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(Circulation. 1995;91:96-102.)
© 1995 American Heart Association, Inc.

Articles

Lack of Evidence for Linkage of the Endothelial Cell Nitric Oxide Synthase Gene to Essential Hypertension

Alain Bonnardeaux, MD; Sophie Nadaud, MSc; Anne Charru, MD; Xavier Jeunemaitre, MD; Pierre Corvol, MD; Florent Soubrier, MD, PhD

From INSERM U36, Collège de France (A.B., S.N., P.C., F.S.), and Hôpital Broussais (A.C., X.J.), Paris, France.

Correspondence to Dr F. Soubrier, INSERM U36, Collège de France, 3 rue d'Ulm, 75005, Paris, France.

	Abstract

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Background The basal release of nitric oxide by the endothelium plays an important role in regulating blood flow and pressure and mediates most of the endothelium-dependent vasodilation. Impairment of nitric oxide production by specific inhibitors increases blood pressure in humans, and several reports suggest that hypertensive subjects have a blunted endothelium-dependent vasodilatation that might be secondary to decreased nitric oxide production from the vessel wall.

Methods and Results To determine whether the endothelial nitric oxide synthase gene is involved in human essential hypertension, we identified informative biallelic and multiallelic markers of this locus and performed case-control and linkage studies in hypertensive subjects and normotensive control subjects. We used the affected sib pair method to test for potential linkage in 145 hypertensive pedigrees (269 sib pairs, 346 subjects) with a highly polymorphic marker of the nitric oxide synthase gene (polymorphism information content of 92%). There was no evidence for linkage among affected siblings. The 95% upper confidence limit of this value suggests that at most 1% of alleles in excess of expected are shared. We also identified two informative biallelic markers of this gene to perform a case-control study on white hypertensive and normotensive subjects. Similar genotype distributions between the two groups were noted for both markers. Estimated haplotype frequencies by maximum likelihood methods combining the two biallelic markers were also similar in both groups.

Conclusions These findings do not suggest that common molecular variants of the endothelial nitric oxide synthase gene are involved in essential hypertension.

Key Words: hereditary diseases • genes • molecular biology • nucleic acids

	Introduction

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Nitric oxide (NO) is a recently identified molecule that has been implicated in neuronal transmission, immunological responses, and vasodilation.¹ Nitric oxide is a diffusible substance with a short half-life that is produced from its precursor L-arginine by a family of enzymes. Three distinct isoforms of NO synthases have been identified in humans. The inducible NO synthase is encoded by a gene comprising 26 exons spanning 37 kb located on chromosome 17cen-q11.2^{2 3} and is expressed after immunological or inflammatory stimuli in macrophages but also in many other cell types. The constitutive neuronal NO synthase is encoded by a 28-exon gene extending over more than 100 kb^{4 5} on chromosome 12q24.2 and is expressed in central and peripheral neurons as well as in skeletal muscle, stomach, uterus, epithelial cells of bronchioli, and beta cells of pancreatic islets. The endothelial NO synthase is expressed in the endothelium and is encoded by a gene located on chromosome 7q35-36 comprising 26 exons that span 21 kb.^{6 7 8}

In the vascular wall, NO diffuses from the endothelium to the vascular smooth muscle cell, where it activates soluble guanylate cyclase, leading to the relaxation of the vascular smooth muscle cell and to vasodilatation. The main physiological stimulus for the release of NO by endothelial cells is shear stress. Other factors are acetylcholine, bradykinin, endothelin, substance P, histamine, and vasopressin.¹ Nitric oxide can also promote vasorelaxation indirectly by inhibiting the release of renin⁹ and norepinephrine,¹⁰ and NO-releasing vasodilators inhibit the proliferation of vascular smooth muscle cells by a cGMP-mediated process.¹¹ The basal release of NO by endothelial cells plays a key role in regulating blood flow and pressure, since inhibition of NO synthesis by a specific inhibitor in humans results in a 50% reduction in basal flow in the infused forearm.¹² Finally, NO is responsible for most of the arterial vasodilation mediated by the endothelium and stimulated by the intra-arterial injection of agents such as acetylcholine.¹³

