+ 163729

NITRIC OXIDE SYNTHASE 3; NOS3


Alternative titles; symbols

NITRIC OXIDE SYNTHASE, ENDOTHELIAL; ENOS


Other entities represented in this entry:

CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO, INCLUDED
ALZHEIMER DISEASE, LATE-ONSET, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION, PREGNANCY-INDUCED, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION RESISTANT TO CONVENTIONAL THERAPY, INCLUDED

HGNC Approved Gene Symbol: NOS3

Cytogenetic location: 7q36.1     Genomic coordinates (GRCh38): 7:150,991,017-151,014,588 (from NCBI)


Gene-Phenotype Relationships
Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
7q36.1 {Alzheimer disease, late-onset, susceptibility to} 104300 AD 3
{Coronary artery spasm 1, susceptibility to} 3
{Hypertension, pregnancy-induced} 189800 AD 3
{Hypertension, susceptibility to} 145500 Mu 3
{Ischemic stroke, susceptibility to} 601367 Mu 3
{Placental abruption} 3

TEXT

Cloning and Expression

Nitric oxide (NO) accounts for the biologic activity of endothelium-derived relaxing factor (EDRF), discovered by Furchgott and Zawadzki (1980) and Rapoport and Murad (1983). NO is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). EDRF is important in regulation of vasomotor tone and blood flow by inhibiting smooth muscle contraction and platelet aggregation. Janssens et al. (1992) isolated a cDNA encoding a human vascular NOS. The translated human protein was 1,203 amino acids long with a predicted molecular mass of 133 kD. They showed that the cDNA encodes a calcium-regulated, constitutively expressed endothelial NOS, capable of producing EDRF in blood vessels. Marsden et al. (1992) likewise cloned and sequenced human endothelial NO synthase. Their cDNA clones predicted a protein of 1,203 amino acids with about 60% identity with the rat brain NO synthase isoform (163731). (Nitric oxide synthases have been assigned to 2 classes: a constitutively expressed, calcium-regulated class identified in brain, neutrophils, and endothelial cells, and a calcium-independent class identified in endotoxin- or cytokine-induced macrophages and endothelial cells (163730).)


Gene Structure

Marsden et al. (1993) isolated genomic clones encoding human endothelial NO synthase and determined the structural organization of the gene. It contains 26 exons spanning approximately 21 kb of genomic DNA and encodes an mRNA of 4,052 nucleotides. Characterization of the 5-prime-flanking region indicated that the NOS3 gene is TATA-less and exhibits proximal promoter elements consistent with a constitutively expressed gene, namely, SP1 and GATA motifs.


Mapping

By Southern blot hybridization of human/rodent somatic cell hybrids, Marsden et al. (1993) assigned the NOS3 gene to chromosome 7 and regionalized it to 7q35-q36 by fluorescence in situ hybridization. By PCR amplification applied to a panel of DNAs from human/rodent somatic cell hybrids, by Southern blot analysis, and by fluorescence in situ hybridization, Robinson et al. (1994) localized the NOS3 gene to 7q36. By analysis of interspecific backcross progeny, Gregg et al. (1995) mapped the mouse homolog to chromosome 5.


Biochemical Features

Crystal Structure

Raman et al. (1998) reported the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (BH4)-free and -bound forms at 1.95-angstrom and 1.9-angstrom resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the BH4-binding site. The unexpected recognition of the substrate L-arginine at the BH4 site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.


Gene Function

Fulton et al. (1999) demonstrated that AKT (164730) directly phosphorylated eNOS and activated the enzyme leading to nitric oxide production, whereas eNOS mutated at a putative AKT phosphorylation site was resistant to phosphorylation and activation by AKT. Activated AKT increased basal nitric oxide release from endothelial cells, and activation-deficient AKT attenuated NO production stimulated by VEGF (192240). Thus, Fulton et al. (1999) concluded the eNOS is an AKT substrate linking signal transduction by AKT to the release of the gaseous second messenger nitric oxide.

Dimmeler et al. (1999) stated that physiologically, the most important stimulus for the continuous formation of nitric oxide is the viscous drag (or shear stress) generated by the streaming blood on the endothelial layer. PI3K (see 601232) and AKT are activated in endothelial cells in response to shear stress. Dimmeler et al. (1999) demonstrated that AKT mediates the activation of eNOS, leading to increased nitric oxide production. Inhibition of the PI3K AKT pathway or mutation of the AKT site on eNOS protein at serine-1177 attenuated the serine phosphorylation and prevented the activation of eNOS. Mimicking the phosphorylation of ser1177 directly enhanced enzyme activity and altered the sensitivity of the enzyme to calcium, rendering its activity maximal at subphysiologic concentrations of calcium. Thus, phosphorylation of eNOS by AKT represented a novel calcium-independent regulatory mechanism for activation of eNOS.

Napolitano et al. (2000) investigated the interactions between ET1 (131240) and the NO system in the feto-placental unit. They examined the mRNA expression of ET1, inducible NOS (163730), and eNOS in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive pregnancies. ET1 expression was increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, was decreased; conversely, eNOS expression was increased. ET1 was able to influence its own expression as well as NOS isoform expression in normal and PE trophoblastic cultured cells. The findings suggested the existence of a functional relationship between ET(s) and NOS isoforms that could constitute the biologic mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation that are characteristic of the pathophysiology of preeclampsia.

Using a yeast 2-hybrid screen, Dedio et al. (2001) found that the oxygenase domain of human ENOS interacted with human NOSIP (616759). Reciprocal coimmunoprecipitation and Western blot analysis confirmed interaction between ENOS and NOSIP in cotransfected COS-7 cells. Deletion analysis showed that NOSIP bound the C-terminal portion of the ENOS oxygenase domain in a region that overlapped the binding site for plasma membrane caveolin (see 601047). Overexpression of NOSIP reduced calcium-stimulated nitric oxide production in ENOS-expressing Chinese hamster ovary (CHO) cells. Overexpression of NOSIP also reduced membrane localization of ENOS in CHO cells, causing a shift from caveolin-rich membrane fractions to intracellular compartments. Dedio et al. (2001) concluded that NOSIP promotes translocation of ENOS from the plasma membrane to intracellular sites, thus inhibiting nitric oxide synthesis.

Autosomal dominant adult polycystic kidney disease (ADPKD; 173900) is associated with altered endothelial-dependent vasodilation and decreased vascular production of NO. Thus, eNOS could have a modifier effect in ADPKD. To test this hypothesis, Persu et al. (2002) genotyped 173 unrelated European ADPKD patients for the glu298-to-asp (163729.0001), intron 4 VNTR, and -786T-C (163729.0002) polymorphisms of ENOS and looked for their influence on the age at end-stage renal disease (ESRD). In 93 males, the glu298-to-asp polymorphism was associated with a lower age at ESRD. This effect was confirmed in a subset of males linked to PKD1 (601313) and reaching ESRD before age 45, and by a cumulative renal survival analysis in PKD1-linked families. Further studies demonstrated that NOS activity was decreased in renal artery samples from PKD males harboring the asp298 allele, in association with posttranslational modifications and partial cleavage of eNOS. No significant effect of the other polymorphisms was found in males, and no polymorphism influenced the age at ESRD in females. Persu et al. (2002) concluded that glu298-to-asp is associated with a 5 year lower mean age at ESRD in a subset of ADPKD males. They hypothesized that the effect could be due to decreased NOS activity and a partial cleavage of eNOS, leading to a further decrease in the vascular production of NO.

Nisoli et al. (2003) found that NO triggers mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of NO was dependent on cGMP and was mediated by the induction of PPARGC1 (604517), a master regulator of mitochondrial biogenesis. Moreover, Nisoli et al. (2003) found that mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of eNOS -/- mice, which had a reduced metabolic rate and accelerated weight gain compared to wildtype mice. Thus, Nisoli et al. (2003) concluded that a NO-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.

Simoncini et al. (2004) demonstrated that tibolone and its estrogenic metabolites activate NO synthesis by recruiting functional estrogen receptors, whereas the progestogenic/androgenic metabolite had no effect. During prolonged exposures, tibolone and the estrogenic compounds enhanced the expression of eNOS. In addition, tibolone was able to induce rapid activation of eNOS, leading to rapid increases in the release of NO. Different from estrogen, rapid activation of eNOS did not rely on recruitment of PI3K but rather on MAPK-dependent cascades.

Robb et al. (2004) found that the 5-prime UTR of the NOS3AS (612205) transcript was complementary to the portion of NOS3 mRNA derived from exons 23 to 26. RT-PCR analysis showed that expression of NOS3 in human umbilical vein endothelial cells (HUVECs) and human aortic vascular smooth muscle cells (HAOVSMCs) was inversely proportional to that of NOS3AS. Both the NOS3 and NOS3AS genes were transcriptionally active; however, NOS3AS appeared to downregulate the steady-state levels of NOS3 mRNA and protein. RT-PCR showed that overexpression of the overlapping region of NOS3AS had little impact on NOS3 mRNA levels in HUVECs; however, Western blot analysis showed that NOS3 protein levels were markedly reduced. NOS3AS also reduced the expression of a chimeric transcript containing the NOS3-overlapping sequence fused to a reporter gene. Inhibition of NOS3AS expression by RNA interference (RNAi) in HAOVSMCs increased NOS3 expression, and inhibition of histone deacetylase (see HDAC1; 601241) in HAOVSMCs increased expression of NOS3AS mRNA prior to decrease in NOS3 mRNA expression. Robb et al. (2004) concluded that NOS3AS participates in the posttranscriptional regulation of NOS3.

Fish et al. (2007) found that NOS3AS was induced by hypoxia in cultured human endothelial and smooth muscle cells and in aortas of hypoxic rats. NOS3AS induction preceded the decrease in NOS3 steady-state mRNA in endothelial cells, and knockdown of NOS3AS by RNAi indicated that NOS3AS downregulated NOS3 expression during hypoxia. Hypoxia did not induce transcription of NOS3AS, but instead stabilized NOS3AS transcripts, predominantly the short NOS3AS variant, leading to elevated NOS3AS levels. RT-PCR of fractionated cells showed that both NOS3AS pre-mRNA and mature NOS3AS mRNA were enriched in the nucleus under basal conditions. Under hypoxic conditions, the cytoplasmic levels of mature NOS3AS mRNA increased more than 30-fold, whereas levels in the nucleus increased only moderately. In contrast, the nuclear/cytoplasmic distribution of NOS3 mRNA was not altered under hypoxic conditions, with NOS3 mRNA levels decreasing in both the cytoplasm and nucleus. NOS3 associated with polyribosomes in normoxic cells, but its association with polyribosomes was attenuated in hypoxic cells, concurrent with association of the short NOS3AS variant with polyribosomes. Fish et al. (2007) concluded that NOS3 expression is regulated by its overlapping NOS3AS transcript in a hypoxia-dependent fashion.

