Association between the NOS3 (−786 T/C) and the ACE (I/D) DNA Genotypes and Early Coronary Artery...

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NITRIC OXIDE: Biology and ChemistryVol. 5, No. 4, pp. 343–348 (2001)doi:10.1006/niox.2001.0351, available online at http://www.idealibrary.com on

Association between the NOS3 (2786 T/C) and the ACEI/D) DNA Genotypes and Early Coronary Artery Disease

Ruth Alvarez,* Pelayo Gonzalez,* Alberto Batalla,† Julian R. Reguero,†ustavo Iglesias-Cubero,† Sergio Hevia,† Arturo Cortina,† Esther Merino,†

gnacio Gonzalez,* Victoria Alvarez,* and Eliecer Coto*,1

*Laboratorio de Genetica Molecular-Instituto de Investigacion Nefrologica and †Servicio de Cardiologıa,ospital Central de Asturias, Oviedo, Spain

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Received July 18, 2000, and in revised form October 24, 2000; pu

DNA polymorphisms at the endothelium constitu-tive nitric oxide synthase gene (NOS3) have beenlinked to the risk of developing coronary arterydisease (CAD). In vitro, a polymorphism in the 5*

region of the NOS3 gene (2786 T/C) influences pro-moter activity. This polymorphism has been associ-ated with coronary spasms among Japanese. Thegenetic variation at the angiotensin-converting en-zyme (ACE) is associated with plasma ACE activi-ties and has also been linked with susceptibility tocardiovascular disease. Our objective was to deter-mine if DNA polymorphisms in the NOS3 and ACEgenes were associated with early CAD. We analyzedthe 2786 T/C polymorphism in the 5* flanking re-gion and the 27-bp repeat polymorphism in NOS3intron 4, as well as the ACE-I/D polymorphism. Atotal of 170 male smokers (CAD patients) youngerthan 50 years and 300 male smokers (healthy con-trols) were genotyped. Frequencies were comparedby the x2 test, and odds ratios (ORs) and their 95%onfidence intervals (CI) were also calculated. Onlyhe 2786 T/C polymorphism in the 5* flanking re-

gion of the NOS3 gene was significantly associatedwith early CAD in our population. The frequency ofthe CC genotype was significantly increased (P 5

0.039) in patients compared to controls (OR 5 1.67;95% CI 5 1.01, 2.72). We found a synergistic effect

1 To whom correspondence should be addressed at Laboratorio
Genetica Molecular, Hospital Central de Asturias, 33006 Oviedo,Spain. Fax: 34 985 27 36 57.
1089-8603/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

d online June 19, 2001

between the NOS3-CC and the ACE-DD genotypes inthe risk of developing early CAD. The frequency ofCC 1 DD was significantly increased among pa-tients (P 5 0.002). Thus, those with a NOS3-CC and

n ACE-DD genotype would have a significantly in-reased risk of suffering an early episode of coro-ary artery disease (OR 5 2.82; 95% CI 5 1.40, 5.70).

Although based on a limited number of patients, ourwork suggests that individuals who are NOS3-CC 1

ACE-DD are at a higher risk for early CAD, probablyas a consequence of increased endothelialdysfunction. © 2001 Academic Press

Key Words: endothelial constitutive nitric oxidesynthase; angiotensin I converting enzyme; coro-nary artery disease.

Nitric oxide (NO, nitrogen monoxide) has a numberof important functions in the vessel wall, such as in-hibition of platelet aggregation and monocyte adhe-sion, prevention of smooth muscle cell proliferation,and modulation of vascular growth. In the vascularwall, NO diffuses from the endothelium to the vascularsmooth muscle cell, where it increases intracellularcGMP concentration, leading to relaxation of the vas-cular smooth muscle cell and to vasodilation (1–3).Thus, impairment of the NO–cGMP pathway can in-duce hypertension (4–7). On the other hand, NO can
also promote vasorelaxation indirectly by inhibitingthe release of renin and norepinephrine (8, 9).
343

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NO is synthesized from the amino acid L-arginineby nitric oxide synthase (NOS).2 Three distinct iso-forms of NOS, neuronal NOS (nNOS), endothelialconstitutive NOS (ecNOS), and inducible NOS(iNOS), have been identified in humans.

