In Silico Analysis of Single Nucleotide Polymorphisms (SNPs) of three Isoforms of Human Nitric Oxide Synthase (nNOS, iNOS, eNOS) Genes

London Journal of Research in Computer Science and Technology
Volume | Issue | Compilation
Authored by Shimaa Mahalah , NA
Classification: J.m
Keywords: nitric oxide synthase (NOS), nitric oxide (NO), single nucleotide polymorphisms (SNPs), bioinformatics softwares.
Language: English

The three isoforms of Nitric Oxide Synthase (NOS) synthesize free radical nitric oxide (NO), which has numerous protein targets in human body. Several vital processes are regulated and/or mediated by NO in nervous, immune and cardiovascular systems. Hence, alteration on NO level leads to pathological conditions in particular cells or tissues. Prior to conduction population genetic research, listing and identifying the most deleterious SNPs that may have strong relation with a particular disease is crucial. Hence, the aim of this study was to determine the functional non-synonymous Single Nucleotide Polymorphisms (nsSNPs) with emphasis on the exon regions for the neuronal, Inducible and Endothelial Nitric Oxide (nNOS, iNOS, eNOS) genes. Data from dbSNP database were functionally and structurally analyzed using different bioinformatics softwares. In the exon region, 222 SNP (from total 5293), 203 SNPs (from 1441) and 195 SNP (from 782) in nNOS, iNOS and eNOS, respectively were analyzed. Results of SIFT and PolyPhen predicted six SNPs in nNOS, iNOS, and seven SNPs in eNOS genes as damaging. Whereas, I-mutant server showed decrease stability of proteins encoded by them. Then CPH modeler 3.2 and Chimera software version 1.2 showed structure of these proteins. Further, Proscan version 1.7 server, Promotor 2.0 prediction server and TSSG prediction program identified cis regulatory elements in the above genes. Interestingly, most deleterious SNPs found in this study have not reported yet, especially in eNOS.

               

In silico analysis of Single Nucleotide Polymorphisms (SNPs) of three isoforms of Human Nitric Oxide Synthase (nNOS, iNOS, eNOS) genes

Shimaa M. S. Mahalahα,  Amina I. Dirarσ, Abubaker H. Mohamedρ, Ozaz Y.MohammedѠ Mohamed M. Hassan¥  &  A. A. Daak§

____________________________________________

 ABSTRACT

The three isoforms of Nitric Oxide Synthase (NOS) synthesize free radical nitric oxide (NO), which has numerous protein targets in human body.Several vital processes are regulated and/or mediated by NO in nervous, immune and cardiovascular systems. Hence, alteration on NO level leads to pathological conditions in particular cells or tissues. Prior to conduction population genetic research, listing and identifying the most deleterious SNPs that may have strong relation with a particular disease is crucial. Hence, the aim of this study was to determine the functional non-synonymous Single Nucleotide Polymorphisms (nsSNPs) with emphasis on the exon regions for the neuronal, Inducible and Endothelial Nitric Oxide (nNOS, iNOS, eNOS) genes. Data from dbSNP database were functionally and structurally analyzed using different bioinformatics softwares. In the exon region, 222 SNP (from total 5293), 203 SNPs (from 1441) and 195 SNP (from 782) in nNOS, iNOS and eNOS, respectively were analyzed. Results of SIFT and PolyPhen predicted six SNPs in nNOS, iNOS, and seven SNPs in eNOS genes as damaging. Whereas, I-mutant server showed decrease stability of proteins encoded by them. Then CPH modeler 3.2 and Chimera software version 1.2 showed structure of these proteins. Further, Proscan version 1.7 server, Promotor 2.0 prediction server and TSSG prediction program identified cis regulatory elements in the above genes. Interestingly, most deleterious SNPs found in this study have not reported yet, especially in eNOS.

Keywords: nitric oxide synthase (NOS), nitric oxide (NO), single nucleotide polymorphisms (SNPs), bioinformatics softwares.

Author α §:  Department of biochemistry -Faculty of Medicine, University of Khartoum (Sudan).

σ: Faculty of Pharmacy, University of Khartoum (Sudan).

ρ: Department of Bioinformatics, Africa city of technology, (Sudan).

Ѡ: Faculty of medical Laboratory Sciences, University of Al-jazeera(Sudan).