Nisoli et al. (2005) reported that calorie restriction for either 3 or 12 months induced eNOS expression and 3-prime/5-prime-cyclic GMP in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and ATP production, and an enhanced expression of Sirt1 (604479). Nisoli et al. (2005) reported these effects were strongly attenuated in eNOS null-mutant mice. Thus, Nisoli et al. (2005) concluded that nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Using human platelets, Ji et al. (2007) demonstrated that polymerization of beta-actin (ACTB; 102630) regulated the activation state of NOS3, and hence NO formation, by altering its binding to heat-shock protein-90 (HSP90, or HSPCA; 140571). NOS3 bound the globular, but not the filamentous, form of beta-actin, and the affinity of NOS3 for globular beta-actin was, in turn, increased by HSP90. Formation of this ternary complex of NOS3, globular beta-actin, and HSP90 increased NOS activity and cyclic GMP, an index of bioactive NO, and increased the rate of HSP90 degradation, thus limiting NOS3 activation. Ji et al. (2007) concluded that beta-actin regulates NO formation and signaling in platelets.

Lim et al. (2008) demonstrated that blocking phosphorylation of the AKT substrate eNOS inhibits tumor initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wildtype Ras proteins (see 190020), which are required throughout tumorigenesis. Lim et al. (2008) suggested that activation of the PI3K-AKT-eNOS-(wildtype) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumor growth.

Chen et al. (2010) showed that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in superoxide generation primarily from the reductase, in which 2 highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. Chen et al. (2010) showed that e-NOS S-glutathionylation in endothelial cells, with loss of nitric oxide and gain of superoxide generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Chen et al. (2010) concluded that S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signaling, endothelial function, and vascular tone.

Straub et al. (2012) reported a model for the regulation of NO signaling by demonstrating that hemoglobin alpha, encoded by the HBA1 (141800) and HBA2 (141850) genes, is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to hemoglobin alpha and is abrogated by its genetic depletion. Mechanistically, endothelial hemoglobin alpha heme iron in the Fe(3+) state permits NO signaling, and this signaling is shut off when hemoglobin alpha is reduced to the Fe(2+) state by endothelial CYB5R3 (613213). Genetic and pharmacologic inhibition of CYB5R3 increases NO bioactivity in small arteries. Straub et al. (2012) concluded that their data revealed a mechanism by which the regulation of the intracellular hemoglobin alpha oxidation state controls NOS signaling in nonerythroid cells. The authors suggested that this model may be relevant to heme-containing globins in a broad range of NOS-containing somatic cells.

Using immunofluorescence analysis, Lechauve et al. (2018) showed that AHSP (ERAF; 605821) was coexpressed with alpha-globin in mouse and human ECs and regulated alpha-globin protein levels. Expression analysis in human coronary artery ECs and experiments with purified human proteins demonstrated that AHSP and eNOS interacted with alpha-globin in a mutually exclusive manner and enhanced its accumulation in cells. However, only AHSP could stabilize oxidized Fe(3+)-alpha-globin. The authors demonstrated that eNOS rapidly reduced AHSP-bound Fe(3+)-alpha-globin via direct electron transfer from its flavin-associated reductase domain.


Molecular Genetics

In a Japanese study of 100 patients with essential hypertension (145500) and 123 patients with normal blood pressure, Nakayama et al. (1997) found that the distribution of allele frequencies for the CA repeat in the NOS3 gene was not significantly different between the 2 groups. However, comparing the allele frequencies in the hypertensive group without left ventricular hypertrophy (LVH) and the normotensive group, the overall distributions were significantly different (p = 0.019). The 33-repeat allele was found more frequently in the hypertensive group without LVH than in the normotensive group.

Bonnardeaux et al. (1995) found that the highly polymorphic (CA)n repeats in intron 13 and 2 biallelic markers in intron 18 of the NOS3 gene are not associated with essential hypertension. Wang et al. (1996) studied a marker closer to the 5-prime end of the NOS3 gene, the 27-bp repeat in intron 4, in relation to coronary artery disease. They identified 2 alleles: a common larger allele (allele frequency, 0.830) and a smaller rare allele (0.170). The larger allele had 5 tandem 27-bp repeats. The smaller allele had only 4 repeats that were apparently missing the third repeat judging by minor a difference in sequence. The distribution of the genotypes appeared to be in Hardy-Weinberg equilibrium and the polymorphism was inherited in a simple mendelian fashion. Wang et al. (1996) found from study of 549 subjects with and 153 without coronary artery disease that in current and ex-cigarette smokers, but not nonsmokers, there was a significant excess of homozygotes for the rare allele in patients with severely stenosed arteries, compared with those with no mild stenosis. This genotype was also associated with a history of myocardial infarction. The authors noted that, since endothelial-dependent vasodilatation is mediated by release of nitric oxide formed by constitutively expressed endothelial nitric oxide synthase, the smoking-dependent excess coronary risk in homozygotes is consistent with a predisposition to endothelial dysfunction.

Coronary spasm plays an important role in the pathogenesis not only of variant angina but also of ischemic heart disease in general. However, the prevalence of coronary spasm appears to be higher in Japanese than in Caucasians (Bertrand et al., 1982; Yasue and Kugiyama, 1990), suggesting that genetic factors may be involved in its pathogenesis. Endothelial-derived nitric oxide has been implicated in the control of vascular tone. Kugiyama et al. (1997) and Motoyama et al. (1997) showed that basal acetylcholine-stimulated and flow-dependent nitric oxide activities are decreased in both coronary and brachial arteries of patients with coronary spasm. Yoshimura et al. (1998) identified a glu298-to-asp variant (E298D; 163729.0001) in exon 7 of the NOS3 gene that was more frequent in patients with coronary spasm. In studies of an elderly population in Australia, Liyou et al. (1998) could find no association of the E298D variant with coronary artery disease.

Nakayama et al. (1999) reported that a -786T-C mutation (163729.0002) in the promoter region of the eNOS gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, Miyamoto et al. (2000) purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1; 179835), known as a single-stranded DNA-binding protein essential for DNA repair, replication, and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotides restored transcription driven by the mutated promoter sequence, whereas overexpression of RPA1 further reduced it. Serum nitrite/nitrate levels among individuals carrying the -786T-C mutation were significantly lower than among those without the mutation. The authors concluded that RPA1 apparently functions as a repressor protein in the -786T-C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.

Pregnancy-induced hypertension (see 189800) may be regarded as a manifestation of endothelial cell dysfunction. Constitutive nitric oxide production in endothelial cells increases during pregnancy and contributes to vasodilatation and blunting of vasopressor response. In women developing pregnancy-induced hypertension, NO generation is inappropriately low, and administration of an NO donor improves flow in the uterine artery in normal early pregnancy and in women at high risk of developing disease. Yallampalli and Garfield (1993) and others had observed that inhibition of NO synthesis in rats during pregnancy produces hypertension, proteinuria, thrombocytopenia, and fetal growth retardation. These considerations prompted Arngrimsson et al. (1997) to study linkage to the NOS3 gene in affected sisters and in multiplex families. A lod score of 3.36 was obtained for D7S505 when a best-fitting model derived from genetic epidemiologic data was used, and lod scores of 2.54 to 4.03 were obtained when various other genetic models were used. The transmission/disequilibrium test (TDT), a model-free estimate of linkage, showed strongest association and linkage with a microsatellite within intron 13 of the NOS3 gene (P = 0.005).

Lewis et al. (1999) were unable to detect linkage of preeclampsia to the NOS3 region on 7q. They studied 2, separately ascertained, affected sister-pairs collections, from Amsterdam and Cambridge (U.K.), that contained 104 sibships. In the Cambridge Centre, a total of 21 extended pedigrees suitable for conventional parametric linkage studies were also identified. The reason for the discrepancy with the results of Arngrimsson et al. (1997) was not clear. Lewis et al. (1999) concluded that although abnormalities in NO production have been observed in preeclampsia, the case for the NOS3 gene or its product, eNOS, having a primary role in the pathophysiology of preeclampsia remained unproved.

Tempfer et al. (2001) performed a prospective case-control study of 105 women with idiopathic recurrent miscarriage and 91 healthy controls. Using PCR, they identified the different alleles of a 27-basepair tandem repeat polymorphism in intron 4 of the NOS3 gene. The wildtype allele was identified in 329 of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B), and 0.5% (A/A). The distribution of genotype frequencies was significantly different between the study and in control groups for allele A/B heterozygotes (36.7 vs 23.8%, P = 0.03, odds ratio 1.6, 95% CI 1.1-3.8). Only 1 person in the study group was A/A.

Tanus-Santos et al. (2001) studied the distribution of genetic variants of 3 clinically relevant NOS3 polymorphisms in 305 ethnically well-characterized DNA samples (100 Caucasians, 100 African Americans, and 105 Asians). They found marked interethnic differences in the distribution of NOS3 variants, in the estimated haplotype frequency, and in the association between variants. The asp298 variant (163729.0001) was more common in Caucasians (34.5%) than in African Americans (15.5%) or Asians (8.6%) (p less than 0.0001). The -786C variant (163729.0002) was also more common in Caucasians (42.0%) than in African Americans (17.5%) or Asians (13.8%) (p less than 0.0001). The 4a VNTR in intron 4 was more common in African Americans (26.5%) than in Caucasians (16.0%) or Asians (12.9%) (p less than 0.0001). The most common predicted haplotype in the 3 groups combined only wildtype variants. Asians had the highest frequency of this haplotype (77% in Asians vs 46% in the other groups). In Caucasians, the asp298 and -786C variants were associated, and this haplotype was predicted to have a frequency of 24%.

Endothelial nitric oxide synthase plays a key role in the regulation of normal function of the vessel wall. Heltianu et al. (2002) found a relatively high frequency of 2 polymorphic variants of NOS3 in males with Fabry disease (301500) and suggested that in addition to mutations in the alpha-galactosidase A gene, variation in NOS3 may be significant in determining the phenotype.

Casas et al. (2004) performed a metaanalysis of 26 case-control studies evaluating the association between the NOS3 polymorphisms E298D, -786T-C, and the intron 4 VNTR and ischemic heart disease (myocardial infarction or angiographic coronary artery occlusion), involving 9,867 cases and 13,161 controls. They found that homozygosity for asp298 or the intron 4 A allele was associated with an increased risk of ischemic heart disease (OR, 1.31 and 1.34, respectively), but no significant association was found with the -786C allele. Casas et al. (2004) suggested that common genetic variations in the NOS3 gene contribute to atherosclerosis susceptibility.

In 110 dizygotic white twin pairs, Persu et al. (2005) identified NOS3 haplotypes based on 3 polymorphisms, E298D, the intron 4 VNTR, and -786T-C, and the intron 13 CA repeat. Haplotype analysis revealed a significant association between NOS3 haplotypes and daytime ambulatory diastolic and systolic blood pressure, with the latter remaining significant after adjustment for multiple testing (p = 0.032) and mainly attributable to 4 haplotypes accounting for 11.9% of all represented haplotypes.

