9 ⫾ 0.1 4.3 ⫾ 0.4
29 ⫾ 6 3.6 ⫾ 0.9
3.5 2.7 4.0 7.7
0.034 0.076 0.048 0.0069
BMI ⫽ body mass index; RFLP ⫽ restriction fragment-length polymorphism; HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein; other abbreviation as in Table 1. Lipid values are mmol/L. M ⫽ major allele; m ⫽ minor allele, of NOS3 polymorphism indicated.
(Glu298Asp) variants revealed nonoccurrence of a T/4a genotype in either group. Only marginal linkage disequilibrium between these markers was observed (4a with G [Glu298], and 4b with T [Asp298]): for the HT group, D⬘ was 1.00, with standardized value for D being 1.82 (P ⫽ .02) and for the NT group, D⬘ was 0.64, D being 2.08 (P ⫽ NS). Comparison of body mass index (BMI) and plasma lipid concentrations across genotypes in HT revealed, for the VNTR, tracking of the 4a allele with lower HDL and higher LDL; for the RFLP, tracking of the T (Asp298) allele with elevation in BMI and a trend for rises in triglyceride were noted (Table 2). A similar spread of medications was seen within each genotype. DISCUSSION We found no associations with essential HT, consistent with findings in other white populations.20,30 In studies by others, genotype frequencies for the VNTR30 and G/T (Glu298Asp) variants in HT23 and CAD34 groups resembled those we saw, but a lower Glu289 frequency in controls (0.56), cf, 0.65 in HT, in the European study may explain their positive (P ⫽ .004) finding.23 Moreover, a lack of association with BP might suggest an artifact of population stratification. To demonstrate a positive association in complex polygenic diseases requires ⱖ 102 per group,35 and our power estimates confirmed the capacity of our study to do this. Our cohort has, moreover, shown strong associations for markers in other genes.31,32 Thus the defect in NO production in HT could involve a gene other than NOS3. A role in other conditions cannot be ruled out. The VNTR 4a allele is associated with smoking-dependent risk of CAD in whites,30 but not with acute myocardial infarction (AMI) in Japanese.36 Moreover, Asp298 homozygotes were elevated in Japanese with AMI,36,37 but more Glu298 homozygotes were seen in French,18 but not Irish,18 British,38 or Australian34 white AMI patients, and no association was seen with CAD.34,38 Because high BMI and dyslipidemia are associated with CAD and AMI,39 it was of interest that we saw tracking of the VNTR 4a allele with higher LDL and lower HDL, and of the Asp298 variant with elevation in triglyceride and BMI, and lower HDL. However,
these could be chance findings due to multiple comparisons, so that a firm conclusion would be premature pending more extensive investigations. Any causative variant will also need to be identified. In this regard an association of the T(⫺786)C variant with reduced promoter activity and coronary spasm in Japanese17 is of interest. ACKNOWLEDGMENTS We thank William Wang for advice with power and linkage disequilibrium analyses, Judith O’Neill for help in patient sample collection, and the Australian Red Cross Blood Bank, Sydney, for NT subjects.
REFERENCES 1.
Rudic RD, Sessa WC: Nitric oxide in endothelial dysfunction and vascular remodelling: clinical correlates and experimental links. Am J Hum Genet 1999;64:673– 677.
2.
Linder L, Kiowski W, Buhler FR, Luscher TF: Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo. Blunted response in essential hypertension. Circulation 1990;81: 1762–1767.
3.
Dominiczak AF, Bohr DF: Nitric oxide and its putative role in hypertension. Hypertension 1995;25:1202–1211.
4.
Forte P, Copland M, Smith LM, Milne E, Sutherland J, Benjamin N: Basal nitric oxide synthesis in essential hypertension. Lancet 1997;349:837– 842.
5.
Node K, Kitakaze M, Yoshikawa H, Kosaka H, Hori M: Reduced plasma concentrations of nitric oxide in individuals with essential hypertension. Hypertension 1997;30:405– 408.
6.
Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE: Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 1990;323:22–27.
7.
Devlin AM, Brosnan J, Graham D, Morton JJ, McPhaden AR, McIntyre M, Hamilton CA, Reid JL, Dominiczak AF: Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo. Am J Physiol 1998;274: H52–H59.
8.
Haynes WG, Noon JP, Walker BR, Webb DJ: Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. J Hypertens 1993;11:1375–1380.
9.
Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC: Hypertension in
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
NITRIC OXIDE SYNTHASE GENETICS
kallikrein as candidate genes for essential hypertension. Clin Exp Pharmacol Physiol 1996;23:564 –566.
mice lacking the gene for endothelial nitric oxide synthase. Nature 1995;377:239 –242. 10.
Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O: Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci USA 1996;93:13176 – 13181.
11.
Go¨decke A, Decking UKM, Ding Z, Hirchenhain J, Bidmon H-J, Go¨decke S, Schrader J: Coronary hemodynamics in endothelial NO synthase knockout mice. Circ Res 1998;82:186 –194.
12.
Stauss HM, Godecke A, Mrowka R, Schrader J, Persson PB: Enhanced blood pressure variability in eNOS knockout mice. Hypertension 1999;33:1359 –1363.
13.
Pravenec M, Zidek V, Simakova M, Kren V, Krenova D, Horky K, Jachymova M, Mikova B, Kazdova L, Aitman TJ, Churchill PC, Webb RC, Hingarh NH, Yang Y, Wang J-M, St. Lezin EM, Kurtz TW: Genetics of Cd36 and the clustering of multiple cardiovascular risk factors in spontaneous hypertension. J Clin Invest 1999; 103:1651–1657.
997
23.
Lacolley P, Gautier S, Poirier O, Pannier B, Cambien F, Benetos A: Nitric oxide synthase gene polymorphisms, blood pressure and aortic stiffness in normotensive and hypertensive subjects. J Hypertens 1997;16:31–35.
24.
Nakayama T, Soma M, Takahashi Y, Izumi Y, Kanmatsuse K, Esumi M: Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension. Clin Genet 1997;51: 26 –30.
25.
Miyamoto Y, Saito Y, Kajiyama N, Yoshimura M, Shimasaki Y, Nakayama M, Kamitani S, Harada M, Ishikawa M, K. K, Ogawa E, Hamanaka I, Takahashi N, Kaneshige T, Teraoka H, Akamizu T, Azuma N, Yoshimasa Y, Yoshimasa T, Itoh H: Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension 1999;32:3– 8.
26.
Yasujima M, Tsutaya S, Shoji M: Endothelial nitric oxide synthase gene polymorphism and hypertension (in Japanese). Rinsho Byori Jap J Clin Pathol 1998;46:1199 – 1204.
14.
Chou TC, Yen MH, Li CY, Ding YA: Alterations in nitric oxide synthase expression with aging and hypertension in rats. Hypertension 1998;31:643– 648.
27.
15.
Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT: Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993;268:17478 –17488.
Kato N, Sugiyama T, Morita H, Nabika T, Kurihara H, Yamori Y, Yazaki Y: Lack of evidence for association between the endothelial nitric oxide synthase gene and hypertension. Hypertension 1999;33:933–936.
28.
Nadaud S, Bonnardeaux A, Lathrop GM, Soubrier F: Gene structure, polymorphism and mapping of the human endothelial nitric oxide synthase gene. Biochem Biophys Res Commun 1994;198:1027–1033.
Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A, Kawaguchi Y, Hosoya T, Igari J: Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun 1998;245:190 –193.
29.
Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, K. N: T-7863 C mutation in the 5⬘flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation 1999;99:2864 –2870.
Uwabo J, Soma M, Nakayama T, Kanmatsuse K: Association of a variable number of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japanese. Am J Hypertens 1998;11:125–128.
30.
Wang XL, Sim AS, Badenhop R, McCredie MR, Wilcken DEL: A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med 1996; 2:41– 45.
31.