In the endothelium, NO is synthesized by theecNOS, and continuously generated NO serves tomaintain basal vascular tone (10–12). The ecNOS isencoded by a gene (NOS3) located on chromosome7q35–q36 (13–15). Recently, a DNA polymorphismin intron 4 of the NOS3 gene has been associatedwith plasma NO levels (16). This polymorphism hasbeen linked to the risk of developing coronary arterydisease (CAD) (17) and hypertension (18). A poly-morphism in the 59 flanking region of the NOS3gene (2786 T/C) has been associated with coronaryspasm among Japanese (19). In addition, a commonvariant of the NOS3 gene (Glu298 3 Asp) has alsobeen linked to the risk for coronary artery disease(20, 21).

Angiotensin I-converting enzyme (ACE) convertsangiotensin I into angiotensin II, a vasoconstrictor,and degrades bradykinin, a vasodilator. Thus, ACEplays a major role in the regulation of the vasomotortone of arteries. A DNA polymorphism at the ACEgene has been associated with plasma ACE activi-ties and with several cardiovascular diseases, in-cluding CAD (22–24).

Here, we studied whether the 2786 T/C and theintron 4 polymorphisms at the NOS3 gene wererelated to the risk for early CAD in the Spanishpopulation. In addition, we analyzed a possible syn-ergistic effect between the NOS3 and the ACE ge-notypes and early CAD.

METHODS

Patients and Controls

A total of 170 male patients who had suffered anepisode of coronary artery disease, defined accordingto the guidelines of the WHO MONICA protocol,were analyzed (25). All were smokers and youngerthan 50 years. All of the patients had at least oneangiographically confirmed diseased vessel (at least

2 Abbreviations used: NOS, nitric oxide synthase; n, neuronal;

344 ALVAR

e, endothelial constitutive; i, inducible; CAD, coronary arterydisease; ACE, angiotensin-converting enzyme.

Copyright © 2001 by Academic Press. All right

one stenosis involving .30% loss of lumen diame-ter). A total of 300 controls (hospital staff, bloodbank donors, and eligible residents) were recruitedfrom the same population. All of the controls weremale smokers and younger than 60 years (averageage 39 6 12 years). None of these controls had hadepisodes of cardiovascular disease. Patients and con-trols were Caucasians and residents of the sameregion (Asturias, Northern Spain), and most of themhad been previously described (26). Informed con-sent was obtained from each individual according tothe recommendations of the World Medical Associ-ation Declaration of Helsinki (27).

NOS3 (27-bp Repeat) Genotyping

Genomic DNA was extracted from peripheralblood leukocytes following a salting out method. Foranalysis of the 27 bp repeat polymorphism in intron4 of the NOS3 gene, we used the primers 59-CTATGGTAGTGCCTTGGCTGGAGG-39 (forward)and 59-ACCGCCCAGGGAACTCCGCT-39 (reverse).The polymerase chain reaction (PCR) was performedin a final volume of 15 ml, containing 100 ng ofgenomic DNA, and consisted of an initial denatur-ation at 95°C for 2 min, followed by 32 cycles of 30 sat 95°C, 1 min at 63°C, and 1 min at 72°C and a finalextension of 5 min at 72°C. PCR products of 195 bp(5 repeat alleles) and 168 bp (4 repeat alleles) wereseparated by electrophoresis on a 3% agarose geland visualized after ethidium bromide staining.

NOS3 (2786 T/C) Genotyping

The T to C change at nucleotide 2786 introducesa site for the restriction enzyme MspI. A sequence of180 bp was PCR-amplified as described for the 27-bprepeat polymorphism, with primers 59 TGGAGAGT-GCTGGTGTACCCCA 39 and 59 GCCTCCAC-CCCCACCCTGTC 39 (annealing at 62°C). After di-gestion with MspI, fragments of 40 bp (constant) and140 (2786 T) or 90 1 50 bp (2786 C) were visual-ized.

ACE (I/D) Genotyping

T AL.

The ACE-I/D polymorphism was analyzed as pre-viously described (28). This analysis included an

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additional PCR to confirm the absence of an I allelein DD individuals (29). The ACE alleles were visu-alized as fragments of 490 bp (I) and 190 bp (D).

Statistical Analysis

The frequencies of the alleles and genotypes inpatients and controls were compared by the Yates x2

test. Odds ratios and the 95% confidence interval(95% CI) were also calculated. Statistical analysiswas done with the BMDP-New Systems statisticalpackage (BMDP Statistical Software Ltd., Cork, Ire-land). P , 0.05 was considered significant.