The therapeutic application of NO in high-altitude (HA) disorders, for the improvement of oxygenation and vasodilation, prompted Ahsan et al. (2005) to investigate the NOS3 gene with respect to high-altitude adaptation. They screened 131 HA monks, 136 HA controls, and 170 lowlanders for the NOS3 894G-T (E298D; 163729.0001) polymorphism and for the 4B/4A polymorphisms. NO levels were estimated and correlated with the polymorphisms. The 3 groups were in Hardy-Weinberg equilibrium for the polymorphisms. Wildtype alleles G and 4B were significantly overrepresented in the HA groups as compared with the lowlanders (p = 0.006 and p = 0.02, respectively). NO levels were highest in HA monks, followed by HA controls, and then lowlanders (p less than 0.0001). Combinations of the GG and BB genotypes were distributed significantly more frequently in the HA monks (p less than 0.0001) and HA controls (p = 0.0005) than in lowlanders. Ahsan et al. (2005) concluded that the genotype combination of NOS3 wildtype homozygotes (GG, BB) occurs more frequently in high-altitude groups that in lowlanders and contributes to higher NO levels associated with high-altitude adaptation.


Animal Model

Pharmacologic blockade of NO production with arginine analogs such as L-nitroarginine or L-N-arginine methylester affects multiple isoforms of nitric oxide synthase and so cannot distinguish their physiologic roles. To study the role of endothelial NOS in vascular function, Huang et al. (1995) disrupted the gene encoding endothelial NOS in mice by homologous recombination. Homozygous mutant mice were found to be viable, fertile, and indistinguishable from wildtype and heterozygous littermates in appearance or routine behavior. Immunoreactive NOS3 protein was not present, as shown by Western blot analysis of the brain, heart, lung, and aorta. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, was absent, and the NOS3 mutant mice were hypertensive. Thus, the author concluded that NOS3 mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggested that nonendothelial isoforms of NOS may be involved in maintaining blood pressure. Huang et al. (1995) suggested that perhaps the renin-angiotensin system and autonomic nervous system evolved to serve primarily as a defense against hypotension, and diminution in their activity is a poor buffer against hypertension. Alternatively, NOS3 may be involved in establishing the baroreceptor set-point. The question of whether subpopulations of humans suffering from hypertension have defects in NOS3 expression awaits an answer from genetic analysis and the development of more selective inhibitors of the NOS isoforms.

Snyder (1995) reviewed the significance of the findings of Huang et al. (1995) in the NOS3 'knockout' mouse because so-called nNOS (NOS1; 163731) occurs in nerves that mediate penile erection and NOS inhibitors block erection. It was thought that the null mutant nNOS mice would not procreate. As it turned out, however, these animals do breed and appear to be generally normal. However, they have dilated stomachs with a constricted pyloric sphincter, and so provide a model for infantile hypertrophic pyloric stenosis. The same mice are resistant to brain damage caused by vascular strokes, confirming that nitric oxide is crucial in mediating stroke damage. Further studies indicated that the nNOS-negative mice are highly aggressive toward other males to the extent that they will kill their wildtype littermates if left unattended, and they display strikingly inappropriate and excessive sexual behavior. Mice homozygous for a knockout in NOS2 (macrophage or inducible NOS, iNOS) have markedly reduced defenses against microorganisms such as Listeria and Leishmania and against the proliferation of lymphoma tumor cells.

Champion et al. (1999) cited work indicating that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of NOS3 could enhance erectile responses, they administered a recombinant adenovirus containing the NOS3 gene into the corpora cavernosum of the aged rat. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue one day after the intracavernosal administration of the beta-gal-marked adenovirus; one day after administration of the construct containing NOS3, transgene expression was confirmed by immunoblot staining of NOS3 protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with NOS3, and erectile responses to acetylcholine and zaprinast were enhanced. Champion et al. (1999) suggested that in vivo gene transfer of NOS3, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.

Steudel et al. (1998) investigated the effects of congenital deficiency of NOS3 on the pulmonary vascular responses to hypoxia. The findings suggested that congenital NOS3 deficiency in mice enhances hypoxic pulmonary vascular remodeling and hypertension, and right ventricular hypertrophy, and that NO production by NOS3 is vital to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress.

To study the role of nitric oxide constitutively produced by NOS3 in the regulation of blood pressure and vascular tone, Ohashi et al. (1998) generated transgenic mice overexpressing bovine NOS3 in the vascular wall using murine preproendothelin-1 (ET1) promoter. Blood pressure was significantly lower in NOS3-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release and basal cGMP levels were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, in addition to the essential role of NOS3 in blood pressure regulation, tonic NO release by NOS3 in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilatators, providing several insights into the pathogenesis of nitrate tolerance.

In the heart, nitric oxide inhibits L-type calcium channels but stimulates sarcoplasmic reticulum calcium release, leading to variable effects on myocardial contractility. Barouch et al. (2002) demonstrated that spatial confinement of specific nitric oxide synthase isoforms regulates this process. Endothelial nitric oxide synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type calcium channels allows nitric oxide to inhibit beta-adrenergic-induced inotropy. Neuronal nitric oxide synthase (NOS1; 163731), however, is targeted to cardiac sarcoplasmic reticulum. NO stimulation of sarcoplasmic reticulum calcium release via the ryanodine receptor (RYR2; 180902) in vitro, suggests that NOS1 has an opposite facilitative effect on contractility. Barouch et al. (2002) demonstrated that Nos1-deficient mice have suppressed inotropic response, whereas Nos3-deficient mice have enhanced contractility, owing to corresponding changes in sarcoplasmic reticulum calcium release. Both Nos1 -/- and Nos3 -/- mice developed age-related hypertrophy, although only Nos3 -/- mice were hypertensive. Nos1/3 -/- double knockout mice had suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodeling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Aicher et al. (2003) demonstrated that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS showed reduced vascular endothelial growth factor (VEGF; 192240)-induced mobilization of endothelial progenitor cells and increased mortality after myelosuppression. Intravenous infusion of wildtype progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3-deficient mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3-null mice. Mechanistically, MMP9 (120361), required for stem cell mobilization, was reduced in the bone marrow of Nos3-null mice. Aicher et al. (2003) concluded that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. The authors suggested that this may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic nitric oxide bioactivity.

Caveolin-3 (CAV3; 601253), a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to NOS3 in cardiac myocytes and NOS1 in skeletal myocytes. Ohsawa et al. (2004) characterized the biochemical and cardiac parameters of P104L (601253.0001)-mutant Cav3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (see RMD2, 606072). Transgenic mouse hearts demonstrated hypertrophic cardiomyopathy, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of Cav3 protein. Cardiac muscle showed activation of NOS3 catalytic activity without increased expression of all NOS isoforms. Ohsawa et al. (2004) suggested that a moderate increase in NOS3 activity associated with loss of Cav3 may result in hypertrophic cardiomyopathy.

Bivalacqua et al. (2004) studied the contribution of RhoA (AHRA; 165390)/Rho kinase (ROCK1; 601702) signaling to erectile dysfunction in streptozotocin (STZ) diabetic rats. Rho kinase and eNOS colocalized in the endothelium of corpus cavernosum, and RhoA and Rho kinase abundance and Mypt1 (602021) phosphorylation were elevated in STZ diabetic rat penis. In addition, eNOS protein expression, cavernosal constitutive NOS activity, and cGMP levels were reduced in STZ diabetic rat penis. Bivalacqua et al. (2004) introduced a dominant-negative RhoA mutant and found that erectile responses in the STZ diabetic rats improved to values similar to controls.

Longo et al. (2005) crossbred Nos3-null and wildtype mice to generate 2 types of heterozygous litters, one with a maternally derived mutation that developed in a Nos3-deficient environment and the other with a paternally derived mutation that developed in a uterine environment similar to wildtype mice. In studies of the in vitro reactivity of carotid and mesenteric artery segments of adult mice to vasoactive agents, the maternally derived heterozygous mice had abnormal vascular reactivity that was similar to that of homozygous knockout mice completely lacking functional NOS3, whereas the paternally derived heterozygous mice had normal vascular reactivity that was not different from that of wildtype mice. Longo et al. (2005) stated that this was the first direct evidence in support of a role for uterine environment in determining vascular function in later life.

In a mouse model of sepsis, Connelly et al. (2005) observed a temporal reduction in iNOS expression and activity in lipopolysaccharide-treated Nos3-knockout mice as compared with wildtype mice, which was reflected in a more stable hemodynamic profile in Nos3-null mice during endotoxemia. In human umbilical vein cells, lipopolysaccharide led to the activation of Nos3 through phosphoinositide 3-kinase (see 171833)- and Akt/protein kinase B (164730)-dependent enzyme phosphorylation. Connelly et al. (2005) concluded that NOS3 has a proinflammatory role in the pathogenesis of sepsis, in which following initial NOS3 activation the resultant NO acts as a costimulus for the expression of iNOS.

Liu et al. (2005) presented evidence suggesting a role for G protein-coupled receptor kinase-2 (GRK2; 109635) in the regulation of NO production and hepatic vascular dynamics in a rat model of liver sinusoidal endothelial injury and portal hypertension induced by bile duct ligation. Sinusoidal endothelial cells isolated from the affected animals had increased levels of GRK2, reduced levels of phosphorylated AKT and eNOS, and decreased levels of NO. Gene silencing of GRK2 using siRNA in injured sinusoidal endothelial cells restored AKT activity and resulted in increased NO production. Liu et al. (2005) also found that heterozygous Grk2 mice had increased levels of phosphorylated Akt and decreased portal hypertension in response to injury compared to wildtype mice. Liu et al. (2005) proposed a mechanism in which upregulation of GRK2 after endothelial cell injury directly inhibits phosphorylation of AKT, leading to reduced activation of eNOS and decreased production of NO, and resulting in portal hypertension.


ALLELIC VARIANTS ( 2 Selected Examples):

.0001 CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO

ALZHEIMER DISEASE, LATE-ONSET, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION, PREGNANCY-INDUCED, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION RESISTANT TO CONVENTIONAL THERAPY, INCLUDED
ISCHEMIC HEART DISEASE, SUSCEPTIBILITY TO, INCLUDED
ISCHEMIC STROKE, SUSCEPTIBILITY TO, INCLUDED
NOS3, GLU298ASP
  
RCV000015053...

Yoshimura et al. (1998) found a glu298-to-asp (E298D) variant (rs1799983) in exon 7 of the NOS3 gene. In a study of 113 patients with coronary spasm, in whom the diagnosis of coronary spasm was made by intracoronary injection of acetylcholine, and 100 control subjects, they found a significant difference in the distribution of the variant; 21.2% of the coronary spasm group and 9.0% of the control group (p = 0.014 for dominant effect) showed the variant. In an elderly population in Australia, Liyou et al. (1998) could demonstrate no difference in the distribution of the E298D alleles in relation to the presence of coronary artery disease.