Benjafield AV, Jeyasingam CL, Nyholt DR, Griffiths LR, Morris BJ: G-protein 3 subunit gene (GNB3) variant in causation of essential hypertension. Hypertension 1998; 32:1094 –1097.
16.
17.
18.
Poirier O, Mao C, Mallet C, Nicaud V, Herrmann SM, Evans A, Ruidavets JB, Arveiler D, Luc G, Tiret L, Soubrier F, Cambien F: Polymorphisms of the endothelial nitric oxide synthase gene—no consistent association with myocardial infarction in the ECTIM study. Eur J Clin Invest 1999;29:284 –290.
19.
Yoshimura M, Yasue H, Nakayama M, Shimasaki Y, Sumida H, Sugiyama S, Kugiyama K, Ogawa H, Ogawa Y, Saito Y, Miyamoto Y, Nakao K: A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese. Hum Genet 1998;103:65– 69.
32.
Morris BJ: Genes for essential hypertension: the first decade of research, in Frossard PM, Parvez SH, Minami M, Parvez S, Saito H (eds): Progress in Hypertension, Vol 4: Molecular, Genetic and Immune Predisposition to Hypertension. Zeist, The Netherlands: VSP International Press, 1999, pp 1– 44.
20.
Bonnardeaux A, Nadaud S, Charru A, Jeunemeitre X, Corvol P, Soubrier F: Lack of evidence for linkage of the endothelial cell nitric oxide synthase gene to essential hypertension. Hypertension 1995;91:96 –102.
33.
Hill WG: Estimation of linkage disequilibrium in randomly mating populations. Heredity 1974;33:229 –239.
34.
Cai H, Wilcken DEL, Wang XL: The Glu-2983 Asp (894G3 T) mutation in exon 7 of the endothelial nitric oxide synthase gene and coronary artery disease. J Mol Med 1999;77:511–514.
35.
Cox NJ, Bell GI: Disease associations. Chance, artifact, or susceptibility genes. Diabetes 1989;38:947–950.
36.
Hibi K, Ishigami T, Tamura K, Mizushima S, Nyui N, Fujita T, Ochiai H, Kosuge M, Watanabe Y, Yoshii Y,
21.
22.
Hunt SC, Williams CS, Sharma AM, Inoue I, Williams RR, Lalouel JM: Lack of linkage between the endothelial nitric oxide synthase gene and hypertension. J Hum Hypertens 1996;10:27–30. Friend LR, Morris BJ, Gaffney PT, Griffiths LR: Examination of the role of nitric oxide synthase and renal
998
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Montheith S, Parsons A, Haydock S, Hopper RV, Stephens NG, O’Shaughnessy KM, Brown MJ: A common variant of the endothelial nitric oxide synthase (Glu2983 Asp) is a major risk factor for coronary artery disease in the UK. Circulation 1999;100:1515–1520.
Kihara M, K. K, Ishii M, Umemura S: Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension 1998;32:521–526. 37.
38.
Shimasaki Y, Yasue H, Yoshimura M, Nakayama M, Kugiyama K, Ogawa H, Harada E, Masuda T, Koyama W, Y. S, Miyamoto Y, Ogawa Y, Nakao K: Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. J Am Coll Cardiol 1998;31:1506 –1510. Hingorani AD, Liang CF, Fatibene J, Lyon A,
39.
Reeder BA, Senthilselvan A, Despres JP, Angel A, Liu L, Wang H, Rabkin SW: The association of cardiovascular disease risk factors with abdominal obesity in Canada. Canadian Heart Health Surveys Research Group. Can Med Assoc J 1997;157:S39 –S45.
