Ambulatory blood pressure, left ventricular mass and vascular ... - Nature

Journal of Human Hypertension (2005) 19, 413–420 & 2005 Nature Publishing Group All rights reserved 0950-9240/05 $30.00 www.nature.com/jhh

ORIGINAL ARTICLE

Ambulatory blood pressure, left ventricular mass and vascular phenotypes in relation to the endothelial nitric oxide synthase gene Glu298Asp and intron 4 polymorphisms in a population-based family study D Czarnecka1, K Kawecka-Jaszcz1, K Stolarz1, A Olszanecka1, A Dembin´ska-Kiec´2 and B Kiec´-Wilk1,2 1

First Cardiac Department, Medical College, Jagiellonian University, Cracow, Poland; 2Department of Clinical Biochemistry, Medical College, Jagiellonian University, Cracow, Poland

Reduced nitric oxide production is associated with pathological changes in the cardiovascular system. In a study of randomly chosen families, we analysed the relationship between two polymorphisms (Glu298Asp and intron 4) of the endothelial nitric oxide synthase (eNOS) and ambulatory blood pressure (ABP), left ventricular mass index (LVMI) and vascular phenotypes. The study population consisted of 127 parents and 167 offspring. All subjects underwent 24 h ABP monitoring using a SpaceLabs 90207 device. 2D and M-mode echocardiograms were obtained. Pulse wave velocity between the common carotid and femoral artery was measured with the Compliors device, and the carotid intima-media thickness (IMT) was assessed by ultrasound. For statistical analysis, covariables and correlations between relatives were taken into account. The frequency of genotypes was as follows: for Glu298Asp:

55.1%—Glu/Glu, 40.1%—Glu/Asp and 4.8%—Asp/Asp; for intron 4: 65.0%—4 b/b, 33.3%—4 b/a and 1.7%—4 a/a, being in Hardy–Weinberg equilibrium (PX0.29). There was no relationship between the eNOS gene polymorphisms and ABP or LVMI either in parents or their offspring. Among parents, carriers of the 298Asp allele had higher IMT values as compared with Glu/Glu homozygotes (0.94 vs 0.70 mm; P ¼ 0.007). Among offspring, there was a similar tendency (0.60 vs 0.53 mm; P ¼ 0.10), which was confirmed by transmission disequilibrium tests for quantitative variables (PX0.07). Our findings indicate that the Glu298Asp polymorphism of eNOS identifies patients with larger carotid IMT, also in younger subjects. Journal of Human Hypertension (2005) 19, 413–420. doi:10.1038/sj.jhh.1001837 Published online 3 March 2005

Keywords: atherosclerosis; blood pressure; genetics; nitric oxide synthase

Introduction Nitric oxide (NO) in endothelial cells is synthesized from L-arginine by a catalyst enzyme—endothelial nitric oxide synthase (eNOS). A constitutive isoform of eNOS gene is expressed in vascular endothelium.1 Mice lacking eNOS gene have higher blood pressure, insulin resistance, hyperlipidaemia2 and advanced atherosclerosis.3 The significant role of

Correspondence: Dr D Czarnecka, First Cardiac Department, Medical College, Jagiellonian University, Kopernika 17 Str, 31-501 Cracow, Poland. E-mail: [email protected] Received 7 October 2004; revised and accepted 16 December 2004; published online 3 March 2005

NO in the regulation of basal and inducible vasorelaxation suggests that abnormal eNOS activity caused by eNOS mutation may be associated with a number of pathologies such as arterial hypertension or atherosclerosis.4 Furthermore, it has been shown that NO release and synthesis are decreased in patients with atherosclerosis.5 The endothelium-derived NO is significantly involved in the regulation of vascular wall tone.6 Disruption of the gene encoding eNOS in mice results in the development of arterial hypertension,7 and humans with hypertension have been found to have impaired NO synthesis.8,9 Experimental inhibition of NO synthesis accelerates formation of early plaque.10 Genetic variability is a highly probable factor predisposing to the development and progression

eNOS and vascular remodelling D Czarnecka et al 414

of atherosclerosis. Functional polymorphisms in the gene encoding eNOS may modulate early stages of this disease. A number of polymorphisms in the eNOS gene have been identified so far. Point mutation in exon 7 of the eNOS gene, resulting in a guanine-thymine substitution (G-T) at position 1917 and leading to a substitution of glutamic acid for aspartic acid (Glu298Asp), seems to affect the function of eNOS protein.11 Furthermore, a 27-base-pair (27-bp) repeat polymorphism in intron 4 has been identified. A larger 4b allele contains five repeats of the 27-bp fragment, whereas the smaller one contains only four and is associated with a lower level of NO metabolites.12,13 As NO seems to have multiple atheroprotective effects, we hypothesized that the eNOS Glu298Asp polymorphism may be a risk factor for susceptibility to cardiovascular alterations. In the present study of families, selected at random in the general population, we analysed the relationship between polymorphisms of the eNOS gene (in exon 7 and intron 4) and ambulatory blood pressure (ABP), left ventricular mass (LVM), intimamedia thickness (IMT) and pulse wave velocity (PWV).