Cai et al. (1999) genotyped 763 white Australians undergoing coronary angiography for the eNOS glu298-to-asp mutation resulting from an 894G-T transversion. The frequencies of the GG, TG, and TT genotypes were not significantly different in men or women with or without coronary artery disease. The mutation was also not associated with myocardial infarction (MI) in males or females, or with the number of significantly stenosed vessels. The T allele frequency (32.5%) was much greater than that reported for the Japanese population (7.8% in controls and 10% in MI patients). Hingorani et al. (1999) investigated the relationship between the glu298-to-asp variant in atherosclerotic coronary artery disease, using 2 independent case-controlled studies. In the first study, cases consisted of 298 unrelated patients with positive coronary angiograms and controls were 138 unrelated healthy individuals ascertained through a population health screen. In the second study, the cases were 249 patients with recent MI and a further 183 unrelated controls. There was an excess of homozygotes for the asp298 variant among patients with angiographic coronary artery disease, and among patients with recent MI when compared with their respective controls (35.9% vs 10.2% in the first study, and 18.1% vs 8.7% in the second study). In comparison to glu298 homozygotes, homozygosity for asp298 was associated with an odds ratio of 4.2 (95% confidence interval, 2.3 to 7.9) for angiographic coronary artery disease and 2.5 (95% confidence interval, 1.3 to 4.2) for MI.

Dahiyat et al. (1999) found a significant association of late-onset Alzheimer disease (AD; 104300) and homozygosity for the glu allele in a study of 122 early-onset and 317 late-onset Alzheimer cases compared to 392 controls. This was independent of apoE status. The authors remarked on the interaction of beta-amyloid with endothelial cells. In contrast, Higuchi et al. (2000) studied 411 Japanese patients with sporadic AD and 2 groups of controls: 350 Japanese controls and 52 Caucasian controls. They found no difference in the glu298-to-asp polymorphism between AD patients and controls, even when stratifying for age of onset and presence of the APOE E4 allele. However, they observed that the glu allele frequency was significantly higher in Japanese controls than in Caucasian controls, suggesting that the association reported by Dahiyat et al. (1999) may be a function of race. Akomolafe et al. (2006) found that the glu298 allele was significantly associated with AD in a group of over 200 African American patients, but not among a similar number of Caucasians. A metaanalysis of 12 previously published similar studies showed a small effect of the glu/glu genotype on AD risk across all studies (OR of 1.15), but also showed significant heterogeneity. Akomolafe et al. (2006) noted that the GG genotype (corresponding to glu/glu) is prevalent in the general population (0.33 to 0.87), suggesting that by itself the polymorphism plays only a modest role in the development of AD and likely interacts with other factors.

In a study of 35 patients with histories of placental abruption and 170 control subjects, Yoshimura et al. (2001) found that the frequency of glu298-to-asp homozygotes and heterozygotes was higher in the placental abruption group than in the control group (40% vs 14%; p less than 0.001).

Kobashi et al. (2001) found in a study in Japan that the frequency of heterozygotes and homozygotes for asp298 in the NOS3 gene was significantly higher in patients with hypertension in pregnancy (0.23) (see 189800) than in controls (0.12) (p less than 0.01). Multivariate analysis showed that a family history of hypertension, the TT genotype of the angiotensinogen gene (AGT; 106150.0001), the GA+AA NOS3 genotype, and a prepregnancy body mass index of more than 24 were independent potent risk factors, after adjustment for maternal age and parity. The odds ratios of these factors were 2.7, 2.3, 2.2, and 2.1, respectively. The results suggested that the asp298 of NOS3 is a potent, independent risk factor for hypertension in pregnancy.

In a study of 150 'coloured' South African patients, 50 with normal pregnancies, 50 with severe preeclampsia, and 50 with abruptio placentae, Hillermann et al. (2005) found that the combined frequency of the GT and TT NOS3 variant genotypes was significantly higher in the abruptio placentae group than in the control group (p = 0.006). Among preeclamptic patients who subsequently developed abruptio placentae, the T allele emerged as a major risk factor for the development of abruptio placentae (p less than 0.0001); the T variant did not seem to affect the risk of preeclampsia itself, however.

Jachymova et al. (2001) found a significantly higher frequency of T alleles (related to the E298D polymorphism) in hypertensives (see 145500) as compared to normotensives. Significant association was found in patients showing resistance to conventional antihypertensive therapy. In well-controlled hypertensives the tendency to a higher frequency of T alleles was observed, but this did not reach statistical significance. The presence of the T allele was thought to be predictive of the patients' therapeutic response.

In a metaanalysis of 14 case-control studies evaluating the association between E298D and ischemic heart disease (myocardial infarction or angiographic coronary artery occlusion) involving 6,036 cases and 6,106 controls, Casas et al. (2004) found that homozygosity for asp298 was associated with an increased risk of ischemic heart disease (OR, 1.31).

Berger et al. (2007) performed 2 large case-control studies involving 1,901 hospitalized stroke patients and 1,747 regional population controls and found that E298D was significantly associated with ischemic stroke (601367) independent of age, gender, hypertension, diabetes, and hypercholesterolemia.


.0002 CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO

NOS3, -786T-C
  
RCV000015059

Nakayama et al. (1999) searched for possible mutations in the endothelial nitric oxide synthase gene in patients with coronary spasm. They found evidence of 3 linked mutations in the 5-prime flanking region of the eNOS gene, among them a -786T-C transition. The incidence of these alleles was significantly greater in 174 patients with coronary artery spasm studied than in 161 controls. Multiple logistic regression analysis with forward stepwise selection using the environmental risk factors and the eNOS gene variant revealed that the most predictive independent risk factor for coronary spasm was the mutant allele. As assessed by luciferase reporter gene assays, the -786T-C mutation resulted in a significant reduction of eNOS gene promoter activity, whereas neither of the other mutations had any effect.


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  52. Rapoport, R. M., Murad, F. Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through CGMP. Circ. Res. 52: 352-357, 1983. [PubMed: 6297832, related citations] [Full Text]

  53. Robb, G. B., Carson, A. R., Tai, S. C., Fish, J. E., Singh, S., Yamada, T., Scherer, S. W., Nakabayashi, K., Marsden, P. A. Post-transcriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript. J. Biol. Chem. 279: 37982-37996, 2004. [PubMed: 15234981, related citations] [Full Text]

  54. Robinson, L. J., Weremowicz, S., Morton, C. C., Michel, T. Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene. Genomics 19: 350-357, 1994. [PubMed: 7514568, related citations] [Full Text]

  55. Simoncini, T., Mannella, P., Fornari, L., Caruso, A., Varone, G., Garibaldi , S., Genazzani, A. R. Tibolone activates nitric oxide synthesis in human endothelial cells. J. Clin. Endocr. Metab. 89: 4594-4600, 2004. [PubMed: 15356068, related citations] [Full Text]

  56. Snyder, S. H. No endothelial NO. Nature 377: 196-197, 1995. [PubMed: 7545786, related citations] [Full Text]

  57. Steudel, W., Scherrer-Crosbie, M., Bloch, K. D., Weimann, J., Huang, P. L., Jones, R. C., Picard, M. H., Zapol, W. M. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J. Clin. Invest. 101: 2468-2477, 1998. [PubMed: 9616218, related citations] [Full Text]

  58. Straub, A. C., Lohman, A. W., Billaud, M., Johnstone, S. R., Dwyer, S. T., Lee, M. Y., Bortz, P. S., Best, A. K., Columbus, L., Gaston, B., Isakson, B. E. Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling. Nature 491: 473-477, 2012. [PubMed: 23123858, images, related citations] [Full Text]

  59. Tanus-Santos, J. E., Desai, M., Flockhart, D. A. Effects of ethnicity on the distribution of clinically relevant endothelial nitric oxide variants. Pharmacogenetics 11: 719-725, 2001. [PubMed: 11692081, related citations] [Full Text]

  60. Tempfer, C., Unfried, G., Zeillinger, R., Hefler, L., Nagele, F., Huber, J. C. Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage. Hum. Reprod. 16: 1644-1647, 2001. [PubMed: 11473956, related citations] [Full Text]

  61. Wang, X. L., Sim, A. S., Badenhop, R. F., McCredie, R. M., Wilcken, D. E. L. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nature Med. 2: 41-45, 1996. [PubMed: 8564837, related citations] [Full Text]

  62. Yallampalli, C., Garfield, R. E. Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia. Am. J. Obstet. Gynec. 169: 1316-1320, 1993. [PubMed: 8238200, related citations] [Full Text]

  63. Yasue, H., Kugiyama, K. Coronary artery spasm: Japanese view. Coron. Artery Dis. 1: 668-673, 1990.

  64. 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. 103: 65-69, 1998. [PubMed: 9737779, related citations] [Full Text]

  65. Yoshimura, T., Yoshimura, M., Tabata, A., Yasue, H., Okamura, H. The missense glu298-to-asp variant of the endothelial nitric oxide synthase gene is strongly associated with placental abruption. Hum. Genet. 108: 181-183, 2001. [PubMed: 11354626, related citations] [Full Text]