2000;13:994 –998
Association Analyses of Endothelial Nitric Oxide Synthase Gene Polymorphisms in Essential Hypertension Adam V. Benjafield and Brian J. Morris
Endothelial nitric oxide synthase (eNOS), encoded by NOS3, is a potent regulator of vasomotor tone and peripheral resistance. Congenic experiments indicate that a chromosomal segment containing the rat eNOS gene contributes to rat spontaneous hypertension (HT). A role for NOS3 in onset of essential hypertension (HT) is, however, controversial. We therefore decided to test NOS3 polymorphisms in a set of patients who have an accentuated ability to show an existing genetic association. The 112 HT subjects had two HT parents and the normotensive (NT) subjects had two NT parents. All were Anglo-Celtic whites. The two most promising polymorphisms, viz, a biallelic variable number of tandem repeats (VNTR) in intron 4 and an exon 7 variant that leads to an amino acid change (Glu298Asp), were genotyped by PCR (and BanII digestion in the case of the latter). Frequency of the minor allele of the VNTR was 0.11 in the NT and 0.10 in the HT subjects (P
ⴝ .9). For the exon 7 variant, Asp298 frequency was 0.30 and 0.32 in each respective group (P ⴝ .6). Tracking was seen for the Asp298 allele with elevation in body mass index (P ⴝ .034), and the minor allele of the VNTR with elevation in LDL (P ⴝ .007) and reduction in HDL (P ⴝ .048). In conclusion, we saw no association of NOS3 markers with HT in the population studied. However, possible genotypic effects on plasma lipids and body mass index might warrant further studies, especially in view of possible associations with heart disease. Am J Hypertens 2000; 13:994 –998 © 2000 American Journal of Hypertension, Ltd.
E
sure (BP),5 endothelium-dependent vasodilation is blunted,6 NO deficiency induces vascular and cardiac hypertrophy,7 and inhibition of eNOS reduces coronary flow and raises BP.8 Deletion of the eNOS gene (NOS3) leads to hypertension9 –11 and BP variability,12 and transfer from a normotensive (NT) strain to a spontaneously hypertensive rat (SHR) background reduced BP 15–20%,13 supporting a role of reduced expression in SHR HT.14 NOS3, on chromosome 7q35– q36,15 is a 21-kb, 26exon gene16 with three single nucleotide polymorphisms (SNP) in 5⬘-flanking DNA (at ⫺788, ⫺924, and ⫺1474 bp),17,18 two SNP in coding DNA (C774T: silent; G894T: Glu298Asp19), and in introns, six SNP,20,18 a variable number of tandem (27-bp) repeats (VNTR)
ndothelial nitric oxide (NO) synthase (eNOS; NOS3), via generation of NO, is the major endothelial-derived arteriolar vasodilator.1 In essential hypertension (HT), NO is reduced,2– 4 NO correlates negatively with blood pres-
Received September 21, 1999. Accepted February 8, 2000. From the Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, The University of Sydney, Sydney, New South Wales, Australia. This study was funded by the National Health and Medical Research Council of Australia. Address correspondence and reprint requests to B. J. Morris, DSc, Hypertension Gene Laboratory, Department of Physiology and Institute for Biomedical Research, Building F13, The University of Sydney, NSW 2006, Australia; e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
KEY WORDS:
Association study, blood pressure, body mass index, chromosome 7, hypertension, lipids, molecular genetics, nitric oxide, endothelial nitric oxide synthase, single nucleotide polymorphism, variable number of tandem repeats polymorphism.
0895-7061/00/$20.00 PII S0895-7061(00)00282-X
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
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TABLE 1. GENOTYPE AND ALLELE FREQUENCIES FOR NOS3 VNTR VARIANT AND RESTRICTION FRAGMENT-LENGTH POLYMORPHISM IN HYPERTENSIVE AND NORMOTENSIVE GROUPS Genotype Frequencies VNTR Group NT
n 184
HT
112
4b/4b 147 (0.80)
4b/4a 35 (0.19)
4a/4a 2 (0.01)
89 (0.79) G/T (Glu298Asp) variant Group n GG NT 149 70 (0.47)
23 (0.21)
2
4a 39 (0.11)
0 (0)
201 (0.90)
23 (0.10)
GT 68 (0.46)
TT 11 (0.07)
G 208 (0.70)
T 90 (0.30)
43 (0.47)
8 (0.09)
123 (0.68)
59 (0.32)
0.29 91
40 (0.44)
Allele Frequencies 4b 329 (0.89)
1.3
HT
P
0.52
0.87
2
P
0.02
0.90
0.26
0.61
VNTR ⫽ variable number of tandem repeats; NT ⫽ normotensive subjects; HT ⫽ hypertensive subjects. Values in parentheses are fractions.