ventricular dysfunction after myocardial infarction (n ¼ 2) or valvular heart disease (n ¼ 1) were excluded from further analysis, and finally 294 subjects were available for analysis. Conventional blood pressure was measured five times consecutively at each of the two separate home visits. Casual blood pressure was taken as a mean of five consecutive measurements at the second home visit. Arterial hypertension was diagnosed if the mean of five measurements was X140 mmHg for systolic and/or 90 mmHg for diastolic blood pressure, or when the participant was taking antihypertensive agents. Oscillometric blood pressure monitors (SpaceLabs 90207, Redmond, WA, USA) were programmed to measure blood pressure every 15 min between, 08:00 and 22:00, and every 30 min between 22:00 and 08:00. In accordance with the most recent recommendations,17 the waking period was from 10:00 to 20:00, and the sleeping period from 24:00 to 06:00. A standardized questionnaire was administered to obtain personal medical history, family history, data on certain beverages, physical activity and chronic consumption of drugs.

Methods

Echocardiographic and vascular measurements

Epidemiological methods

Echocardiograms were performed using a HewlettPackard Sonos 2000 with a 3.5 MHz transducer. Mmode echocardiographic measurements of the left ventricle were obtained in the parasternal long axis guided by 2D echocardiography, with a crosssectional axis below the mitral valve at the level of the posterior tendinous chord. According to the recommendations of the American Society of Echocardiography, the following measurements were performed: left ventricular internal dimension (LVID), interventricular septal (IVS) and posterior wall thickness (PWT).18,19 An average of measurements obtained during three consecutive cardiac cycles was taken for statistical analysis. Left ventricular end-diastolic dimensions were used to calculate LVM19 according to an anatomically defined formula.20 Left ventricular mass index (LVMI) was obtained by dividing LVM by body surface area.20 Carotid artery examinations were performed with a Hewlett-Packard Sonos 2000 scanner equipped with a 7.5 MHz transducer. The IMT was measured on both body sides, on the proximal and distal common carotid artery wall about 2.0 cm from the bifurcation.21 The same observer (AO) provided all echocardiographic as well as carotid IMT measurements. The intraobserver intrasession reproducibility coefficient was 2.5% for LVM and 3.2% for carotid IMT. PWV was measured with a Compliors device (Complior, Colson, Garges les Genosse). The TY-36Fukuda pressure transducers were placed over the common carotid artery and the femoral artery on the

The study population was recruited in the European Project on Genes in Hypertension (EPOGH).14 The epidemiological methods used in the EPOGH study were previously described in detail.15 In addition to the standardized EPOGH protocol, the Polish participants underwent echocardiography, vascular measurements and genotyping for eNOS gene polymorphisms. The study was conducted in accordance with the Declaration of Helsinki recommendations,16 and it was approved by the Bioethics Committee at Jagiellonian University Medical College in Krako´w. All participants gave their written consent to the study. Nuclear (two-generation) families, consisting of at least one parent and at least two siblings, all family members ranging in age from 18 to 60 years, were recruited from the general population. Based on the voting lists, the index persons were randomly selected and enquiries were made about their family structure and willingness to participate in the study. Of the 158 nuclear families fulfilling the inclusion criteria, members of 54.5% (n ¼ 86) families agreed to participate in the study. The index persons who did not agree to participate in the study were slightly older than those who were willing to participate (mean age 38.575.2 vs 34.274.8 years, Po0.05). Epidemiological data were obtained from 326 subjects. Those (1) who did not agree to undergo echocardiographic and vascular examinations (n ¼ 27), (2) in whom the quality of echocardiograms was insufficient (n ¼ 2) and (3) who had left Journal of Human Hypertension


right side. PWV was calculated by dividing the distance between the transducers by the distance between the peaks of carotid and femoral pulse waves. The result was averaged from 20 measurements. Genetic measurements

Genome DNA was extracted from peripheral blood leucocytes using the Qiagen DNA kit. Genotypes were determined by polymerase chain reaction (PCR) using PCR kit Qiagen and Tag polymerase Qiagen Company. The VNRT polymorphisms in intron 4 of the eNOS gene were determined according to Wang et al,22 using the 27-bp primers: sense 50 -GGC CCT ATG GTA GTG CCT TT-30 and antisense 50 -TCT CTT AGT GCT GTG GTC AC-30 (DNA-Gdansk Company). The PCR products were separated on 3% agarose gel.22 The lengths of the separated fragments were 441 bp (five repeats—4b) and 414 bp (four repeats—4a) allowing the identification of three genotypes: b/b, b/a, a/a. The Glu298Asp polymorphism of eNOS gene was determined by the polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). The specific sense and antisense primers 50 -AAG GCA GGA GAC AGT GGA TGGA-30 and 50 -CCC AGT CAA TCC CTT TGG TGC TCA-30 (DNA-Gdansk Company) were used in the PCR reaction. The PCR products were digested with restriction enzyme BanII (GibcoBRL); the mutant allele (T) has no BanII cutting side. The digestion products were separated on a 2% agarose gel and visualized by ethidium bromide staining.23 Statistical analysis