Bao Lige - updated : 01/23/2019
Patricia A. Hartz - updated : 01/15/2016
Ada Hamosh - updated : 12/14/2012
Ada Hamosh - updated : 3/29/2011
Marla J. F. O'Neill - updated : 6/22/2010
Patricia A. Hartz - updated : 5/6/2009
Ada Hamosh - updated : 4/16/2008
Patricia A. Hartz - updated : 1/16/2008
Marla J. F. O'Neill - updated : 8/21/2007
Cassandra L. Kniffin - updated : 8/25/2006
George E. Tiller - updated : 2/17/2006
Marla J. F. O'Neill - updated : 12/7/2005
Marla J. F. O'Neill - updated : 11/7/2005
Ada Hamosh - updated : 10/25/2005
Cassandra L. Kniffin - updated : 10/4/2005
Victor A. McKusick - updated : 8/19/2005
Marla J. F. O'Neill - updated : 7/19/2005
John A. Phillips, III - updated : 4/13/2005
Patricia A. Hartz - updated : 10/27/2004
Ada Hamosh - updated : 10/29/2003
Ada Hamosh - updated : 2/21/2003
Victor A. McKusick - updated : 1/14/2003
George E. Tiller - updated : 9/18/2002
Victor A. McKusick - updated : 9/9/2002
Cassandra L. Kniffin - updated : 6/14/2002
Ada Hamosh - updated : 4/8/2002
Victor A. McKusick - updated : 3/19/2002
Victor A. McKusick - updated : 10/5/2001
Victor A. McKusick - updated : 8/29/2001
John A. Phillips, III - updated : 5/10/2001
Victor A. McKusick - updated : 4/6/2001
George E. Tiller - updated : 1/25/2001
Ada Hamosh - updated : 5/29/2000
Orest Hurko - updated : 12/2/1999
Victor A. McKusick - updated : 10/21/1999
Ada Hamosh - updated : 6/23/1999
Victor A. McKusick - updated : 3/3/1999
Victor A. McKusick - updated : 2/16/1999
Stylianos E. Antonarakis - updated : 2/4/1999
Victor A. McKusick - updated : 12/18/1998
Victor A. McKusick - updated : 8/19/1998
Victor A. McKusick - updated : 6/26/1998
Victor A. McKusick - updated : 9/24/1997
Victor A. McKusick - updated : 4/24/1997
Creation Date:
Victor A. McKusick : 9/14/1992
mgross : 01/23/2019
carol : 09/27/2018
alopez : 08/04/2016
mgross : 01/15/2016
carol : 3/12/2015
tpirozzi : 10/1/2013
carol : 4/4/2013
alopez : 12/17/2012
terry : 12/14/2012
alopez : 8/3/2012
carol : 6/7/2012
terry : 5/3/2011
terry : 4/29/2011
alopez : 3/31/2011
terry : 3/29/2011
wwang : 8/6/2010
wwang : 6/28/2010
terry : 6/22/2010
alopez : 1/13/2010
mgross : 5/15/2009
terry : 5/6/2009
joanna : 4/10/2009
alopez : 5/13/2008
terry : 4/16/2008
mgross : 1/25/2008
terry : 1/16/2008
wwang : 8/27/2007
wwang : 8/27/2007
terry : 8/21/2007
wwang : 9/1/2006
ckniffin : 8/25/2006
alopez : 3/28/2006
wwang : 3/9/2006
terry : 2/17/2006
carol : 12/7/2005
terry : 12/7/2005
wwang : 11/7/2005
alopez : 10/26/2005
terry : 10/25/2005
carol : 10/11/2005
ckniffin : 10/4/2005
wwang : 9/2/2005
wwang : 8/25/2005
terry : 8/19/2005
wwang : 7/25/2005
terry : 7/19/2005
carol : 5/25/2005
joanna : 5/25/2005
wwang : 5/11/2005
wwang : 4/13/2005
mgross : 10/27/2004
carol : 7/6/2004
alopez : 11/7/2003
cwells : 11/5/2003
alopez : 10/29/2003
terry : 10/29/2003
alopez : 2/24/2003
terry : 2/21/2003
alopez : 1/15/2003
terry : 1/14/2003
cwells : 9/18/2002
alopez : 9/9/2002
carol : 6/17/2002
ckniffin : 6/14/2002
alopez : 4/8/2002
alopez : 4/8/2002
cwells : 4/5/2002
cwells : 3/21/2002
terry : 3/19/2002
carol : 1/4/2002
carol : 11/13/2001
mcapotos : 10/15/2001
mcapotos : 10/10/2001
terry : 10/5/2001
cwells : 9/14/2001
cwells : 8/30/2001
terry : 8/29/2001
mgross : 5/10/2001
mgross : 5/10/2001
mcapotos : 4/16/2001
terry : 4/6/2001
mcapotos : 2/1/2001
mcapotos : 1/25/2001
alopez : 6/1/2000
terry : 5/29/2000
carol : 12/3/1999
terry : 12/2/1999
carol : 10/22/1999
terry : 10/21/1999
carol : 9/20/1999
alopez : 6/23/1999
mgross : 3/17/1999
carol : 3/10/1999
terry : 3/3/1999
carol : 2/16/1999
terry : 2/16/1999
carol : 2/4/1999
carol : 1/13/1999
terry : 12/18/1998
dkim : 9/10/1998
carol : 8/24/1998
terry : 8/19/1998
carol : 6/30/1998
terry : 6/26/1998
terry : 9/30/1997
terry : 9/24/1997
terry : 4/24/1997
terry : 4/21/1997
terry : 2/6/1996
mark : 1/5/1996
terry : 1/3/1996
terry : 10/30/1995
mark : 9/20/1995
carol : 2/15/1994
carol : 9/21/1993
carol : 9/13/1993
carol : 1/11/1993

+ 163729

NITRIC OXIDE SYNTHASE 3; NOS3


Alternative titles; symbols

NITRIC OXIDE SYNTHASE, ENDOTHELIAL; ENOS


Other entities represented in this entry:

CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO, INCLUDED
ALZHEIMER DISEASE, LATE-ONSET, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION, PREGNANCY-INDUCED, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION RESISTANT TO CONVENTIONAL THERAPY, INCLUDED

HGNC Approved Gene Symbol: NOS3

Cytogenetic location: 7q36.1     Genomic coordinates (GRCh38): 7:150,991,017-151,014,588 (from NCBI)


Gene-Phenotype Relationships

Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
7q36.1 {Alzheimer disease, late-onset, susceptibility to} 104300 Autosomal dominant 3
{Coronary artery spasm 1, susceptibility to} 3
{Hypertension, pregnancy-induced} 189800 Autosomal dominant 3
{Hypertension, susceptibility to} 145500 Multifactorial 3
{Ischemic stroke, susceptibility to} 601367 Multifactorial 3
{Placental abruption} 3

TEXT

Cloning and Expression

Nitric oxide (NO) accounts for the biologic activity of endothelium-derived relaxing factor (EDRF), discovered by Furchgott and Zawadzki (1980) and Rapoport and Murad (1983). NO is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). EDRF is important in regulation of vasomotor tone and blood flow by inhibiting smooth muscle contraction and platelet aggregation. Janssens et al. (1992) isolated a cDNA encoding a human vascular NOS. The translated human protein was 1,203 amino acids long with a predicted molecular mass of 133 kD. They showed that the cDNA encodes a calcium-regulated, constitutively expressed endothelial NOS, capable of producing EDRF in blood vessels. Marsden et al. (1992) likewise cloned and sequenced human endothelial NO synthase. Their cDNA clones predicted a protein of 1,203 amino acids with about 60% identity with the rat brain NO synthase isoform (163731). (Nitric oxide synthases have been assigned to 2 classes: a constitutively expressed, calcium-regulated class identified in brain, neutrophils, and endothelial cells, and a calcium-independent class identified in endotoxin- or cytokine-induced macrophages and endothelial cells (163730).)


Gene Structure

Marsden et al. (1993) isolated genomic clones encoding human endothelial NO synthase and determined the structural organization of the gene. It contains 26 exons spanning approximately 21 kb of genomic DNA and encodes an mRNA of 4,052 nucleotides. Characterization of the 5-prime-flanking region indicated that the NOS3 gene is TATA-less and exhibits proximal promoter elements consistent with a constitutively expressed gene, namely, SP1 and GATA motifs.


Mapping

By Southern blot hybridization of human/rodent somatic cell hybrids, Marsden et al. (1993) assigned the NOS3 gene to chromosome 7 and regionalized it to 7q35-q36 by fluorescence in situ hybridization. By PCR amplification applied to a panel of DNAs from human/rodent somatic cell hybrids, by Southern blot analysis, and by fluorescence in situ hybridization, Robinson et al. (1994) localized the NOS3 gene to 7q36. By analysis of interspecific backcross progeny, Gregg et al. (1995) mapped the mouse homolog to chromosome 5.


Biochemical Features

Crystal Structure

Raman et al. (1998) reported the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (BH4)-free and -bound forms at 1.95-angstrom and 1.9-angstrom resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the BH4-binding site. The unexpected recognition of the substrate L-arginine at the BH4 site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.


Gene Function

Fulton et al. (1999) demonstrated that AKT (164730) directly phosphorylated eNOS and activated the enzyme leading to nitric oxide production, whereas eNOS mutated at a putative AKT phosphorylation site was resistant to phosphorylation and activation by AKT. Activated AKT increased basal nitric oxide release from endothelial cells, and activation-deficient AKT attenuated NO production stimulated by VEGF (192240). Thus, Fulton et al. (1999) concluded the eNOS is an AKT substrate linking signal transduction by AKT to the release of the gaseous second messenger nitric oxide.

Dimmeler et al. (1999) stated that physiologically, the most important stimulus for the continuous formation of nitric oxide is the viscous drag (or shear stress) generated by the streaming blood on the endothelial layer. PI3K (see 601232) and AKT are activated in endothelial cells in response to shear stress. Dimmeler et al. (1999) demonstrated that AKT mediates the activation of eNOS, leading to increased nitric oxide production. Inhibition of the PI3K AKT pathway or mutation of the AKT site on eNOS protein at serine-1177 attenuated the serine phosphorylation and prevented the activation of eNOS. Mimicking the phosphorylation of ser1177 directly enhanced enzyme activity and altered the sensitivity of the enzyme to calcium, rendering its activity maximal at subphysiologic concentrations of calcium. Thus, phosphorylation of eNOS by AKT represented a novel calcium-independent regulatory mechanism for activation of eNOS.

Napolitano et al. (2000) investigated the interactions between ET1 (131240) and the NO system in the feto-placental unit. They examined the mRNA expression of ET1, inducible NOS (163730), and eNOS in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive pregnancies. ET1 expression was increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, was decreased; conversely, eNOS expression was increased. ET1 was able to influence its own expression as well as NOS isoform expression in normal and PE trophoblastic cultured cells. The findings suggested the existence of a functional relationship between ET(s) and NOS isoforms that could constitute the biologic mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation that are characteristic of the pathophysiology of preeclampsia.

Using a yeast 2-hybrid screen, Dedio et al. (2001) found that the oxygenase domain of human ENOS interacted with human NOSIP (616759). Reciprocal coimmunoprecipitation and Western blot analysis confirmed interaction between ENOS and NOSIP in cotransfected COS-7 cells. Deletion analysis showed that NOSIP bound the C-terminal portion of the ENOS oxygenase domain in a region that overlapped the binding site for plasma membrane caveolin (see 601047). Overexpression of NOSIP reduced calcium-stimulated nitric oxide production in ENOS-expressing Chinese hamster ovary (CHO) cells. Overexpression of NOSIP also reduced membrane localization of ENOS in CHO cells, causing a shift from caveolin-rich membrane fractions to intracellular compartments. Dedio et al. (2001) concluded that NOSIP promotes translocation of ENOS from the plasma membrane to intracellular sites, thus inhibiting nitric oxide synthesis.

Autosomal dominant adult polycystic kidney disease (ADPKD; 173900) is associated with altered endothelial-dependent vasodilation and decreased vascular production of NO. Thus, eNOS could have a modifier effect in ADPKD. To test this hypothesis, Persu et al. (2002) genotyped 173 unrelated European ADPKD patients for the glu298-to-asp (163729.0001), intron 4 VNTR, and -786T-C (163729.0002) polymorphisms of ENOS and looked for their influence on the age at end-stage renal disease (ESRD). In 93 males, the glu298-to-asp polymorphism was associated with a lower age at ESRD. This effect was confirmed in a subset of males linked to PKD1 (601313) and reaching ESRD before age 45, and by a cumulative renal survival analysis in PKD1-linked families. Further studies demonstrated that NOS activity was decreased in renal artery samples from PKD males harboring the asp298 allele, in association with posttranslational modifications and partial cleavage of eNOS. No significant effect of the other polymorphisms was found in males, and no polymorphism influenced the age at ESRD in females. Persu et al. (2002) concluded that glu298-to-asp is associated with a 5 year lower mean age at ESRD in a subset of ADPKD males. They hypothesized that the effect could be due to decreased NOS activity and a partial cleavage of eNOS, leading to a further decrease in the vascular production of NO.

Nisoli et al. (2003) found that NO triggers mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of NO was dependent on cGMP and was mediated by the induction of PPARGC1 (604517), a master regulator of mitochondrial biogenesis. Moreover, Nisoli et al. (2003) found that mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of eNOS -/- mice, which had a reduced metabolic rate and accelerated weight gain compared to wildtype mice. Thus, Nisoli et al. (2003) concluded that a NO-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.