¥: Faculty of medical Laboratory Sciences, University of Medical Science and Technology (Sudan).

  1. INTRODUCTION

Single Nucleotide Polymorphisms (SNPs) represent the most frequent form of polymorphism in the human genome.[1,2]. Nitric oxide synthases (NOSs) synthesize the metastable free radical nitric oxide (NO), it is an unorthodox messenger molecule, has numerous targets enzymes and proteins [3,4]. There are three isoforms of  NOS: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS)[5]. Neuronal NOS (nNOS) gene located within chr12, reference Sequence: NG_011991.2. It is calcium-dependent and produces low level of NO as a cell signaling molecule. It constitutively expressed in specific neurons of the brain .It has been identified by immunohistochemistry in: spinal cord, sympathetic ganglia, peripheral nitrergic nerves, epithelial cells of various organs, kidney (macula densa cells), adrenal glands, islet cells of pancreatic, and  vascular smooth muscle. The major source of nNOS in mammalians, is the skeletal muscle.nNOS has been implicated in modulating learning, memory, and neurogenesis. Disturbance in NO signaling , contribute to a variety of neurodegenerative pathologies such as excitotoxicity following stroke, multiple sclerosis, Alzheimer’s, and Parkinson’s diseases.Also Freudenberg  mentioned in his review , some variants of nNOS associated with development of Psychiatric diseases such as Major depression, bipolar disorders, Autistic Spectrum disorder (ASD), Obsessive Compulsive disorder(OCD), Anxiety disorders, and Schizophrenia (Freudenberg et, al 2015[6-10].  In addition there are two researches done in 2012, and 2013 showed that here is significant association between some SNPs in nNOS and ischemic stroke [11,12]. Inducible (iNOS) gene located in: chr17, reference Sequence NG_011470.1. It encode calcium-independent enzyme that produces large amounts of NO.It identified basically in macrophages and express virtually in any cell or tissue that can be cytotoxic on parasitic microorganisms as well as on some tumor cells [13].Also can be expressed in cells when induced by bacterial lipopolysaccharide, cytokines, and other  inducing agents . So, non-immune cells can  be induced and affect the adjacent cells, for example ,Cytokine-activated endothelial cells, have been shown to lyse tumor cells, and induced hepatocytes kill malaria sporozoites . Also, it involved in non-specific allograft  rejection[7,14-17]. Moreover excessive release of NO by iNOS plays a major role in septic shock, it lower the blood pressure predominantly due to it is effect in the vascular wall[18]. Interestingly , genetic variations in iNOS are involved in resistance to malaria as mentioned in research done by Maria de Jesus Trovoada, and her colleges in 2014 [19].

Endothelial NOS (eNOS) gene located in chr7, reference sequence NG_011992.1(NCBI). It encodes calcium-dependent NOS that produce low levels of NO, mostly it expressed in endothelial cells, and has been detected in cardiac myocytes, platelets, certain neurons of the brain, in syncytio-trophoblasts of the human placenta and in LLC-PK1 kidney tubular epithelial cells [7,17].Physiological functions of eNOS include: vasodilation, inhibition of platelet aggregation and adhesion, inhibition of leucocyte adhesion,vascular inflammation, and Control of vascular smooth muscle proliferation. Furthermore, NO has been shown to inhibit DNA synthesis, mitogenesis, and proliferation of vascular smooth muscle cells [20,21], and has a role in Stimulation of angiogenesis postnatal, also had been found to be critical for collateral formation and angiogenesis post-ischemia [22]. One of the meta-analysis revealed that eNOS 4b/polymorphisms could be a risk factor for coronary artery disease, particularly in African populations [23]. Other research conclude that, (A-->G) change in eNOS is one of the most important variant associated with susceptibility to essential hypertension [24].Moreover, NOS is known to decrease the bioavailability of NO in sickle patients and play a role in different phenotypic presentation in patients, so every deleterious SNPs may affect the severity and outcome of the disease in sickle patients.