A number of observations indirectly suggest that the NO/cGMP pathway might be impaired in hypertension, for which the endothelial NO synthase represents an interesting candidate gene. First, decreased NO production is a potential primary hypertensive process, since inhibition of NO synthesis by the injection of N^G-monomethyl-L-arginine (L-NMMA), a specific antagonist of NO synthase, results in increased blood pressure in humans¹⁴ and animals.^{15 16} Second, the endothelium-dependent arterial vasodilation induced by acetylcholine, an indirect method that assesses endothelial NO production and action on the vascular smooth muscle, is blunted in patients with untreated essential hypertension^{13 17} as well as in several animal models.^{18 19 20 21} This is apparently not explained by decreased substrate availability in hypertensive subjects, since contrary to the endothelial dysfunction of hypercholesterolemia,^{22 23} administration of L-arginine does not restore the impaired vascular responses to endothelium-dependent agents.^{24 25} It does not seem to be secondary to decreased peripheral action of NO, since the arterial response to the NO donor nitroprusside is normal in these subjects.^{13 17} Furthermore, the vascular responses to the intra-arterial infusion of L-NMMA with and without concurrent administration of acetylcholine are also blunted in hypertensive subjects,^{25 26} thereby implying impaired basal and stimulated release of NO by the endothelium, which is therefore less affected by inhibition of its synthesis. Normalization of blood pressure does not appear to restore the impaired vascular responses in patients with essential hypertension,²⁷ but it does so in rats.²⁸ This suggests that either the endothelial dysfunction in essential hypertensive subjects is a primary phenomenon and plays a causal role or the hypertensive process irreversibly injures the endothelium and causes the endothelial dysfunction secondarily. Finally, the NO-cGMP pathway seems to be implicated in the adaptive response to salt loading, an important mechanism for blood pressure regulation.²⁹ In the deoxycorticosterone acetate–salt (DOCA-salt)³⁰ and Dahl/Rapp rats but not in the salt-sensitive Dahl/Rapp rat,³¹ salt loading increases NO production, thereby maintaining normal pressure. Furthermore, salt-sensitive hypertension is prevented by the administration of L-arginine in the salt-sensitive Dahl/Rapp rat but not in the spontaneously hypertensive rat,³¹ pointing to a possible defect in the NO-mediated vasodilator pathway in the former strain. From these studies we can conclude that (1) impairment of the NO-cGMP pathway is a process that can induce hypertension in animals and humans, and (2) the NO-cGMP pathway is altered in essential hypertension, although it is still unclear whether this is a primary phenomenon that would indirectly point to the endothelial NO synthase as a likely candidate gene or a process secondary to the hypertensive state itself.

Therefore, the present study was conducted to determine whether the endothelial NO synthase gene might be involved in essential hypertension. After cloning the endothelial NO synthase gene and identifying a highly informative microsatellite,^{7 8} we characterized biallelic markers and performed association and linkage studies of this locus, using hypertension as a qualitative trait.

	Methods

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Patient Population
Hypertensive Index Cases and Sibships With Multiple Hypertensive Subjects
This study was approved by an institutional review committee, and the subjects recruited gave informed consent. A total of 198 Caucasian index cases (97 men, 101 women) were selected at the Hypertension Clinic of the Broussais Hospital in Paris (n=165) and at several centers in the Bordeaux and Toulouse areas (n=33) according to the following criteria: (1) age >20 years; (2) onset of hypertension <60 years; (3) established hypertension as defined either by chronic treatment or by a diastolic BP >95 mm Hg on two consecutive visits for those untreated; (4) absence of secondary forms of hypertension through extensive workup when indicated; and (5) family history of hypertension (occurring before 60 years of age), with at least one parent and one sibling being affected. Subjects with a history of alcohol intake (more than three drinks per day), oral contraceptive therapy, diabetes mellitus, or renal failure were excluded. Blood pressure was measured in the supine position with a sphygmomanometer. Phase V Korotkoff sounds were taken as diastolic BP.