The SAS, version 8.2 software (SAS Institute Inc., Cary, NC, USA), was used to create database and perform most statistical analyses. A traditional test of association and family-based analysis were performed in the study group. Association analysis was based on the GEE method (generalized estimating equations) allowing for consideration of covariables and within-family relationships.24 Quantitative transmission disequilibrium tests (QTDT) were performed in family-based analysis using three methods: an orthogonal model proposed by Abecasis et al25,26 in the QTDT software, version 2.3 (http://www.sph.umich.edu/csg/abecasis/QTDT), Allison model27 and a logistic regression model.28

Results The study population (n ¼ 294) consisted of 127 parents and 167 offspring. Subject characteristics are summarized in Table 1. Mean age (7s.d.) was 50.674.8 years for parents and 24.074.7 years for offspring. Body mass index (28.774.8 vs 22.873.2 kg/m2), blood pressure for the entire 24 h (124.1711.4/76.477.7 vs 117.578.5/67.97

6.4 mmHg), LVMI (103.3729.5 vs 91.3717.9 g/m2), carotid arterial IMT (0.8070.44 vs 0.5770.27 cm) and PWV (10.571.6 vs 8.371.2 m/s) were higher in parents than in offspring (Po0.001 for all variables). Arterial hypertension diagnosed from conventional blood pressure measurements was significantly more frequent in parents than in offspring (55.9 vs 13.2%, P ¼ 0.001). Among parents, 38 subjects (29.9%) were regular cigarette smokers at the time of the study, and 25 subjects (24.6%) admitted alcohol consumption equal to or exceeding 5 g of pure ethanol daily. Among offspring, the numbers corresponded, respectively, to 41 (24.6%) and 33 subjects (19.8%). Differences between sexes showed a similar tendency in both groups and are summarized in Table 1. The frequencies of Glu298Asp (P ¼ 0.68) and intron 4 (P ¼ 0.29) polymorphisms of the eNOS gene were in Hardy–Weinberg equilibrium. The frequencies of genotypes and alleles did not differ between hypertensives and normotensives (Table 2). The following variables were considered in statistical analysis of blood pressure: age, sex, body mass index, antihypertensive treatment, smoking and regular alcohol intake. Analysis of LVM and IMT included age, sex, conventional systolic blood pressure, antihypertensive treatment and the above-mentioned lifestyle factors. Analysis of PWV additionally included heart rate at the time of the study. Analysis of offspring additionally included within-family relationships between phenotypes and genotypes. There was no relationship between the Glu298Asp polymorphisms of eNOS gene and ABP either in parents or their offspring (Tables 3 and 4). Similarly, LVMI did not differ between genotypes of Glu298Asp polymorphisms in both study groups. Among parents, carriers of the 298Asp allele had higher IMT values as compared with Glu/Glu homozygotes (0.94 vs 0.70 mm; P ¼ 0.007; Table 3). Among offspring, there was a similar tendency (0.60 vs 0.53 mm; P ¼ 0.10; Table 4). Neither in parents nor in offspring did we observe any association between the Glu298Asp polymorphism of eNOS and PWV. The 27-bp repeat polymorphism in intron 4 was not associated with the study phenotypes either (Tables 3 and 4). An orthogonal model proposed by Abecasis did not show stratification of the study population for any phenotype (PX0.45). Among offspring, the three QTDT models revealed a tendency towards the relationship between the Glu298Asp polymorphism of eNOS gene and carotid IMT (Table 5). There was no relationship between the investigated eNOS gene polymorphisms in intron 4 and the study phenotypes (Table 5).

Discussion The present study was undertaken to assess phenotypes of the Glu298Asp and intron 4 polymorphisms of eNOS gene. The leading finding among parents Journal of Human Hypertension


Table 1 Characteristics of the study population by generation and sex Fathers (N ¼ 51)

Mothers (N ¼ 76)

Sons (N ¼ 84)

Clinical characteristics Age (years) Body mass index (kg/m2) Systolic blood pressure (mmHg)a Diastolic blood pressure (mmHg)a Hypertension (%) Diabetes mellitus (%) Obesity (%) Antihypertensive treatment (%) Lipid-lowering drugs (%) Glucose-lowering drugs (%) Smokers (%) Ethanol X5 g/day (%)

51.2 28.2 136.2 86.6 28 8 17 14 2 2 19 21

(4.3) (3.7) (17.3) (11.4) (54.9%) (15.4%) (33.3%) (27.5%) (3.9%) (3.9%) (37.3%) (41.2%)