Simoncini et al. (2004) demonstrated that tibolone and its estrogenic metabolites activate NO synthesis by recruiting functional estrogen receptors, whereas the progestogenic/androgenic metabolite had no effect. During prolonged exposures, tibolone and the estrogenic compounds enhanced the expression of eNOS. In addition, tibolone was able to induce rapid activation of eNOS, leading to rapid increases in the release of NO. Different from estrogen, rapid activation of eNOS did not rely on recruitment of PI3K but rather on MAPK-dependent cascades.

Robb et al. (2004) found that the 5-prime UTR of the NOS3AS (612205) transcript was complementary to the portion of NOS3 mRNA derived from exons 23 to 26. RT-PCR analysis showed that expression of NOS3 in human umbilical vein endothelial cells (HUVECs) and human aortic vascular smooth muscle cells (HAOVSMCs) was inversely proportional to that of NOS3AS. Both the NOS3 and NOS3AS genes were transcriptionally active; however, NOS3AS appeared to downregulate the steady-state levels of NOS3 mRNA and protein. RT-PCR showed that overexpression of the overlapping region of NOS3AS had little impact on NOS3 mRNA levels in HUVECs; however, Western blot analysis showed that NOS3 protein levels were markedly reduced. NOS3AS also reduced the expression of a chimeric transcript containing the NOS3-overlapping sequence fused to a reporter gene. Inhibition of NOS3AS expression by RNA interference (RNAi) in HAOVSMCs increased NOS3 expression, and inhibition of histone deacetylase (see HDAC1; 601241) in HAOVSMCs increased expression of NOS3AS mRNA prior to decrease in NOS3 mRNA expression. Robb et al. (2004) concluded that NOS3AS participates in the posttranscriptional regulation of NOS3.

Fish et al. (2007) found that NOS3AS was induced by hypoxia in cultured human endothelial and smooth muscle cells and in aortas of hypoxic rats. NOS3AS induction preceded the decrease in NOS3 steady-state mRNA in endothelial cells, and knockdown of NOS3AS by RNAi indicated that NOS3AS downregulated NOS3 expression during hypoxia. Hypoxia did not induce transcription of NOS3AS, but instead stabilized NOS3AS transcripts, predominantly the short NOS3AS variant, leading to elevated NOS3AS levels. RT-PCR of fractionated cells showed that both NOS3AS pre-mRNA and mature NOS3AS mRNA were enriched in the nucleus under basal conditions. Under hypoxic conditions, the cytoplasmic levels of mature NOS3AS mRNA increased more than 30-fold, whereas levels in the nucleus increased only moderately. In contrast, the nuclear/cytoplasmic distribution of NOS3 mRNA was not altered under hypoxic conditions, with NOS3 mRNA levels decreasing in both the cytoplasm and nucleus. NOS3 associated with polyribosomes in normoxic cells, but its association with polyribosomes was attenuated in hypoxic cells, concurrent with association of the short NOS3AS variant with polyribosomes. Fish et al. (2007) concluded that NOS3 expression is regulated by its overlapping NOS3AS transcript in a hypoxia-dependent fashion.

Nisoli et al. (2005) reported that calorie restriction for either 3 or 12 months induced eNOS expression and 3-prime/5-prime-cyclic GMP in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and ATP production, and an enhanced expression of Sirt1 (604479). Nisoli et al. (2005) reported these effects were strongly attenuated in eNOS null-mutant mice. Thus, Nisoli et al. (2005) concluded that nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Using human platelets, Ji et al. (2007) demonstrated that polymerization of beta-actin (ACTB; 102630) regulated the activation state of NOS3, and hence NO formation, by altering its binding to heat-shock protein-90 (HSP90, or HSPCA; 140571). NOS3 bound the globular, but not the filamentous, form of beta-actin, and the affinity of NOS3 for globular beta-actin was, in turn, increased by HSP90. Formation of this ternary complex of NOS3, globular beta-actin, and HSP90 increased NOS activity and cyclic GMP, an index of bioactive NO, and increased the rate of HSP90 degradation, thus limiting NOS3 activation. Ji et al. (2007) concluded that beta-actin regulates NO formation and signaling in platelets.

Lim et al. (2008) demonstrated that blocking phosphorylation of the AKT substrate eNOS inhibits tumor initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wildtype Ras proteins (see 190020), which are required throughout tumorigenesis. Lim et al. (2008) suggested that activation of the PI3K-AKT-eNOS-(wildtype) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumor growth.

Chen et al. (2010) showed that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in superoxide generation primarily from the reductase, in which 2 highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. Chen et al. (2010) showed that e-NOS S-glutathionylation in endothelial cells, with loss of nitric oxide and gain of superoxide generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Chen et al. (2010) concluded that S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signaling, endothelial function, and vascular tone.

Straub et al. (2012) reported a model for the regulation of NO signaling by demonstrating that hemoglobin alpha, encoded by the HBA1 (141800) and HBA2 (141850) genes, is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to hemoglobin alpha and is abrogated by its genetic depletion. Mechanistically, endothelial hemoglobin alpha heme iron in the Fe(3+) state permits NO signaling, and this signaling is shut off when hemoglobin alpha is reduced to the Fe(2+) state by endothelial CYB5R3 (613213). Genetic and pharmacologic inhibition of CYB5R3 increases NO bioactivity in small arteries. Straub et al. (2012) concluded that their data revealed a mechanism by which the regulation of the intracellular hemoglobin alpha oxidation state controls NOS signaling in nonerythroid cells. The authors suggested that this model may be relevant to heme-containing globins in a broad range of NOS-containing somatic cells.

Using immunofluorescence analysis, Lechauve et al. (2018) showed that AHSP (ERAF; 605821) was coexpressed with alpha-globin in mouse and human ECs and regulated alpha-globin protein levels. Expression analysis in human coronary artery ECs and experiments with purified human proteins demonstrated that AHSP and eNOS interacted with alpha-globin in a mutually exclusive manner and enhanced its accumulation in cells. However, only AHSP could stabilize oxidized Fe(3+)-alpha-globin. The authors demonstrated that eNOS rapidly reduced AHSP-bound Fe(3+)-alpha-globin via direct electron transfer from its flavin-associated reductase domain.


Molecular Genetics

In a Japanese study of 100 patients with essential hypertension (145500) and 123 patients with normal blood pressure, Nakayama et al. (1997) found that the distribution of allele frequencies for the CA repeat in the NOS3 gene was not significantly different between the 2 groups. However, comparing the allele frequencies in the hypertensive group without left ventricular hypertrophy (LVH) and the normotensive group, the overall distributions were significantly different (p = 0.019). The 33-repeat allele was found more frequently in the hypertensive group without LVH than in the normotensive group.

Bonnardeaux et al. (1995) found that the highly polymorphic (CA)n repeats in intron 13 and 2 biallelic markers in intron 18 of the NOS3 gene are not associated with essential hypertension. Wang et al. (1996) studied a marker closer to the 5-prime end of the NOS3 gene, the 27-bp repeat in intron 4, in relation to coronary artery disease. They identified 2 alleles: a common larger allele (allele frequency, 0.830) and a smaller rare allele (0.170). The larger allele had 5 tandem 27-bp repeats. The smaller allele had only 4 repeats that were apparently missing the third repeat judging by minor a difference in sequence. The distribution of the genotypes appeared to be in Hardy-Weinberg equilibrium and the polymorphism was inherited in a simple mendelian fashion. Wang et al. (1996) found from study of 549 subjects with and 153 without coronary artery disease that in current and ex-cigarette smokers, but not nonsmokers, there was a significant excess of homozygotes for the rare allele in patients with severely stenosed arteries, compared with those with no mild stenosis. This genotype was also associated with a history of myocardial infarction. The authors noted that, since endothelial-dependent vasodilatation is mediated by release of nitric oxide formed by constitutively expressed endothelial nitric oxide synthase, the smoking-dependent excess coronary risk in homozygotes is consistent with a predisposition to endothelial dysfunction.

Coronary spasm plays an important role in the pathogenesis not only of variant angina but also of ischemic heart disease in general. However, the prevalence of coronary spasm appears to be higher in Japanese than in Caucasians (Bertrand et al., 1982; Yasue and Kugiyama, 1990), suggesting that genetic factors may be involved in its pathogenesis. Endothelial-derived nitric oxide has been implicated in the control of vascular tone. Kugiyama et al. (1997) and Motoyama et al. (1997) showed that basal acetylcholine-stimulated and flow-dependent nitric oxide activities are decreased in both coronary and brachial arteries of patients with coronary spasm. Yoshimura et al. (1998) identified a glu298-to-asp variant (E298D; 163729.0001) in exon 7 of the NOS3 gene that was more frequent in patients with coronary spasm. In studies of an elderly population in Australia, Liyou et al. (1998) could find no association of the E298D variant with coronary artery disease.

Nakayama et al. (1999) reported that a -786T-C mutation (163729.0002) in the promoter region of the eNOS gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, Miyamoto et al. (2000) purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1; 179835), known as a single-stranded DNA-binding protein essential for DNA repair, replication, and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotides restored transcription driven by the mutated promoter sequence, whereas overexpression of RPA1 further reduced it. Serum nitrite/nitrate levels among individuals carrying the -786T-C mutation were significantly lower than among those without the mutation. The authors concluded that RPA1 apparently functions as a repressor protein in the -786T-C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.

Pregnancy-induced hypertension (see 189800) may be regarded as a manifestation of endothelial cell dysfunction. Constitutive nitric oxide production in endothelial cells increases during pregnancy and contributes to vasodilatation and blunting of vasopressor response. In women developing pregnancy-induced hypertension, NO generation is inappropriately low, and administration of an NO donor improves flow in the uterine artery in normal early pregnancy and in women at high risk of developing disease. Yallampalli and Garfield (1993) and others had observed that inhibition of NO synthesis in rats during pregnancy produces hypertension, proteinuria, thrombocytopenia, and fetal growth retardation. These considerations prompted Arngrimsson et al. (1997) to study linkage to the NOS3 gene in affected sisters and in multiplex families. A lod score of 3.36 was obtained for D7S505 when a best-fitting model derived from genetic epidemiologic data was used, and lod scores of 2.54 to 4.03 were obtained when various other genetic models were used. The transmission/disequilibrium test (TDT), a model-free estimate of linkage, showed strongest association and linkage with a microsatellite within intron 13 of the NOS3 gene (P = 0.005).

Lewis et al. (1999) were unable to detect linkage of preeclampsia to the NOS3 region on 7q. They studied 2, separately ascertained, affected sister-pairs collections, from Amsterdam and Cambridge (U.K.), that contained 104 sibships. In the Cambridge Centre, a total of 21 extended pedigrees suitable for conventional parametric linkage studies were also identified. The reason for the discrepancy with the results of Arngrimsson et al. (1997) was not clear. Lewis et al. (1999) concluded that although abnormalities in NO production have been observed in preeclampsia, the case for the NOS3 gene or its product, eNOS, having a primary role in the pathophysiology of preeclampsia remained unproved.

Tempfer et al. (2001) performed a prospective case-control study of 105 women with idiopathic recurrent miscarriage and 91 healthy controls. Using PCR, they identified the different alleles of a 27-basepair tandem repeat polymorphism in intron 4 of the NOS3 gene. The wildtype allele was identified in 329 of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B), and 0.5% (A/A). The distribution of genotype frequencies was significantly different between the study and in control groups for allele A/B heterozygotes (36.7 vs 23.8%, P = 0.03, odds ratio 1.6, 95% CI 1.1-3.8). Only 1 person in the study group was A/A.