Affected siblings of hypertensive index cases were screened according to the criteria mentioned above; the study sample comprised 346 hypertensive sibs (169 men and 177 women), yielding a total of 145 sibships composed of 99 pairs, 38 trios, 6 quartets, and 2 quintets representing 269 sib pairs.

Control Subjects
A group of 106 white normotensive control subjects previously described³² was selected from the Broussais transfusion center (n=30) and from patients examined in preventive medicine centers in Paris (n=76).

Genotyping of CA Alleles at the Endothelial NO Synthase Locus
The genotypes for the multiallelic repeat were established by amplifying enzymatically a 160-bp fragment comprising a highly informative dinucleotide repeat of the CA/GT type located in intron 13 of the endothelial NO synthase gene, as previously described.⁷

Identification and Detection of Polymorphisms of the Endothelial NO Synthase Gene
Enzymatic Amplification of Segments of the Endothelial NO Synthase Gene and Detection of Single-Strand Conformation Polymorphism
From the known genomic structure of the NO synthase gene,⁷ we amplified five fragments of 200 to 600 bp from 10 subjects (see Table 1 for location and primer sequences) using 50 ng of DNA from 20 hypertensive subjects in a total volume of 25 µL containing 50 mmol/L KCl, 5 mmol/L Tris-HCl (pH 8.3), 0.01% gelatin, 1.5 mmol/L MgCl₂, 50 µmol/L dNTPs, 10 pmol each primer, and 0.5 U Taq polymerase (Boehringer Mannheim). All primers were located inside introns except for the lower primer used to amplify exons 7 and 8. For single-strand conformation polymorphism, 0.3 µL of [-³²P]dCTP was added to the reaction. Polymerase chain reaction (PCR) products were enzyme-restricted overnight by addition of 5 U of one or more appropriate enzymes (Table 1) to yield fragments of approximately 150 bp and subsequently resolved by electrophoresis under nondenaturing conditions.³³

View this table: [in this window] [in a new window]   Table 1. Primers Used for PCR-SSCP of the Endothelial NO Synthase Gene

Direct Sequencing of Electrophoretic Variants
DNA from patients presenting variant electrophoretic patterns was reamplified by PCR using the above conditions (unlabeled primers). PCR products were purified by 2% agarose gel electrophoresis and eluted with Geneclean (Bio 101). Asymmetric PCR reactions (45 cycles) were performed with each primer (sense and antisense) using 0.1 of the original double-stranded template. The single-stranded template was purified with a Centricon 30 column (Amicon). Sequencing was performed in five rounds of PCR with [-³²P]dATP end-labeled primers with a direct sequencing kit (Circumvent, New England Biolabs).

Allele-Specific Oligonucleotide Hybridizations
To determine the genotypes for each biallelic marker, we performed allele-specific oligonucleotide hybridizations as previously described.³⁴ The sequences of the probes (5' to 3') and the final washing temperatures (in 1x SSC, 0.1% sodium dodecyl sulfate) used for these experiments were as follows: for polymorphism A²⁷C of intron 18, A²⁷-CAGGGGTTGGGGGGC (reverse strand) and C²⁷-GCCCCCCCACCCCTG at 54°C and 56°C, respectively, and for polymorphism G¹⁰T of intron 23, G¹⁰-TTTAGTCCCCAGCCT (reverse strand) and T¹⁰-AGGCTGGTGACTAAA at 40°C for both.