in intron 4,15 and a (CA)-repeat in intron 13.15 In whites, linkage20,21 and association22 involving the latter have proved negative for HT, as have intronic SNP.20,23 In Japanese an association was seen in HT without left ventricular hypertrophy.24 Although the exon 7 (Glu298Asp) variant, detected as a BanII restriction fragment-length polymorphism (RFLP), has shown an association with HT in two25,26 of three27 Japanese studies and in one study of whites,23 this concerned contrasting alleles. The intron 4 VNTR was associated with plasma nitrite and nitrate,28 and with HT in one Japanese study,29 but not in two others,25,26 nor with HT in whites with coronary artery disease (CAD).30 The present study tested the two most promising polymorphisms in a well-studied cohort having two HT parents and early-onset, moderate to severe disease.31,32
0.1 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT), 63 mmol/L KCl, 13 mmol/L Tris-HCl, pH 8.3, 2 mmol/Lp MgCl2). PCR products of 393 bp (4a allele) and 420 bp (4b allele) were seen on 3% agarose gel electrophoresis. Genotypes for the G/T (Glu298Asp) variant were determined by PCR using primers (sense) 5⬘-TCC CTG AGG AGG GCA TGA GGC T-3⬘, (antisense) 5⬘-TGA GGG TCA CAC AGG TTC CT-3⬘, synthesized by GeneWorks. After 94°C for 5 min, 30 cycles of 94°C, 61°C, and 72°C for 1 min each were performed in 20 L (100 ng genomic DNA, 13.5 pmol each primer, 3 mmol/L each dNTP, 0.1 U AmpliTaq DNA polymerase, 63 mmol/L KCl, 13 mmol/L Tris-HCl, pH 8.3, 2 mmol/L MgCl2). PCR products (20 L) were digested with 2 U BanII (Promega, Madison, WI) at 37°C for 8 h. With T (Asp298) present the 457-bp PCR product was not cut, but was for the G (Glu298) allele, to fragments of 137 and 320 bp.
MATERIALS AND METHODS
Statistical Analyses 2 and ANOVA was performed with StatView (Abacus Concepts, Berkeley, CA). Linkage disequilibrium was tested by the method of Hill.33
Subjects These, their characteristics, and methods for determination of various parameters were as described previously.31,32 Genotyping DNA was isolated from whole blood using a QIAamp DNA Mini Kit (Qiagen). Genotypes for the VNTR ‘4a/b’ polymorphism were determined by polymerase chain reaction (PCR) using the primers (sense) 5⬘-AGG CCC TAT GGT AGT GCC TTT-3⬘, (antisense) 5⬘-TCT CTT AGT GCT GTG GTC AC-3⬘, synthesized by GeneWorks (Adelaide). Steps were: 94°C for 5 min, then 35 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min in 20 L (100 ng genomic DNA, 13.5 pmol each primer, 3 mmol/L each dNTP,
RESULTS Genotype data for each variant are shown in Table 1. Genotype frequencies did not deviate from HardyWeinberg expectations. No significant association with HT was seen. For a relative risk of 1.6, our study had 97% power to detect significant association of the Glu298Asp variant with HT, given the P value we obtained, and for the VNTR marker was 96%. For the VNTR, no HT subjects were homozygous for the 4a allele, which is rare: frequency ⫽ 3% in whites.30 Examination of haplotypes of the VNTR and G/T
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TABLE 2. GENOTYPIC ASSOCIATIONS WITH BODY MASS INDEX AND PLASMA LIPIDS Parameter 2
BMI (kg/m ) Triglycerides HDL cholesterol LDL cholesterol
Variant
MM
Mm
mm
F
P
RFLP RFLP VNTR VNTR
25 ⫾ 4 2.2 ⫾ 0.2 1.2 ⫾ 0.1 3.4 ⫾ 0.1
27 ⫾ 4 2.8 ⫾ 0.4 0.9 ⫾ 0.1 4.3 ⫾ 0.4
29 ⫾ 6 3.6 ⫾ 0.9
3.5 2.7 4.0 7.7
0.034 0.076 0.048 0.0069
BMI ⫽ body mass index; RFLP ⫽ restriction fragment-length polymorphism; HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein; other abbreviation as in Table 1. Lipid values are mmol/L. M ⫽ major allele; m ⫽ minor allele, of NOS3 polymorphism indicated.