Tanus-Santos et al. (2001) studied the distribution of genetic variants of 3 clinically relevant NOS3 polymorphisms in 305 ethnically well-characterized DNA samples (100 Caucasians, 100 African Americans, and 105 Asians). They found marked interethnic differences in the distribution of NOS3 variants, in the estimated haplotype frequency, and in the association between variants. The asp298 variant (163729.0001) was more common in Caucasians (34.5%) than in African Americans (15.5%) or Asians (8.6%) (p less than 0.0001). The -786C variant (163729.0002) was also more common in Caucasians (42.0%) than in African Americans (17.5%) or Asians (13.8%) (p less than 0.0001). The 4a VNTR in intron 4 was more common in African Americans (26.5%) than in Caucasians (16.0%) or Asians (12.9%) (p less than 0.0001). The most common predicted haplotype in the 3 groups combined only wildtype variants. Asians had the highest frequency of this haplotype (77% in Asians vs 46% in the other groups). In Caucasians, the asp298 and -786C variants were associated, and this haplotype was predicted to have a frequency of 24%.

Endothelial nitric oxide synthase plays a key role in the regulation of normal function of the vessel wall. Heltianu et al. (2002) found a relatively high frequency of 2 polymorphic variants of NOS3 in males with Fabry disease (301500) and suggested that in addition to mutations in the alpha-galactosidase A gene, variation in NOS3 may be significant in determining the phenotype.

Casas et al. (2004) performed a metaanalysis of 26 case-control studies evaluating the association between the NOS3 polymorphisms E298D, -786T-C, and the intron 4 VNTR and ischemic heart disease (myocardial infarction or angiographic coronary artery occlusion), involving 9,867 cases and 13,161 controls. They found that homozygosity for asp298 or the intron 4 A allele was associated with an increased risk of ischemic heart disease (OR, 1.31 and 1.34, respectively), but no significant association was found with the -786C allele. Casas et al. (2004) suggested that common genetic variations in the NOS3 gene contribute to atherosclerosis susceptibility.

In 110 dizygotic white twin pairs, Persu et al. (2005) identified NOS3 haplotypes based on 3 polymorphisms, E298D, the intron 4 VNTR, and -786T-C, and the intron 13 CA repeat. Haplotype analysis revealed a significant association between NOS3 haplotypes and daytime ambulatory diastolic and systolic blood pressure, with the latter remaining significant after adjustment for multiple testing (p = 0.032) and mainly attributable to 4 haplotypes accounting for 11.9% of all represented haplotypes.

The therapeutic application of NO in high-altitude (HA) disorders, for the improvement of oxygenation and vasodilation, prompted Ahsan et al. (2005) to investigate the NOS3 gene with respect to high-altitude adaptation. They screened 131 HA monks, 136 HA controls, and 170 lowlanders for the NOS3 894G-T (E298D; 163729.0001) polymorphism and for the 4B/4A polymorphisms. NO levels were estimated and correlated with the polymorphisms. The 3 groups were in Hardy-Weinberg equilibrium for the polymorphisms. Wildtype alleles G and 4B were significantly overrepresented in the HA groups as compared with the lowlanders (p = 0.006 and p = 0.02, respectively). NO levels were highest in HA monks, followed by HA controls, and then lowlanders (p less than 0.0001). Combinations of the GG and BB genotypes were distributed significantly more frequently in the HA monks (p less than 0.0001) and HA controls (p = 0.0005) than in lowlanders. Ahsan et al. (2005) concluded that the genotype combination of NOS3 wildtype homozygotes (GG, BB) occurs more frequently in high-altitude groups that in lowlanders and contributes to higher NO levels associated with high-altitude adaptation.


Animal Model

Pharmacologic blockade of NO production with arginine analogs such as L-nitroarginine or L-N-arginine methylester affects multiple isoforms of nitric oxide synthase and so cannot distinguish their physiologic roles. To study the role of endothelial NOS in vascular function, Huang et al. (1995) disrupted the gene encoding endothelial NOS in mice by homologous recombination. Homozygous mutant mice were found to be viable, fertile, and indistinguishable from wildtype and heterozygous littermates in appearance or routine behavior. Immunoreactive NOS3 protein was not present, as shown by Western blot analysis of the brain, heart, lung, and aorta. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, was absent, and the NOS3 mutant mice were hypertensive. Thus, the author concluded that NOS3 mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggested that nonendothelial isoforms of NOS may be involved in maintaining blood pressure. Huang et al. (1995) suggested that perhaps the renin-angiotensin system and autonomic nervous system evolved to serve primarily as a defense against hypotension, and diminution in their activity is a poor buffer against hypertension. Alternatively, NOS3 may be involved in establishing the baroreceptor set-point. The question of whether subpopulations of humans suffering from hypertension have defects in NOS3 expression awaits an answer from genetic analysis and the development of more selective inhibitors of the NOS isoforms.

Snyder (1995) reviewed the significance of the findings of Huang et al. (1995) in the NOS3 'knockout' mouse because so-called nNOS (NOS1; 163731) occurs in nerves that mediate penile erection and NOS inhibitors block erection. It was thought that the null mutant nNOS mice would not procreate. As it turned out, however, these animals do breed and appear to be generally normal. However, they have dilated stomachs with a constricted pyloric sphincter, and so provide a model for infantile hypertrophic pyloric stenosis. The same mice are resistant to brain damage caused by vascular strokes, confirming that nitric oxide is crucial in mediating stroke damage. Further studies indicated that the nNOS-negative mice are highly aggressive toward other males to the extent that they will kill their wildtype littermates if left unattended, and they display strikingly inappropriate and excessive sexual behavior. Mice homozygous for a knockout in NOS2 (macrophage or inducible NOS, iNOS) have markedly reduced defenses against microorganisms such as Listeria and Leishmania and against the proliferation of lymphoma tumor cells.

Champion et al. (1999) cited work indicating that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of NOS3 could enhance erectile responses, they administered a recombinant adenovirus containing the NOS3 gene into the corpora cavernosum of the aged rat. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue one day after the intracavernosal administration of the beta-gal-marked adenovirus; one day after administration of the construct containing NOS3, transgene expression was confirmed by immunoblot staining of NOS3 protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with NOS3, and erectile responses to acetylcholine and zaprinast were enhanced. Champion et al. (1999) suggested that in vivo gene transfer of NOS3, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.

Steudel et al. (1998) investigated the effects of congenital deficiency of NOS3 on the pulmonary vascular responses to hypoxia. The findings suggested that congenital NOS3 deficiency in mice enhances hypoxic pulmonary vascular remodeling and hypertension, and right ventricular hypertrophy, and that NO production by NOS3 is vital to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress.

To study the role of nitric oxide constitutively produced by NOS3 in the regulation of blood pressure and vascular tone, Ohashi et al. (1998) generated transgenic mice overexpressing bovine NOS3 in the vascular wall using murine preproendothelin-1 (ET1) promoter. Blood pressure was significantly lower in NOS3-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release and basal cGMP levels were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, in addition to the essential role of NOS3 in blood pressure regulation, tonic NO release by NOS3 in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilatators, providing several insights into the pathogenesis of nitrate tolerance.

In the heart, nitric oxide inhibits L-type calcium channels but stimulates sarcoplasmic reticulum calcium release, leading to variable effects on myocardial contractility. Barouch et al. (2002) demonstrated that spatial confinement of specific nitric oxide synthase isoforms regulates this process. Endothelial nitric oxide synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type calcium channels allows nitric oxide to inhibit beta-adrenergic-induced inotropy. Neuronal nitric oxide synthase (NOS1; 163731), however, is targeted to cardiac sarcoplasmic reticulum. NO stimulation of sarcoplasmic reticulum calcium release via the ryanodine receptor (RYR2; 180902) in vitro, suggests that NOS1 has an opposite facilitative effect on contractility. Barouch et al. (2002) demonstrated that Nos1-deficient mice have suppressed inotropic response, whereas Nos3-deficient mice have enhanced contractility, owing to corresponding changes in sarcoplasmic reticulum calcium release. Both Nos1 -/- and Nos3 -/- mice developed age-related hypertrophy, although only Nos3 -/- mice were hypertensive. Nos1/3 -/- double knockout mice had suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodeling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Aicher et al. (2003) demonstrated that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS showed reduced vascular endothelial growth factor (VEGF; 192240)-induced mobilization of endothelial progenitor cells and increased mortality after myelosuppression. Intravenous infusion of wildtype progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3-deficient mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3-null mice. Mechanistically, MMP9 (120361), required for stem cell mobilization, was reduced in the bone marrow of Nos3-null mice. Aicher et al. (2003) concluded that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. The authors suggested that this may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic nitric oxide bioactivity.

Caveolin-3 (CAV3; 601253), a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to NOS3 in cardiac myocytes and NOS1 in skeletal myocytes. Ohsawa et al. (2004) characterized the biochemical and cardiac parameters of P104L (601253.0001)-mutant Cav3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (see RMD2, 606072). Transgenic mouse hearts demonstrated hypertrophic cardiomyopathy, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of Cav3 protein. Cardiac muscle showed activation of NOS3 catalytic activity without increased expression of all NOS isoforms. Ohsawa et al. (2004) suggested that a moderate increase in NOS3 activity associated with loss of Cav3 may result in hypertrophic cardiomyopathy.

Bivalacqua et al. (2004) studied the contribution of RhoA (AHRA; 165390)/Rho kinase (ROCK1; 601702) signaling to erectile dysfunction in streptozotocin (STZ) diabetic rats. Rho kinase and eNOS colocalized in the endothelium of corpus cavernosum, and RhoA and Rho kinase abundance and Mypt1 (602021) phosphorylation were elevated in STZ diabetic rat penis. In addition, eNOS protein expression, cavernosal constitutive NOS activity, and cGMP levels were reduced in STZ diabetic rat penis. Bivalacqua et al. (2004) introduced a dominant-negative RhoA mutant and found that erectile responses in the STZ diabetic rats improved to values similar to controls.

Longo et al. (2005) crossbred Nos3-null and wildtype mice to generate 2 types of heterozygous litters, one with a maternally derived mutation that developed in a Nos3-deficient environment and the other with a paternally derived mutation that developed in a uterine environment similar to wildtype mice. In studies of the in vitro reactivity of carotid and mesenteric artery segments of adult mice to vasoactive agents, the maternally derived heterozygous mice had abnormal vascular reactivity that was similar to that of homozygous knockout mice completely lacking functional NOS3, whereas the paternally derived heterozygous mice had normal vascular reactivity that was not different from that of wildtype mice. Longo et al. (2005) stated that this was the first direct evidence in support of a role for uterine environment in determining vascular function in later life.