RESULTS

We genotyped 170 male patients for the NOS3 andACE polymorphisms. All of the patients were male,younger than 50 years (average age 42 6 6 years),and smokers. In addition, 10% were also diabetics,31% were hypertensives, and 19% had hypercholes-terolemia (total cholesterol .250 mg/dl). Table Isummarizes the anthropometric characteristics andaverage lipoprotein values in these patients. We alsogenotyped 300 controls (all healthy male smokers).

Allele and genotype frequencies for the NOS3 andACE polymorphisms are summarized in Table I. The2786 T/C polymorphism was weakly associatedwith early CAD in our population. Individuals ho-mozygous for the C allele were at a significantlyincreased frequency (P 5 0.039) among patientscompared to controls (22% vs 14%). Allele and geno-type frequencies did not differ between hypertensive

TABLE I

Clinical Characteristics of and Average BiochemicalValues in the 170 Patients with Early CAD

and in the 300 Controls

Patients Controls

verage age 42 6 6 years 39 6 12mokers 170 (100%) 300 (100%)iabetics 17 (10%) 0ypertensives 52 (31%) 0otal cholesterol (mg/dl) 220 6 51 199 6 42DL (mg/dl) 32 6 8 54 6 13riglycerides (mg/dl) 183 6 91 120 6 72

NOS3 AND AC

and normotensive patients.The NOS3 intron 4 polymorphism was in Hardy-


Weinberg equilibrium, and allele and genotype fre-quencies did not differ between patients and con-trols (Table II). Gene and genotype frequencies didnot differ between the hypertensive and normoten-sive patients.

The ACE-DD genotype was associated with asligthly but not significantly increased risk of devel-oping early CAD (P 5 0.084; OR 5 1.39; 95% CI 5.95, 2.03). We also investigated a possible synergis-ic effect between the NOS3 and the ACE genotypesTable III). Among those with a DD genotype, 12% ofhe controls were CC (15/123) compared to 26% ofhe patients (22/85) (P 5 0.011). The frequency ofOS3-CC 1 ACE-DD individuals was significantly

increased (P 5 0.002) in total patients (13%; 22/170)compared to total controls (5%; 15/300). In our pop-ulation, carriers of both genotypes have a higherrisk of suffering an early episode of CAD (OR 5 2.82;95% CI 5 1.40, 5.70).

DISCUSSION

Early CAD is strongly associated with tobaccoconsumption in our population. According to ourdata, 95% of the patients who suffered an early (,50years old) episode of myocardial infarction weresmokers. Hypercholesterolemia (total cholesterol.250 mg/dl) was less frequent among our patients(19%) than among patients from Northern Europeanpopulations. Thus, it is likely that those geneticfactors that interact with smoking modulate the riskfor early CAD in our population, while genes in-volved in fat metabolism would be less important. Inagreement with this, the APOE-«4 allele (which isstrongly associated with CAD in Northern Europeanpopulations) did not confer an increased risk forearly CAD in our population (30).

Smoking induces changes in the expression ofecNOS and ACE, and DNA polymorphisms at theNOS3 and ACE genes could modulate the risk ofCAD among smokers (17, 31–33). Recently,genotype-dependent and cigarette-specific effects onecNOS expression and enzyme activity have beendescribed (34). We analyzed two DNA polymor-phisms at the NOS3 gene that have been linked tohypertension, CAD among smokers, and coronary

345GENOTYPES

spasm (17–19). According to Nakayama et al., the2786 C allele would be associated with a signifi-


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CP

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cantly reduced ecNOS promoter activity (19). Thereduced endothelial production of NO in the coro-nary arteries would predispose carriers of the Callele to coronary spasm. The same authors sug-gested that coronary spasm might be more severeand prolonged in CC homozygotes, increasing therisk of suffering acute myocardial infarction. Accord-ing to our results, the 2786 NOS3-CC genotypeonfers an increased risk of suffering an early epi-ode of coronary artery disease. In contrast with thisesult, we failed to confirm the previously described

TA

Genotype and Allele Frequencies of the NOS3 a

TT TC

Controls 120 (40) 137 (46)Patients 52 (31) 81 (47)

55 45

ontrols 222 (73) 72 (24)atients 131 (77) 37 (22)

DD ID

ontrols 123 (41) 132 (44)atients 85 (54) 60 (35)

Note. Parentheses Indicate %.*P 5 0.038; OR 5 1.67 (95% CI 5 1.01, 2.72) CC vs CT/TT.