50.2 29.0 135.3 85.6 43 3 26 36 1 1 19 4

(5.1) (5.4) (18.7) (9.1) (56.6%) (3.9%)* (34.2%) (47.4%)* (1.3%) (1.3%) (25.0%) (5.3%)***

23.3 23.0 125.1 76.6 18

Ambulatory blood pressure monitoring Systolic BP—entire 24 h (mmHg) Diastolic BP—entire 24 h (mmHg) Systolic BP—day (mmHg) Diastolic BP—day (mmHg) Systolic BP—night (mmHg) Diastolic BP—night (mmHg)

126.2 78.8 131.9 83.7 116.5 70.8

(10.2) (7.4) (11.0) (7.7) (12.5) (9.7)

122.7 74.8 129.5 80.7 111.5 65.4

(12.0) (7.5)** (12.4) (7.7)* (13.1)* (8.9)**

121.3 68.6 129.1 75.0 109.6 58.6

Echocardiographic measurements Left ventricular mass (g) Left ventricular mass index (g/m2)

228.5 (72.8) 114.5 (37.6)

Vascular measurements Carotid artery IMT (mm) Pulse wave velocity (m/s)

0.78 (0.33) 10.5 (1.4)

171.8 (40.8)*** 95.7 (19.3)***

1 7 27 29

Daughters (N ¼ 83)

(4.5) (3.0) (11.9) (10.6) (21.4%) 0 (1.2%) (8.3%) 0 0 (32.1%) (34.5%)

24.8 22.7 113.6 71.2 4

(7.5) (6.9) (8.0) (7.9) (8.8) (7.6)

113.7 67.1 120.8 74.2 102.8 56.6

2 2 14 4

(4.9)* (3.5) (10.8)*** (7.2)*** (4.8%)** 0 (2.4%) (2.4%)* 0 0 (16.9%)* (4.8%)*** (7.7)*** (5.9) (8.7)*** (7.0) (7.8)*** (5.6)*

186.1 (42.4) 97.3 (19.2)

143.7 (28.9)*** 85.3 (14.3)***

0.58 (0.33) 8.6 (1.1)

0.55 (0.19) 8.0 (1.3)**

0.82 (0.51) 10.5 (1.7)

Results are given as arithmetic means (s.d.) or numbers of participants (%). BP: blood pressure; IMT: intima-media thickness. Statistical significance of differences between sexes within each generation: *Po0.05, **Po0.01 and ***Po0.001. a Average of five measurements obtained at second visit.

Table 2 Frequency of genotypes and alleles Polymorphism

Genotypes

Pa

Alleles

Glu/Glu

Glu/Asp

Asp/Asp

Glu

Asp

162 (55.1%)

118 (40.1%)

14 (4.8%)

442 (75.2%)

146 (24.8%)

Hypertension Normal BP

51 (54.8%) 111 (55.2%)

40 (43.0%) 78 (38.8%)

2 (2.2%) 12 (6.0%)

142 (76.3%) 300 (74.0%)

44 (23.7%) 102 (26.0%)

Intron 4 (4a/4b) All subjects

4 b/b 191 (65.0%)

4 b/a 98 (33.3%)

4 a/a 5 (1.7%)

4b 480 (81.7%)

4a 108 (18.3%)

Hypertension Normal BP

59 (63.4%) 132 (65.7%)

33 (35.5%) 65 (32.3%)

1 (1.1%) 4 (2.0%)

151 (81.2%) 329 (81.8%)

35 (18.8%) 73 (18.2%)

Glu298Asp All subjects

0.54

0.85

a

Hypertension vs normal BP.

was more advanced carotid artery remodelling measured from the IMT in carriers of the 298Asp allele. A similar tendency, later confirmed by family structure analysis, was observed in offspring. There was no relationship between the polymorphisms and ABP, LVMI or PWV. Atherosclerosis is the leading cause of mortality in industrialized countries.29 Dysfunction and morphological endothelial changes appear to be critical Journal of Human Hypertension

for the occurrence and progression of clinical manifestation of atherosclerosis.30 NO is one of the most important endothelium-derived substances that affects importantly the function and morphology of the vascular wall. For this reason, eNOS gene is a target gene for atherosclerosis. Computer-aided analysis of eNOS protein revealed that mutation of the Glu298Asp modifies the particle conformation,31 suggesting that the presence of the 298Asp allele


Table 3 Cardiovascular phenotypes by eNOS genotype—associations among parents Polymorphism

Genotypes Statistics (s.e.)