As Goldstein DB said in his paper, much attention of researches focused to date has focused on three polymorphisms in the eNOS gene which are: (-786T>C (rs2070744), intron 4 27-base-pair repeat, and Glu298Asp (rs1799983)), and this limits the study of eNOS to an isolated‘‘candidate polymorphism’’ rather than a ‘‘candidate gene [25]. Therefore , identifying other pathological variants is required to carry out other genetic researches -the Same approach has been used to investigate the effect of nsSNP of HLA–DRB1 and HLA-DQB1 genes[26], so this study was done to identify the most deleterious SNPs in each isoform, and to determine the effect of them in structure and function and stability of proteins, that may contribute with other molecules to cause diseases.

  1. MATERIAL AND METHODS

Softberry (http://www.softberry.com/),and is the most The data of SNPs of the 3 isoform of human NOS genes was collected from National Center for Biological Information (NCBI) web site. The information of the SNP (i.e protein accession number and SNP ID) of these isoform was retrieved from NCBI dbSNP. (http://www. ncbi.nlm.nih.gov/snp/) and Swiss Prot databases (http://expasy.org/),

2.1  In silico analysis of the functional impact of coding nsSNPs using

(SIFT) software (Sorting Tolerant From Intolerant),(http://blocks.fhcrc.org/sift/SIFT.html)  is an online bioinformatics tool used to predict whether an amino acid substitution will affect the protein function or not.[27]. The main underlying principle of this program is that it generates alignments with a large number of homologous sequences, and assigns scores to each residue ranging from zero to one. Scores close to zero indicate evolutionary conservation of the genes and intolerance to substitution, while scores close to one indicate tolerance to substitution only [28].

(PolyPhen) software: Is an online bioinformatics program (http://genetics.Bwh.harvard.edu/ pph2/),  automatically predict the consequence of an amino acid change on the structural and functional protein level. The program search for  protein 3D structures, do multiple alignments of homologous sequences and amino acid contact information in several protein structure databases, then calculate position-specific independent count scores (PSIC) for each of two variants, and then computes the PSIC scores difference between two variants. The higher PSIC score difference, indicate that the functional impact of particular amino acid substitution is likely to occur [29, 30].

2.2  Identification of cis regulatory elements using

  1. PROSCAN version 1.7 Web Promoter Scan Service

It predicts promoter regions based on homologies with putative eukaryotic Pol II promoter sequences. The site is serviced and maintained by Dr. Dan Prestridge at the Advanced Biosciences Computing Center, University of Minnesota. (http://bimas.dcrt.nih.gov/molbio/proscan/)

  • Promoter 2.0 Prediction Server

It predicts transcription start sites of vertebrate Pol II promoters in DNA sequences. It has been developed as a frequently updated database of simulated transcription factors that interact with sequences in promoter regions. It builds on principles that are common to neural networks and genetic algorithms. The site is serviced and maintained by Steen Knudsen at The Center for Biological Sequence Analysis at the Technical University of Denmark. (http:/www.cbs.Dtu. dk/services/Promoter/)

  • TSSG

This is the tool that used in Recognition of human Pol II promoter regions and transcription start sites, located in accurate mammalian cis element prediction program.  Also has the fewest false positive predictions[31].

2.3  Prediction of the protein stability using

  1. I-Mutant server

To support the results and to predict the stability of deleterious SNPs, I-Mutant2.0 online software has been used (http://folding.biofold. org/i- mutant/i-mutant2.0.html). It is a web server using for  automatic prediction of protein stability changes upon single-site mutations, the result is a sign of DDG (the free energy change), positive sign (+) indicates increase of stability, negative sing (-) indicates decrease of stability.

2.4  Modeling of the deleterious SNPs using

  1.  CPH model 3.2 server

It has been used to predict the 3D structure for those proteins with an unknown 3D structure model. It is a protein homology modeling server, where the template recognition is based on profile to profile alignment, guided by secondary structure and exposure predictions. http://www.cbs.dtu.dk/services/CPHmodels/.

2)  Chimera software version 1.8.1

This is a homology modeling software that has been used to generate the mutated models of each of the selected PDB (protein database) entries. This software is used to browse respectively locate the 3D structure of the specific protein and then alter the native amino acid with a mutated one to then look for structural effect that may produce. The outcome is then a graphic model depicting the mutation [32].