Statistical Analysis
Analysis of Linkage in Hypertensive Sib Pairs
Linkage analysis was performed according to the affected sib pair method, a nonparametric test based on the analysis of affected members of a pedigree. Allele frequencies were determined by genotyping the hypertensive index cases and normotensive control subjects, and we used identity-by-state methods to calculate the expected proportions of alleles shared.³⁵ The comparison between the observed and the expected mean number of alleles shared by the siblings of each sibship was performed with Student's t test. A one-sided test was performed, since it is difficult to imagine a disease in which affected members of a pedigree would have a deficit in allele sharing.³⁵ The weighting of each sibship size was performed according to Hodge.³⁶

Analysis of Genotype and Allele Frequencies for Endothelial NO Synthase Gene Polymorphisms
Allele frequencies were calculated from genotype frequencies in the hypertensive and normotensive groups. Deviation from Hardy Weinberg equilibrium was assessed by a ² test with 1 df. Since the genotypes from the parents were not generally available, we used maximum-likelihood methods³⁷ to estimate haplotype frequencies. Differences in genotype distributions between hypertensive subjects and normotensive control subjects were tested by a ² test with 2 df.

	Results

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Absence of Linkage of the Endothelial NO Synthase Gene in Hypertensive Sibships
A total of 145 sibships representing 346 hypertensive subjects (269 sib pairs) were analyzed. Clinical characteristics are shown in Table 2. A highly informative genetic marker based on a variable number of tandem repeats of the CA motif located within intron 13 of the endothelial NO synthase gene was characterized in all study subjects.⁷ We detected a total number of 24 alleles yielding a polymorphism information content of 92% in 304 unrelated individuals (198 index cases and 106 normotensive controls). Allele frequencies were similar in hypertensive versus normotensive subjects (Figure). The observed number of alleles shared between affected siblings at the endothelial NO synthase locus was not different from that expected from random segregation (Table 3). The 95% upper confidence limit of this value suggests that at most 1% of alleles in excess of expected are shared.

View this table: [in this window] [in a new window]   Table 2. Clinical Parameters of Hypertensive and Normotensive Subjects

View larger version (15K): [in this window] [in a new window]   Figure 1. Bar graph showing distribution of the alleles of the CA repeat polymorphism of the endothelial NO synthase gene in hypertensive (closed bars) and normotensive (open bars) subjects. Similar distributions were observed between the two groups (2=7.4, P=.69, 10 df).

View this table: [in this window] [in a new window]   Table 3. Sib Pair Linkage Analysis at the Endothelial NO Synthase Locus

Absence of Association of Polymorphisms of the Endothelial NO Synthase Gene With Hypertension
Since linkage studies using the sib pair approach might have limited power compared with association studies,³⁸ we also performed a case-control study to detect the presence of a weak susceptibility locus that would not be resolved by the linkage approach. The clinical characteristics of cases and controls are shown in Table 2. Since potential functional variants of the endothelial NO synthase gene might be distributed over many alleles of the CA repeat and render a simple association test between microsatellite alleles and hypertension negative (Figure), we also screened 8 exons of the endothelial NO synthase gene by single-strand conformation polymorphism to find informative biallelic markers (Table 1). We found two substitutions within introns 18 (A²⁷C) and 23 (G¹⁰T) and compared allele frequencies between hypertensive subjects and normotensive control subjects. As shown in Table 4, there were no differences in allele and genotype frequencies between hypertensive subjects and normotensive control subjects for either mutation. The odds ratios performed on allele frequencies for polymorphisms A²⁷C and G¹⁰T were 0.88 (²=0.53; 95% CI, 0.60 to 1.27) and 0.86 (²=0.70; 95% CI, 0.60 to 1.24), respectively. To increase the number of alleles and because parental genotypes were mostly unavailable for this study, we determined haplotype frequencies in both groups by maximum-likelihood methods, after prior verification that genotypes for both polymorphisms in both groups as well as the combined data set were in Hardy-Weinberg equilibrium. A strong linkage disequilibrium was found between the two markers (standardized linkage disequilibrium coefficient of 0.34, ²=13.2, P<.0001). As shown in Table 5, the four possible haplotypes were represented, with important differences in their frequencies. However, haplotype distributions were similar for hypertensive subjects and normotensive control subjects (²=2.4, P=.50, 3 df).