PANOVA

P

Glu298Asp Systolic BP—day (mmHg) Diastolic BP—day (mmHg) Systolic BP—night (mmHg) Diastolic BP—night (mmHg) Left ventricular mass index (g/m2) Carotid intima-media thickness (mm) Pulse wave velocity (m/s)

Glu/Glu (N ¼ 73) 130.9 (1.3) 81.8 (0.9) 113.9 (1.4) 67.2 (1.0) 105.4 (3.1) 0.70 (0.05) 10.4 (0.2)

Glu/Asp (N ¼ 46) 129.2 (1.7) 82.1 (1.1) 112.5 (1.8) 67.8 (1.3) 98.3 (3.9) 0.92 (0.07) 10.6 (0.3)

Asp/Asp (N ¼ 8) 133.9 (4.0) 82.1 (2.7) 115.6 (4.3) 69.0 (3.1) 112.7 (9.5) 1.10 (0.18) 10.6 (0.7)

0.48 0.98 0.74 0.83 0.22 0.02 0.78

Asp carriers vs Glu/Glu 0.59 0.83 0.68 0.63 0.30 0.007 0.48

Intron 4 (a/b) Systolic BP—day (mmHg) Diastolic BP—day (mmHg) Systolic BP—night (mmHg) Diastolic BP—night (mmHg) Left ventricular mass index (g/m2) Carotid intima-media thickness (mm) Pulse wave velocity (m/s)

4 b/b (n ¼ 83) 129.7 (1.2) 81.5 (0.8) 113.9 (1.3) 67.8 (0.9) 105.8 (2.9) 0.84 (0.05) 10.6 (0.2)

4 b/a (n ¼ 43) 131.5 (1.7) 82.5 (1.2) 112.5 (1.9) 66.8 (1.3) 99.0 (4.1) 0.73 (0.08) 10.4 (0.3)

4 a/a (n ¼ 1) 146.7 (11.3) 88.0 (7.7) 123.4 (12.2) 74.7 (8.7) 75.6 (27.0) 0.96 (0.43) 10.7 (1.5)

0.25 0.59 0.62 0.60 0.25 0.51 0.87

4a carriers vs 4 b/b 0.32 0.44 0.66 0.62 0.15 0.30 0.63

Results are given as means (s.e.) adjusted for sex, age, antihypertensive medication, smoking and regular alcohol consumption. Additionally, blood pressure phenotypes are adjusted for body mass index, left ventricular mass index and vascular phenotypes for systolic blood pressure and pulse wave velocity for heart rate.

Table 4 Cardiovascular phenotypes by eNOS genotype—associations among offspring Polymorphism

Genotypes Statistics (s.e.)

PANOVA

P

Glu298Asp Systolic BP—day (mmHg) Diastolic BP—day (mmHg) Systolic BP—night (mmHg) Diastolic BP—night (mmHg) Left ventricular mass index (g/m2) Carotid intima-media thickness (mm) Pulse wave velocity (m/s)

Glu/Glu (N ¼ 89) 124.4 (0.9) 74.2 (0.8) 106.2 (0.9) 57.9 (0.7) 92.2 (1.8) 0.53 (0.04) 8.2 (0.1)

Glu/Asp (N ¼ 72) 125.4 (1.0) 75.2 (0.8) 106.0 (1.0) 57.4 (0.8) 90.8 (2.0) 0.55 (0.04) 8.5 (0.2)

Asp/Asp (N ¼ 6) 129.5 (3.4) 73.0 (2.9) 108.9 (3.8) 73.0 (2.9) 84.4 (6.9) 0.60 (0.17) 8.9 (0.8)

0.25 0.31 0.69 0.49 0.10 0.33 0.26

Asp carriers vs Glu/Glu 0.20 0.25 0.65 0.86 0.25 0.10 0.14

Intron 4 (a/b) Systolic BP—day (mmHg) Diastolic BP—day (mmHg) Systolic BP—night (mmHg) Diastolic BP—night (mmHg) Left ventricular mass index (g/m2) Carotid intima-media thickness (mm) Pulse wave velocity (m/s)

4 b/b (n ¼ 108) 125.5 (0.8) 74.1 (0.7) 105.7 (0.8) 57.2 (0.6) 91.9 (1.6) 0.57 (0.03) 8.4 (0.1)

4 b/a (n ¼ 55) 126.6 (1.1) 76.0 (0.9) 107.4 (1.1) 58.5 (0.8) 90.2 (2.3) 0.57 (0.05) 8.2 (0.2)

4 a/a (n ¼ 4) 117.4 (4.1) 69.4 (3.5) 101.7 (4.3) 56.6 (3.2) 91.4 (8.6) 0.62 (0.20) 7.7 (0.8)

0.18 0.18 0.28 0.45 0.68 0.34 0.58

4a carriers vs 4 b/b 0.28 0.33 0.35 0.29 0.70 0.98 0.44

Results are given as means (s.e.) adjusted for sex, age, antihypertensive medication, smoking and regular alcohol consumption. Additionally, blood pressure phenotypes are adjusted for body mass index, left ventricular mass index and vascular phenotypes for systolic blood pressure and pulse wave velocity for heart rate.

may decrease the activity of eNOS. This is in accordance with the findings of Philip et al,32 who demonstrated increased vasoconstriction in response to phenylephrine in homozygotes Asp/Asp, which may be accounted for by impaired NOderived modulation of adrenergic-dependent vasoconstriction. Also Leeson et al33 observed marked endothelial dysfunction in male smokers, carriers of the 298Asp allele of eNOS gene. Guzik et al34 demonstrated in isolated vessels that there is a relationship between the Glu298Asp polymorphism and NO-mediated endothelial function.