(Glu298Asp) variants revealed nonoccurrence of a T/4a genotype in either group. Only marginal linkage disequilibrium between these markers was observed (4a with G [Glu298], and 4b with T [Asp298]): for the HT group, D⬘ was 1.00, with standardized value for D being 1.82 (P ⫽ .02) and for the NT group, D⬘ was 0.64, D being 2.08 (P ⫽ NS). Comparison of body mass index (BMI) and plasma lipid concentrations across genotypes in HT revealed, for the VNTR, tracking of the 4a allele with lower HDL and higher LDL; for the RFLP, tracking of the T (Asp298) allele with elevation in BMI and a trend for rises in triglyceride were noted (Table 2). A similar spread of medications was seen within each genotype. DISCUSSION We found no associations with essential HT, consistent with findings in other white populations.20,30 In studies by others, genotype frequencies for the VNTR30 and G/T (Glu298Asp) variants in HT23 and CAD34 groups resembled those we saw, but a lower Glu289 frequency in controls (0.56), cf, 0.65 in HT, in the European study may explain their positive (P ⫽ .004) finding.23 Moreover, a lack of association with BP might suggest an artifact of population stratification. To demonstrate a positive association in complex polygenic diseases requires ⱖ 102 per group,35 and our power estimates confirmed the capacity of our study to do this. Our cohort has, moreover, shown strong associations for markers in other genes.31,32 Thus the defect in NO production in HT could involve a gene other than NOS3. A role in other conditions cannot be ruled out. The VNTR 4a allele is associated with smoking-dependent risk of CAD in whites,30 but not with acute myocardial infarction (AMI) in Japanese.36 Moreover, Asp298 homozygotes were elevated in Japanese with AMI,36,37 but more Glu298 homozygotes were seen in French,18 but not Irish,18 British,38 or Australian34 white AMI patients, and no association was seen with CAD.34,38 Because high BMI and dyslipidemia are associated with CAD and AMI,39 it was of interest that we saw tracking of the VNTR 4a allele with higher LDL and lower HDL, and of the Asp298 variant with elevation in triglyceride and BMI, and lower HDL. However,
these could be chance findings due to multiple comparisons, so that a firm conclusion would be premature pending more extensive investigations. Any causative variant will also need to be identified. In this regard an association of the T(⫺786)C variant with reduced promoter activity and coronary spasm in Japanese17 is of interest. ACKNOWLEDGMENTS We thank William Wang for advice with power and linkage disequilibrium analyses, Judith O’Neill for help in patient sample collection, and the Australian Red Cross Blood Bank, Sydney, for NT subjects.
REFERENCES 1.
Rudic RD, Sessa WC: Nitric oxide in endothelial dysfunction and vascular remodelling: clinical correlates and experimental links. Am J Hum Genet 1999;64:673– 677.
2.
Linder L, Kiowski W, Buhler FR, Luscher TF: Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo. Blunted response in essential hypertension. Circulation 1990;81: 1762–1767.
3.
Dominiczak AF, Bohr DF: Nitric oxide and its putative role in hypertension. Hypertension 1995;25:1202–1211.
4.