In a mouse model of sepsis, Connelly et al. (2005) observed a temporal reduction in iNOS expression and activity in lipopolysaccharide-treated Nos3-knockout mice as compared with wildtype mice, which was reflected in a more stable hemodynamic profile in Nos3-null mice during endotoxemia. In human umbilical vein cells, lipopolysaccharide led to the activation of Nos3 through phosphoinositide 3-kinase (see 171833)- and Akt/protein kinase B (164730)-dependent enzyme phosphorylation. Connelly et al. (2005) concluded that NOS3 has a proinflammatory role in the pathogenesis of sepsis, in which following initial NOS3 activation the resultant NO acts as a costimulus for the expression of iNOS.

Liu et al. (2005) presented evidence suggesting a role for G protein-coupled receptor kinase-2 (GRK2; 109635) in the regulation of NO production and hepatic vascular dynamics in a rat model of liver sinusoidal endothelial injury and portal hypertension induced by bile duct ligation. Sinusoidal endothelial cells isolated from the affected animals had increased levels of GRK2, reduced levels of phosphorylated AKT and eNOS, and decreased levels of NO. Gene silencing of GRK2 using siRNA in injured sinusoidal endothelial cells restored AKT activity and resulted in increased NO production. Liu et al. (2005) also found that heterozygous Grk2 mice had increased levels of phosphorylated Akt and decreased portal hypertension in response to injury compared to wildtype mice. Liu et al. (2005) proposed a mechanism in which upregulation of GRK2 after endothelial cell injury directly inhibits phosphorylation of AKT, leading to reduced activation of eNOS and decreased production of NO, and resulting in portal hypertension.


ALLELIC VARIANTS 2 Selected Examples):

.0001   CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO

ALZHEIMER DISEASE, LATE-ONSET, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION, PREGNANCY-INDUCED, SUSCEPTIBILITY TO, INCLUDED
HYPERTENSION RESISTANT TO CONVENTIONAL THERAPY, INCLUDED
ISCHEMIC HEART DISEASE, SUSCEPTIBILITY TO, INCLUDED
ISCHEMIC STROKE, SUSCEPTIBILITY TO, INCLUDED
NOS3, GLU298ASP
SNP: rs1799983, gnomAD: rs1799983, ClinVar: RCV000015053, RCV000015054, RCV000015055, RCV000015056, RCV000015057, RCV000015058, RCV000455688, RCV000677305, RCV001689570, RCV002504788

Yoshimura et al. (1998) found a glu298-to-asp (E298D) variant (rs1799983) in exon 7 of the NOS3 gene. In a study of 113 patients with coronary spasm, in whom the diagnosis of coronary spasm was made by intracoronary injection of acetylcholine, and 100 control subjects, they found a significant difference in the distribution of the variant; 21.2% of the coronary spasm group and 9.0% of the control group (p = 0.014 for dominant effect) showed the variant. In an elderly population in Australia, Liyou et al. (1998) could demonstrate no difference in the distribution of the E298D alleles in relation to the presence of coronary artery disease.

Cai et al. (1999) genotyped 763 white Australians undergoing coronary angiography for the eNOS glu298-to-asp mutation resulting from an 894G-T transversion. The frequencies of the GG, TG, and TT genotypes were not significantly different in men or women with or without coronary artery disease. The mutation was also not associated with myocardial infarction (MI) in males or females, or with the number of significantly stenosed vessels. The T allele frequency (32.5%) was much greater than that reported for the Japanese population (7.8% in controls and 10% in MI patients). Hingorani et al. (1999) investigated the relationship between the glu298-to-asp variant in atherosclerotic coronary artery disease, using 2 independent case-controlled studies. In the first study, cases consisted of 298 unrelated patients with positive coronary angiograms and controls were 138 unrelated healthy individuals ascertained through a population health screen. In the second study, the cases were 249 patients with recent MI and a further 183 unrelated controls. There was an excess of homozygotes for the asp298 variant among patients with angiographic coronary artery disease, and among patients with recent MI when compared with their respective controls (35.9% vs 10.2% in the first study, and 18.1% vs 8.7% in the second study). In comparison to glu298 homozygotes, homozygosity for asp298 was associated with an odds ratio of 4.2 (95% confidence interval, 2.3 to 7.9) for angiographic coronary artery disease and 2.5 (95% confidence interval, 1.3 to 4.2) for MI.

Dahiyat et al. (1999) found a significant association of late-onset Alzheimer disease (AD; 104300) and homozygosity for the glu allele in a study of 122 early-onset and 317 late-onset Alzheimer cases compared to 392 controls. This was independent of apoE status. The authors remarked on the interaction of beta-amyloid with endothelial cells. In contrast, Higuchi et al. (2000) studied 411 Japanese patients with sporadic AD and 2 groups of controls: 350 Japanese controls and 52 Caucasian controls. They found no difference in the glu298-to-asp polymorphism between AD patients and controls, even when stratifying for age of onset and presence of the APOE E4 allele. However, they observed that the glu allele frequency was significantly higher in Japanese controls than in Caucasian controls, suggesting that the association reported by Dahiyat et al. (1999) may be a function of race. Akomolafe et al. (2006) found that the glu298 allele was significantly associated with AD in a group of over 200 African American patients, but not among a similar number of Caucasians. A metaanalysis of 12 previously published similar studies showed a small effect of the glu/glu genotype on AD risk across all studies (OR of 1.15), but also showed significant heterogeneity. Akomolafe et al. (2006) noted that the GG genotype (corresponding to glu/glu) is prevalent in the general population (0.33 to 0.87), suggesting that by itself the polymorphism plays only a modest role in the development of AD and likely interacts with other factors.

In a study of 35 patients with histories of placental abruption and 170 control subjects, Yoshimura et al. (2001) found that the frequency of glu298-to-asp homozygotes and heterozygotes was higher in the placental abruption group than in the control group (40% vs 14%; p less than 0.001).

Kobashi et al. (2001) found in a study in Japan that the frequency of heterozygotes and homozygotes for asp298 in the NOS3 gene was significantly higher in patients with hypertension in pregnancy (0.23) (see 189800) than in controls (0.12) (p less than 0.01). Multivariate analysis showed that a family history of hypertension, the TT genotype of the angiotensinogen gene (AGT; 106150.0001), the GA+AA NOS3 genotype, and a prepregnancy body mass index of more than 24 were independent potent risk factors, after adjustment for maternal age and parity. The odds ratios of these factors were 2.7, 2.3, 2.2, and 2.1, respectively. The results suggested that the asp298 of NOS3 is a potent, independent risk factor for hypertension in pregnancy.

In a study of 150 'coloured' South African patients, 50 with normal pregnancies, 50 with severe preeclampsia, and 50 with abruptio placentae, Hillermann et al. (2005) found that the combined frequency of the GT and TT NOS3 variant genotypes was significantly higher in the abruptio placentae group than in the control group (p = 0.006). Among preeclamptic patients who subsequently developed abruptio placentae, the T allele emerged as a major risk factor for the development of abruptio placentae (p less than 0.0001); the T variant did not seem to affect the risk of preeclampsia itself, however.

Jachymova et al. (2001) found a significantly higher frequency of T alleles (related to the E298D polymorphism) in hypertensives (see 145500) as compared to normotensives. Significant association was found in patients showing resistance to conventional antihypertensive therapy. In well-controlled hypertensives the tendency to a higher frequency of T alleles was observed, but this did not reach statistical significance. The presence of the T allele was thought to be predictive of the patients' therapeutic response.

In a metaanalysis of 14 case-control studies evaluating the association between E298D and ischemic heart disease (myocardial infarction or angiographic coronary artery occlusion) involving 6,036 cases and 6,106 controls, Casas et al. (2004) found that homozygosity for asp298 was associated with an increased risk of ischemic heart disease (OR, 1.31).

Berger et al. (2007) performed 2 large case-control studies involving 1,901 hospitalized stroke patients and 1,747 regional population controls and found that E298D was significantly associated with ischemic stroke (601367) independent of age, gender, hypertension, diabetes, and hypercholesterolemia.


.0002   CORONARY ARTERY SPASM 1, SUSCEPTIBILITY TO

NOS3, -786T-C
SNP: rs2070744, gnomAD: rs2070744, ClinVar: RCV000015059

Nakayama et al. (1999) searched for possible mutations in the endothelial nitric oxide synthase gene in patients with coronary spasm. They found evidence of 3 linked mutations in the 5-prime flanking region of the eNOS gene, among them a -786T-C transition. The incidence of these alleles was significantly greater in 174 patients with coronary artery spasm studied than in 161 controls. Multiple logistic regression analysis with forward stepwise selection using the environmental risk factors and the eNOS gene variant revealed that the most predictive independent risk factor for coronary spasm was the mutant allele. As assessed by luciferase reporter gene assays, the -786T-C mutation resulted in a significant reduction of eNOS gene promoter activity, whereas neither of the other mutations had any effect.


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Contributors:
Bao Lige - updated : 01/23/2019
Patricia A. Hartz - updated : 01/15/2016
Ada Hamosh - updated : 12/14/2012
Ada Hamosh - updated : 3/29/2011
Marla J. F. O'Neill - updated : 6/22/2010
Patricia A. Hartz - updated : 5/6/2009
Ada Hamosh - updated : 4/16/2008
Patricia A. Hartz - updated : 1/16/2008
Marla J. F. O'Neill - updated : 8/21/2007
Cassandra L. Kniffin - updated : 8/25/2006
George E. Tiller - updated : 2/17/2006
Marla J. F. O'Neill - updated : 12/7/2005
Marla J. F. O'Neill - updated : 11/7/2005
Ada Hamosh - updated : 10/25/2005
Cassandra L. Kniffin - updated : 10/4/2005
Victor A. McKusick - updated : 8/19/2005
Marla J. F. O'Neill - updated : 7/19/2005
John A. Phillips, III - updated : 4/13/2005
Patricia A. Hartz - updated : 10/27/2004
Ada Hamosh - updated : 10/29/2003
Ada Hamosh - updated : 2/21/2003
Victor A. McKusick - updated : 1/14/2003
George E. Tiller - updated : 9/18/2002
Victor A. McKusick - updated : 9/9/2002
Cassandra L. Kniffin - updated : 6/14/2002
Ada Hamosh - updated : 4/8/2002
Victor A. McKusick - updated : 3/19/2002
Victor A. McKusick - updated : 10/5/2001
Victor A. McKusick - updated : 8/29/2001
John A. Phillips, III - updated : 5/10/2001
Victor A. McKusick - updated : 4/6/2001
George E. Tiller - updated : 1/25/2001
Ada Hamosh - updated : 5/29/2000
Orest Hurko - updated : 12/2/1999
Victor A. McKusick - updated : 10/21/1999
Ada Hamosh - updated : 6/23/1999
Victor A. McKusick - updated : 3/3/1999
Victor A. McKusick - updated : 2/16/1999
Stylianos E. Antonarakis - updated : 2/4/1999
Victor A. McKusick - updated : 12/18/1998
Victor A. McKusick - updated : 8/19/1998
Victor A. McKusick - updated : 6/26/1998
Victor A. McKusick - updated : 9/24/1997
Victor A. McKusick - updated : 4/24/1997

Creation Date:
Victor A. McKusick : 9/14/1992

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carol : 2/15/1994
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carol : 1/11/1993