Carotid IMT is related to the frequency of cardiovascular diseases and is a risk factor for cardiovascular events.35,36 The higher risk for the development of symptoms of vascular remodelling and early atherosclerosis in carriers of the 298Asp allele of eNOS gene in the present study may suggest that the associated altered eNOS protein structure aggravates atherosclerotic mechanisms. Increased IMT might be also associated with older age, male gender or higher systolic blood pressure. In our study, however, the Glu298Asp genotypes were not different with regard to age, gender distribution or Journal of Human Hypertension


Table 5 Results of QTDT Model

n, probands/ offspring

Systolic BP day

Diastolic BP day

Systolic BP night

Diastolic BP night

LVMI

Carotid IMT

w2

P

w2

P

w2

P

w2

P

w2

P

w2

P

PWV

w2

P

Glu298Asp Orthogonal Allison Logistic

75/167 54/167 71

1.52 2.40 0.39

0.22 0.30 0.53

0.64 1.66 0.29

0.42 0.50 0.59

0.34 2.06 0.44

0.56 0.36 0.51

0.58 4.40 0.08

0.45 0.10 0.77

1.76 2.31 1.58

0.18 0.31 0.21

2.53 3.25 3.51

0.08 0.07 0.08

0.88 0.35 0.89 0.35 0.51 0.48

Intron4 b/a Orthogonal Allison Logistic

91/167 63/167 42

0.19 0.09 1.18

0.66 0.77 0.28

0.01 0.90 1.02

0.93 0.34 0.31

0.78 0.02 0.05

0.38 0.96 0.83

0.88 0.18 0.01

0.35 0.67 0.94

0.07 0.07 0.12

0.79 0.80 0.73

0.01 0.59 1.28

0.98 0.44 0.26

0.04 0.84 0.01 0.94 0.31 0.58

BP: blood pressure; LVMI: left ventricular mass index; IMT: intima-media thickness; PWV: pulse wave velocity.

blood pressure level. We may only speculate that increased carotid IMT in carriers of the 298Asp allele of eNOS gene reflects their susceptibility to overreaction with vascular remodelling even to blood pressure values similar to 298Glu homozygotes. The Glu298Asp polymorphism of eNOS and its contribution to atherosclerosis has been extensively studied, yielding controversial results in various populations. As in our study, Lembo et al37 found in a group of patients screened for hypertension an excess of Asp/Asp homozygotes among subjects who had atherosclerotic plaques on their carotid arteries compared with subjects without carotid plaques. Conversely, the SAPHIR study and observations in the Finnish population did not reveal the effect of the Glu298Asp polymorphism on the carotid arterial IMT.38,39 Similarly Markus et al40 found that this particular polymorphism was not associated with the presence of transient cerebral attacks (TIA), stroke or carotid arterial stenosis in the British population. Our findings are also in concordance with the observations in eNOS knockout mice, which have been found to have increased IMT.41 In the present study, polymorphism in intron 4 of eNOS gene was not associated with carotid IMT. Conversely, Japanese investigators in haemodialysed patients found the odds ratio for carotid plaque was increased to 3.72 by the 4a allele of the intron 4 polymorphism.42 In the present study, among offspring, we have not observed any association between the Glu298Asp polymorphism and PWV. Similarly, other investigators did not find the relationship between this particular polymorphism and arterial stiffness.43 ABP as a continuous variable was not associated with Glu298Asp or intron 4 polymorphisms of eNOS in the present study. There were no differences in the frequency of genotypes and alleles between hypertensives and normotensives. Several studies have been undertaken to determine the relationship between eNOS gene and arterial hypertension, demonstrating an association between the Journal of Human Hypertension