Forte P, Copland M, Smith LM, Milne E, Sutherland J, Benjamin N: Basal nitric oxide synthesis in essential hypertension. Lancet 1997;349:837– 842.
5.
Node K, Kitakaze M, Yoshikawa H, Kosaka H, Hori M: Reduced plasma concentrations of nitric oxide in individuals with essential hypertension. Hypertension 1997;30:405– 408.
6.
Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE: Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 1990;323:22–27.
7.
Devlin AM, Brosnan J, Graham D, Morton JJ, McPhaden AR, McIntyre M, Hamilton CA, Reid JL, Dominiczak AF: Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo. Am J Physiol 1998;274: H52–H59.
8.
Haynes WG, Noon JP, Walker BR, Webb DJ: Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. J Hypertens 1993;11:1375–1380.
9.
Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC: Hypertension in
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
NITRIC OXIDE SYNTHASE GENETICS
kallikrein as candidate genes for essential hypertension. Clin Exp Pharmacol Physiol 1996;23:564 –566.
mice lacking the gene for endothelial nitric oxide synthase. Nature 1995;377:239 –242. 10.
Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O: Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci USA 1996;93:13176 – 13181.
11.
Go¨decke A, Decking UKM, Ding Z, Hirchenhain J, Bidmon H-J, Go¨decke S, Schrader J: Coronary hemodynamics in endothelial NO synthase knockout mice. Circ Res 1998;82:186 –194.
12.
Stauss HM, Godecke A, Mrowka R, Schrader J, Persson PB: Enhanced blood pressure variability in eNOS knockout mice. Hypertension 1999;33:1359 –1363.
13.
Pravenec M, Zidek V, Simakova M, Kren V, Krenova D, Horky K, Jachymova M, Mikova B, Kazdova L, Aitman TJ, Churchill PC, Webb RC, Hingarh NH, Yang Y, Wang J-M, St. Lezin EM, Kurtz TW: Genetics of Cd36 and the clustering of multiple cardiovascular risk factors in spontaneous hypertension. J Clin Invest 1999; 103:1651–1657.
997
23.
Lacolley P, Gautier S, Poirier O, Pannier B, Cambien F, Benetos A: Nitric oxide synthase gene polymorphisms, blood pressure and aortic stiffness in normotensive and hypertensive subjects. J Hypertens 1997;16:31–35.
24.
Nakayama T, Soma M, Takahashi Y, Izumi Y, Kanmatsuse K, Esumi M: Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension. Clin Genet 1997;51: 26 –30.
25.
Miyamoto Y, Saito Y, Kajiyama N, Yoshimura M, Shimasaki Y, Nakayama M, Kamitani S, Harada M, Ishikawa M, K. K, Ogawa E, Hamanaka I, Takahashi N, Kaneshige T, Teraoka H, Akamizu T, Azuma N, Yoshimasa Y, Yoshimasa T, Itoh H: Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension 1999;32:3– 8.
26.
Yasujima M, Tsutaya S, Shoji M: Endothelial nitric oxide synthase gene polymorphism and hypertension (in Japanese). Rinsho Byori Jap J Clin Pathol 1998;46:1199 – 1204.
14.
Chou TC, Yen MH, Li CY, Ding YA: Alterations in nitric oxide synthase expression with aging and hypertension in rats. Hypertension 1998;31:643– 648.
27.
15.
Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT: Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993;268:17478 –17488.
Kato N, Sugiyama T, Morita H, Nabika T, Kurihara H, Yamori Y, Yazaki Y: Lack of evidence for association between the endothelial nitric oxide synthase gene and hypertension. Hypertension 1999;33:933–936.
28.
Nadaud S, Bonnardeaux A, Lathrop GM, Soubrier F: Gene structure, polymorphism and mapping of the human endothelial nitric oxide synthase gene. Biochem Biophys Res Commun 1994;198:1027–1033.
Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A, Kawaguchi Y, Hosoya T, Igari J: Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun 1998;245:190 –193.
29.
Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, K. N: T-7863 C mutation in the 5⬘flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation 1999;99:2864 –2870.