View this table: [in this window] [in a new window]   Table 4. Comparison of Genotype and Allele Frequencies for Polymorphisms A27C (Intron 18) and G10T (Intron 23) in hypertensive and Normotensive Subjects

View this table: [in this window] [in a new window]   Table 5. Estimation of Haplotype Frequencies Using Both Biallelic Markers of the Endothelial NO Synthase Locus in Hypertensive Subjects and Normotensive Control Subjects

	Discussion

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The results of the present study demonstrate an absence of linkage and association of the endothelial NO synthase gene with human essential hypertension in a Caucasian population. To perform these studies, we developed specific tools, ie, various polymorphic DNA markers, to test the hypothesis of the involvement of this gene in hypertension. The endothelial NO synthase gene appeared to be a potential candidate from previously published data in humans showing (1) that basal NO production from the endothelium is important for the maintenance of a continuous vasodilator tone in vessels,¹² (2) that inhibition of NO production increases blood pressure,¹⁴ and (3) that hypertensive subjects might have a primary decrease in the production of NO by the endothelium, explaining the abnormal endothelium-dependent responses to vasoactive substances.^{13 17}

To detect linkage, we used the affected sib pair method. This nonparametric method compares the number of alleles shared at a given locus between affected siblings of a pedigree and the theoretical value under the hypothesis of independent segregation of trait and locus. It does not require any a priori hypothesis as to the mode of transmission of the disease or penetrance, and it accommodates genetic heterogeneity. In addition, entire pedigrees need not be available for testing, an advantage in the study of late-onset diseases. Its main disadvantage, however, is the lack of power and its critical dependence on the informativity of the marker used.³⁹ In this study, the informativity of the (CA)_n repeat polymorphism that was characterized (polymorphism information content of 92%) gave a high statistical power, as demonstrated by the upper limit of the 95% CI of the observed number of alleles shared, suggesting that at most 1% of alleles are shared in excess of expected. This provides adequate confidence in stating that variants of the endothelial NO synthase gene are not a common cause of essential hypertension.

In addition to the linkage study, we also performed a case-control study of the endothelial NO synthase locus using the index cases from our pedigrees and normotensive control subjects. This method relies on the presence of a linkage disequilibrium between known marker polymorphism(s) and putative functional variant(s) to reveal an association between locus and disease and is more sensitive than sib pair studies to detect weak susceptibility genes in polygenic diseases. Indeed, sib pair studies can be negative when a disease susceptibility allele of a gene conferring a weak increase in the relative risk is common and likely to be represented more than once in the parental alleles. This was the case for the insulin gene in insulin-dependent diabetes mellitus, in which affected members of a pedigree frequently inherited different parental haplotypes (explaining the absence of linkage) but associated with the same disease susceptibility allele.⁴⁰ Alternatively, linkage studies can be negative because of the heterogeneity of polygenic diseases such as hypertension (if the segregating susceptibility loci are not the same between different pedigrees and among affected members of a given pedigree). To perform this association study, we identified two additional informative base substitutions located in introns 18 and 23 of the gene and unlikely to be functional by themselves and compared the genotypes for these 2 polymorphisms in cases and controls. The distribution of genotypes was similar in both groups for both mutations, yielding odds ratios calculated on allele frequencies that were not significantly different from 1 (95% CI, 0.60 to 1.2). Estimation of the frequencies of haplotypes combining both markers by maximum likelihood methods revealed the existence of the four possible haplotypes with strong linkage disequilibriums between alleles of both markers. Similar frequencies of haplotypes were noted for both groups. Thus, we confirmed the findings of the linkage study, suggesting that functional variants of the endothelial NO synthase gene do not seem to be involved in essential hypertension.