Glu298Asp polymorphism of eNOS and hypertension. However, studies in various ethnic populations produced conflicting results. In the French population, the 298Glu allele was more common in hypertensives as compared with the controls,43 whereas the population-based Suita Study in Japan involving 4055 subjects did not find differences in blood pressure with respect to the Glu298Asp polymorphism.44 There are also negative findings in the white Caucasian populations.45,46 As regards the 4a allele of the eNOS gene, Pulkkinen et al47 demonstrated an association with elevated blood pressure. The association of this particular allele with elevated blood pressure was also found in the Japanese population.48,49 Conversely, Anglo-Celtic Australians did not show the association between the 4a allele of eNOS and arterial hypertension.46 In the present study, there was no relationship between any of the polymorphisms of eNOS and LVM. In accord with our findings, Karvonen et al45 did not find the association between the Glu298Asp polymorphism of eNOS and LVM in the Finnish population. The association between the Glu298Asp polymorphism and LVM has been found only in the Japanese population.50 The present findings should be interpreted after consideration of possible study limitations. The data obtained in our study might only be generalized for the subjects with age ranging from 18 to 60 years. The discrepancies between our findings and those of other groups might depend on several factors, including selection of subjects. Although our study is population-based, we only recruited members of two-generation families, whereas other studies had either the case–control37,38,40 or population-based cross-sectional39 design. However, in our study, the two-generation groups had increased IMT in the carotid artery in carriers of the 298Asp allele of eNOS, confirmed by QTDT tests (Table 6). The current findings suggest that screening for the 298Asp allele of the eNOS gene might detect subjects specially prone to vascular remodelling and early development of atherosclerosis.


Table 6 Already available and added information on the role of the Glu298Asp polymorphism of the eNOS gene in carotid atherosclerosis Status

Information

Already known

Glu298Asp mutation modifies the eNOS particle 298Asp variant is associated with endothelial dysfunction Asp/Asp genotype more frequent in patients with atherosclerotic plaques in carotid arteries eNOS knockout mice have increased IMT

Novel findings

298Asp variant is associated with increased carotid IMT in subjects aged 18–60 years

In conclusion, our study shows that the Glu298Asp polymorphism of the eNOS gene identifies patients with increased carotid arterial IMT, even in younger age groups. However, there is no relationship between this particular polymorphism, LVM or PWV. The 27-bp repeat polymorphism in intron 4 of eNOS was not related to cardiovascular phenotypes in our population.

Acknowledgements Nuclear families were recruited for the European Project on Genes in Hypertension, supported by the European Union (contract no. IC15-CT98-0329EPOGH). The present study was supported by a grant from the State Committee for Research (Warsaw, Poland), Grant No. 6PO5B 134 21.

References 1 Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993; 329: 2002–2012. 2 Duplain H, Burcelin R, Sartori C. Insulin resistance, hyperlipidaemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation 2001; 104: 342–345. 3 Kuhlencordt PJ, Gyurko R, Han F. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation 2001; 104: 448–454. 4 Soubrier F. Nitric oxide synthase genes. Candidate genes among many others. Hypertension 1999; 33: 924–926. 5 Oemar BS et al. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 1998; 97: 2494–2498. 6 Vallance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989; 2: 997–1000. 7 Huang PL et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995; 377: 239–242. 8 Forte P et al. Basal nitric oxide synthesis in essential hypertension. Lancet 1997; 349: 837–842. 9 Taddei S et al. Defective L-arginine–nitric oxide pathway in offspring of essential hypertensive patients. Circulation 1996; 94: 1298–1303.

Reference(s) 31 33,34 37 41

10 Cayatte A, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb 1994; 14: 753–759. 11 Tesauro M et al. Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary disease: cleavage of proteins with aspartate vs glutamate at position 298. Proc Natl Acad Sci USA 2000; 97: 2832–2835. 12 Tsukada T et al. Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Com 1998; 245: 190–193. 13 Wang XL et al. 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. 14 Kuznetsova T et al. Quality control of the blood pressure phenotype in the European Project on Genes in Hypertension. Blood Pressure Monit 2002; 7: 215–224. 15 Staessen JA, Fagard R, Amery A. Life style as a determinant of blood pressure in the general population. Am J Hypertens 1994; 7: 685–694. 16 41st World Medical Assembly. Declaration of Helsinki: recommendations guiding physicians in biomedical research involving human subjects. Bull Pan Am Health Organ 1990; 24: 606–609. 17 van Ittersum FJ, IJzerman RG, Stehouwer CDA, Donker AJM. Analysis of twenty-four-hour ambulatory blood pressure monitoring: what time period to assess blood pressure during waking and sleeping. J Hypertens 1995; 13: 1053–1058. 18 Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978; 58: 1072–1083. 19 Schiller NB et al. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American society of echocardiography committee on standards, subcommittee on quantitation of twodimensional echocardiograms. J Am Soc Echocardiogr 1989; 2: 358–367. 20 Devereux RB et al. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings. Am J Cardiol 1986; 57: 450–458. 21 Wendelhag I, Wiklund O, Wikstrand J. Atherosclerotic changes in the femoral and carotid arteries in familial hypercholesterolemia. Ultrasonographic assessement of intima-media thickness and plaque occurence. Arterioscler Thromb 1993; 13: 1404–1411. 22 Wang XL et al. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the Journal of Human Hypertension