Uwabo J, Soma M, Nakayama T, Kanmatsuse K: Association of a variable number of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japanese. Am J Hypertens 1998;11:125–128.
30.
Wang XL, Sim AS, Badenhop R, McCredie MR, Wilcken DEL: A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med 1996; 2:41– 45.
31.
Benjafield AV, Jeyasingam CL, Nyholt DR, Griffiths LR, Morris BJ: G-protein 3 subunit gene (GNB3) variant in causation of essential hypertension. Hypertension 1998; 32:1094 –1097.
16.
17.
18.
Poirier O, Mao C, Mallet C, Nicaud V, Herrmann SM, Evans A, Ruidavets JB, Arveiler D, Luc G, Tiret L, Soubrier F, Cambien F: Polymorphisms of the endothelial nitric oxide synthase gene—no consistent association with myocardial infarction in the ECTIM study. Eur J Clin Invest 1999;29:284 –290.
19.
Yoshimura M, Yasue H, Nakayama M, Shimasaki Y, Sumida H, Sugiyama S, Kugiyama K, Ogawa H, Ogawa Y, Saito Y, Miyamoto Y, Nakao K: A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese. Hum Genet 1998;103:65– 69.
32.
Morris BJ: Genes for essential hypertension: the first decade of research, in Frossard PM, Parvez SH, Minami M, Parvez S, Saito H (eds): Progress in Hypertension, Vol 4: Molecular, Genetic and Immune Predisposition to Hypertension. Zeist, The Netherlands: VSP International Press, 1999, pp 1– 44.
20.
Bonnardeaux A, Nadaud S, Charru A, Jeunemeitre X, Corvol P, Soubrier F: Lack of evidence for linkage of the endothelial cell nitric oxide synthase gene to essential hypertension. Hypertension 1995;91:96 –102.
33.
Hill WG: Estimation of linkage disequilibrium in randomly mating populations. Heredity 1974;33:229 –239.
34.
Cai H, Wilcken DEL, Wang XL: The Glu-2983 Asp (894G3 T) mutation in exon 7 of the endothelial nitric oxide synthase gene and coronary artery disease. J Mol Med 1999;77:511–514.
35.
Cox NJ, Bell GI: Disease associations. Chance, artifact, or susceptibility genes. Diabetes 1989;38:947–950.
36.
Hibi K, Ishigami T, Tamura K, Mizushima S, Nyui N, Fujita T, Ochiai H, Kosuge M, Watanabe Y, Yoshii Y,
21.
22.
Hunt SC, Williams CS, Sharma AM, Inoue I, Williams RR, Lalouel JM: Lack of linkage between the endothelial nitric oxide synthase gene and hypertension. J Hum Hypertens 1996;10:27–30. Friend LR, Morris BJ, Gaffney PT, Griffiths LR: Examination of the role of nitric oxide synthase and renal
998
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
BENJAFIELD AND MORRIS
Montheith S, Parsons A, Haydock S, Hopper RV, Stephens NG, O’Shaughnessy KM, Brown MJ: A common variant of the endothelial nitric oxide synthase (Glu2983 Asp) is a major risk factor for coronary artery disease in the UK. Circulation 1999;100:1515–1520.
Kihara M, K. K, Ishii M, Umemura S: Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension 1998;32:521–526. 37.
38.
Shimasaki Y, Yasue H, Yoshimura M, Nakayama M, Kugiyama K, Ogawa H, Harada E, Masuda T, Koyama W, Y. S, Miyamoto Y, Ogawa Y, Nakao K: Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. J Am Coll Cardiol 1998;31:1506 –1510. Hingorani AD, Liang CF, Fatibene J, Lyon A,
39.
Reeder BA, Senthilselvan A, Despres JP, Angel A, Liu L, Wang H, Rabkin SW: The association of cardiovascular disease risk factors with abdominal obesity in Canada. Canadian Heart Health Surveys Research Group. Can Med Assoc J 1997;157:S39 –S45.