As with any genetic study, these findings apply only to the population studied, and we cannot rule out that variants of this gene are involved in selected pedigrees, in distinct populations, or under different environmental conditions. This hospital-recruited group of pedigrees has been carefully selected for a strong familial component of hypertension and the exclusion of interacting factors such as obesity and diabetes to increase the genetic predisposition for the disease and has previously allowed us to demonstrate the role of angiotensinogen in hypertension in a collaborative study.³⁴ Nevertheless, it will be of interest to perform other linkage and association studies involving hypertensive sibships selected according to other criteria or with different ethnic backgrounds. A second possible limitation to such studies is that we have treated hypertension as a dichotomous trait, ie, hypertension/no hypertension, and more power could be gained if blood pressure were to be analyzed as a quantitative trait. However, this type of study would require blood pressure measurements under standardized conditions, particularly regarding drug treatment and salt intake, which is difficult to perform in large-scale human studies. A third caveat to this study is that although we have excluded the endothelial NO synthase as a candidate gene for essential hypertension, this does not rule out the presence of common functional variants of the endothelial NO synthase gene affecting endothelium-dependent responses in hypertensive or normotensive populations. To exclude the presence of such variants, one would need to correlate the phenotype (endothelium-dependent response to vasoactive agents) with the different endothelial NO synthase genotypes (markers and haplotypes identified in this study), using association or linkage/segregation methodology. However, from the results of this study, it is unlikely that such variants would impart a strong effect on blood pressure, if discovered.

Several mechanisms other than impaired NO synthesis from a defective variant of the endothelial NO synthase could explain the blunted endothelium-dependent vasodilation in hypertension, thus representing additional candidate mechanisms/genes for essential hypertension. These include decreased production of NO from impaired intracellular availability of L-arginine or of one of the cofactors of endothelial NO synthase, abnormal release of NO by endothelial cells, impaired diffusion of NO between the endothelium and the vascular smooth muscle, increased production of an endothelium-derived contracting factor⁴¹ such as prostaglandin endoperoxide or impaired release of an endothelium-derived hyperpolarizing factor,⁴² and increased degradation of NO from the release of oxygen-derived free radicals. The inducible NO synthase also represents an interesting candidate because it is expressed in rat and human immunostimulated vascular smooth muscle cells^{2 43} but also in vascular smooth muscle cells from terminal afferent arterioles of normal unstimulated rat kidneys.⁴⁴ Microperfusion of L-NMMA in the rabbit afferent arteriole decreases its diameter and increases angiotensin II–induced constriction,⁴⁵ thus providing potential mechanisms of decreased renal perfusion, glomerular filtration rate, and sodium excretion. Indeed, the inducible NO synthase seems necessary for the adaptation to salt loading in rats and potentially deficient in Dahl/Rapp salt-sensitive rats.⁴⁶ Linkage studies of this locus in genetic crosses involving the Dahl/Rapp salt-sensitive strain and eventually in humans should help clarify its potential implication in hypertension.

Further studies will also be needed to determine whether the blunted endothelium-dependent response in hypertensive subjects is a cause or merely a consequence of hypertension. Since several disease states such as secondary forms of hypertension,⁴¹ microvascular angina,⁴⁷ hypercholesterolemia,⁴⁸ atherosclerosis,^{49 50} and insulin-dependent diabetes mellitus⁵¹ are also characterized by blunted endothelium-dependent vasodilation, one would be inclined to postulate that this is a marker of conditions affecting the vessel wall and not a process responsible for elevating blood pressure per se. Finally, in contrast to previous reports, a recent study challenges the abnormal endothelium-dependent vasodilation in essential hypertension.⁵²

In conclusion, this extensive linkage and association study of the endothelial nitric oxide synthase gene does not suggest that common molecular variants of this gene are involved in human essential hypertension. The markers developed for this study could be useful for future studies of this gene in conditions associated with abnormal endothelium-dependent responses.

	Acknowledgments

 
This work was supported by Institut National de la Santé et de la Recherche Médicale and in part by the Groupement de Recherche et d'Etudes sur les Génomes. Dr Bonnardeaux is the recipient of a fellowship from the Medical Research Council of Canada. We thank Isabelle Féry for excellent technical help.

Received April 19, 1994; accepted July 31, 1994.

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