23

24 25 26 27 28

29 30 31 32

33

34

35 36 37

38

endothelial nitric oxide synthase gene. Nat Med 1996; 2: 41–45. Shimasaki Y et al. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. J Am Coll Cardiol 1998; 31: 1506–1510. The SAS Institute. The GENMOD Procedure, SAS Online Doc Version 7.1: SAS/STAT. The SAS Institute Inc.: Cary, NC, 2000, pp 1311–1411. Abecasis GR, Cardon LR, Cookson WOC. A general test of association for quantitative traits in nuclear families. Am J Hum Genet 2000; 66: 279–292. Abecasis GR, Cookson WOC, Cardon LR. Pedigree tests of transmission disequilibrium. Eur J Hum Genet 2000; 8: 545–551. Allison DB. Transmission-disequilibrium test for quantitative traits. Am J Hum Genet 1997; 60: 676–690. Waldman ID, Robinson BF, Rowe DC. A logistic regression based extension of the TDT for continuous and categorical traits. Ann Hum Genet 1999; 63: 329–340. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990 s. Nature 1993; 362: 801–809. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340: 115–126. Cooke JP. Role of nitric oxide in progression and regression of atherosclerosis. West J Med 1996; 164: 419–424. Philip I et al. G894 T polymorphism in the endothelial nitric oxide synthase gene is associated with an enhanced vascular responsiveness to phenilephrine. Circulation 1999; 99: 3096–3098. Leeson CPM et al. Glu298Asp endothelial nitric oxide synthase gene polymorphism interacts with environmental and dietary factors to influence endothelial function. Circ Res 2002; 90: 1153–1158. Guzik TJ, Black E, West NE. Relationship between the G894T polymorphism (Glu298Asp variant) in endothelial nitric oxide synthase and nitric-oxide mediated endothelial function in human atherosclerosis. Am J Med Genet 2001; 100: 130–137. Bots ML et al. Common carotid intima-media thickness and risk of stroke and myocardial infarction. Circulation 1997; 96: 1432–1437. Hodis HN et al. The role of carotid arterial intimamedia thickness in predicting clinical coronary events. Ann Intern Med 1998; 128: 262–269. Lembo G et al. A common variant of endothelial nitric oxide synthase (Glu298Asp) is an independent risk factor for carotid atherosclerosis. Stroke 2001; 32: 735–740. Karvonen J et al. Endothelial nitric oxide synthase gene Glu298Asp polymorphisms and blood pressure, left ventricular mass and carotid artery atherosclerosis

Journal of Human Hypertension

39

40

41 42

43

44

45

46

47

48 49

50

in a population-based cohort. J Intern Med 2002; 251: 102–110. Schmoelzer I et al. Lack of association of the Glu298Asp polymorphism of endothelial nitric oxide synthase with manifest coronary artery disease, carotid atherosclerosis and forearm vascular reactivity in two Austrian populations. Eur J Clin Invest 2003; 33: 191–198. Markus HS, Ruigrok Y, Ali N, Powell JF. Endothelial nitric oxide synthase exon 7 polymorphism and ischaemic cerebrovascular disease and carotid atheroma. Stroke 1998; 32: 735–740. Rudic RD et al. Direct evidence for the importance of endothelial-derived nitric oxide in vascular remodeling. J Clin Invest 1998; 101: 731–736. Asakimori Y, Yorioka N, Tanaka J, Kohno N. Effect of polymorphism of the endothelial nitric oxide synthase and apolipoprotein E genes on carotid atherosclerosis in hemodialysis patients. Am J Kidney Dis 2003; 41: 822–832. Lacolley P et al. Nitric oxide synthase gene polymorphisms, blood pressure and aortic stiffness in normotensive and hypertensive subjects. J Hypertens 1998; 16: 31–35. Tsujita Y et al. Association analyses between genetic polymorphisms of endothelial nitric oxide synthase gene and hypertension in Japanese: The Suita Study. J Hypertens 2001; 19: 1941–1948. Karvonen J et al. Endothelial nitric oxide synthase gene Glu298Asp polymorphisms and blood pressure, left ventricular mass and carotid artery atherosclerosis in a population-based cohort. J Intern Med 2002; 251: 102–110. Benjafield AV, Morris BJ. Association analyses of endothelial nitric oxide synthase gene polymorphisms in essential hypertension. Am J Hypertens 2000; 13: 994–998. Pulkkinen A et al. Intron 4 polymorphism of the endothelial nitric oxide synthase gene is associated with elevated blood pressure in type 2 diabetic patients with coronary heart disease. J Mol Med 2000; 78: 372–379. Morita T et al. Effect of polymorphism of endothelial nitric oxide synthase gene in Japanese patients with IgA nephropathy. Clin Nephrol 1999; 52: 203–209. Uwabo J, Soma M, Nakayama T, Kanmatsuse K. Association of a variable number of tandem repeats in the endothelial nitric oxide synthase gene with essential hypertension in Japanese. Am J Hypertens 1998; 11: 125–128. Takaoka M et al. Association of the missense Glu298Asp variant of the eNOS gene with left ventricular hypertrophy and carotid atherosclerosis in patients with essential hypertension. Atherosclerosis 2001; 2: 42–43.