**P 5 0.53; 55 vs 45/45.***P 5 0.084; OR 5 1.39 (95% CI 5 0.95, 2.03); DD vs ID/II.

TABLE III

Distribution of NOS3 Genotypes (2786 T/C) Accordingto ACE (I/D) Genotypes

ACE-DD ACE-ID/II

Controls Patients Controls Patients

OS3 CC 15 (12) 22 (26) 28 (16) 15 (18)TT1TC 108 (88) 63 (74) 149 (84) 70 (82)

P 5 0.001 P 5 0.38

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ec

Note. Parentheses indicate % relative to the number of patientsr controls.


association between the 27-bp repeat polymorphismand CAD (17).

The ACE-DD genotype (which is associated withthe highest ACE activities in plasma) was slightlyassociated with early CAD. However, the ACE-DD 1

OS3-CC genotypes significantly increased the riskf suffering an early episode of CAD. ACE and NOlay a major role in the regulation of the vascularone, and changes in the synthesis of ACE and NOre associated with hypertension and coronary ar-ery disease. The synergistic effect between theCE-DD and the NOS3-CC genotypes could be aonsequence of the fact that ACE and ecNOS inter-ct in the cardiovascular system. There is growingvidence for a “cross talk” between ACE and ecNOSctivities by means of a feedback regulation (35).hus, inhibition of ecNOS upregulates vascular andardiac ACE activity (36). Stimulated endothelialO release from rat carotid arteries reduced the

onversion of angiotensin I to angiotensin II (37).here is clear evidence that NO-dependent vasodi-

ation is impaired among smokers (38, 39), and ACE

I

E-I/D Polymorphisms in Patients and Controls

NOS3 (2786 T/C)*

CC T C

43 (14) 377 (63) 223 (37)37 (22) 185 (55) 155 (45)

OS3 (27-bp repeat)**

44 5 4

6 (2) 516 (86) 84 (14)2 (1) 299 (88) 41 (12)

ACE (I/D)***

II D I

45 (15) 378 (63) 222 (37)25 (15) 230 (68) 110 (32)

T AL.

BLE I

nd AC

N

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xpression is increased when ecNOS activity is de-reased. Because the 2786 NOS3-C allele is associ-


1

1

1

1

1

1

1

1

E DNA

ated with a reduced NOS3 promoter activity, whilethe ACE-DD genotype shows the highest ACE activ-ities, smokers who are CC 1 DD could display thehighest endothelial dysfunction, and be at a higherrisk for developing early coronary artery disease.

Our work has several limitations. To maximizethe contribution of the genetic variation at theNOS3 and ACE genes to the risk of developing CAD,we analyzed patients with an early onset of thedisease and an angiographically confirmed athero-sclerotic lesion. The sample was therefore small.However, these patients were from the same homo-geneous Caucasian population (Asturias, NorthernSpain, total population 1 million), thus excludingthe effect of admixture of individuals from differentethnical or geographical origin. We cannot excludethe presence of diseased vessels among the controls.However, because CAD was strongly associated withtobacco consumption in our population, a group ofsmokers without a history of cardiovascular diseasewas used as controls. In addition, all groups were inHardy–Weinberg equilibrium, suggesting that theobserved frequencies corresponded to the NOS3 andACE allele and genotypes frequencies in our popu-lation.

Our aim was to define a role for the NOS3 andACE polymorphisms in the risk of developing CADand to analyze the existence of an association withclassical risk factors. Thus, our work can only beseen as suggestive of a synergistic effect between theNOS3 and the ACE genotypes in the risk of devel-oping early CAD, and additional studies on otherpopulations are required to confirm these results.

ACKNOWLEDGMENTS

This work was suported by a grant from the Spanish Fondo deInvestigaciones Sanitarias (FIS 99/0924). R.A. is the recipient ofa fellowship from Fondo de Investigaciones Sanitarias (FIS 98/9019).

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NOS3 AND AC

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Association between the NOS3 (−786 T/C) and the ACE (I/D) DNA Genotypes and Early Coronary Artery...

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