2.5  Analysis of the deleterious SNPs using project hope

It is online software (http://www.Cmbi. ru.nl/hope) has been used to build an automatic mutant analysis server that analyzes the structural and functional effects of point mutations, it is easy to understand and deal with and provide attractive results for the researchers [33]. It evaluate the effect of the mutation on the following features: Contacts made by the mutated residue, structural domains in which the residue is located, modifications on this residue and known variants for this residue.

  1.  RESULTS

Table 1: shows numbers of  SNPs in regions of nNOS, iNOS ,and eNOS genes (based on the dbSNP database)

 

 

Number of SNPs

 

sNPS

nNOS

iNOS

eNOS

Exons

222

203

195

5UTR

19

29

2

3UTR

145

43

8

Introns and other

4907

1166

477

Figure 1: Bar chart represents the distribution of 3′ UTR, 5′ UTR, Exons and intronic and other SNPs for nNOS, iNOS and eNOS genes (based on the dbSNP database)

Table 2: shows List of deleterious nsSNPs of iNOS that were analyzed using SIFT and Polyphen software’s, And also result of I-Mutant server

Rs id nNOS

Position

Sift Score

Prediction

Poly2 Score

Predioction in PolyPhen

Stability

rs 56308341

R23H

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 76839820

TIo5M

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 78402290

R19c

0.01

DAMAGINE

1

Probably damaging

Decrease

rs80348085

R48S

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 78422671

R1129H

0.02

DAMAGINE

0.903

Probably damaging

Decrease

rs 55922940

L216P

0.01

DAMAGINE

0.814

Possible damaging

Decrease

Rs id iNOS

Position

Sift Score

Prediction in Sift Score

Poly2 Score

Predioction in PolyPhen

Stability

rs 28944201

R1009C

0.02

DAMAGINE

0.984

Probably damaging

Decrease

rs 406104261

R506W

0.02

DAMAGINE

0.996

Probably damaging

Decrease

rs 112588673

R452Q

0.01

DAMAGINE

0.99

Probably damaging

Decrease

rs 143835443

L720F

0.05

DAMAGINE

0.97

Probably damaging

Decrease

rs 150704221

R750H

0.01

DAMAGINE

0.821

Possible damaging

Decrease

145383683

V1037l

0.02

DAMAGINE

0.456

Possible damaging

Decrease

Rs id eNOS

Position

Sift Score

Prediction in Sift Score

Poly2 Score

Predioction in PolyPhen

Stability

rs 146141837

R1172C

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 14178 7079

R530Q

0.01

DAMAGINE

0.984

Probably damaging

Decrease

rs 143324164

R128W

0.01

DAMAGINE

0.993

Probably damaging

Decrease

rs 145711802

F172L

0.03

DAMAGINE

0.937

Possible damaging

Decrease

rs 141456642

E156K

0.03

DAMAGINE

0.935

Possible damaging

Decrease

rs 145000830

Q411H

0.02

DAMAGINE

0.591

Possible damaging

Decrease

rs 3918232

V827M

0.04

DAMAGINE

0.476

Possible damaging

Decrease

Table 3: Shows results of proscan version 1.7 and tssg of nNOSgene

No

Strand

Promoter Location

No.

Stand

Promoter Location

1

Forward

5317 to 5567

16

Forward

140126 to140376

2

Forward

5591 to 5841

17

Forward

145087 to 145337

3

Forward

15511 to 15761

18

Forward

149576 to 149826

4

Forward

22122 to 22372

19

Forward

154991 to 155241

5

Forward

34103 to 34335

20

Reverse

157321 to 157071

6

Forward

35351 to 35601

21

Reverse

142595 to142345

7

Forward

37430 to 37680

22

Reverse

118101 to 117851

8

Forward

40608to 40858

23

Reverse

98357 to 98107

9

Forward

42576 to 42826

24

Reverse

89682 to 89432

10

Forward

43442to 43692

25

Reverse

56773 to 56523

11

Forward

72314 to 72564

26

Reverse

35798 to 35548

12

Forward

106229 to 106479

27

Reverse

18620 to 18370

13

Forward

114649 to 114899

28

Reverse

5845 to 5595

14

Forward

128185 to 128435

29

Reverse

5411 to 5161

15

Forward

131052 to 131302

 

 

 

 Table 4: Shows result of proscan version 1.7 and tssg of eNOS and iNOS genes

eNOS Promoter

iNOS Promoter

No

Strand

Location

Stand

Location

1

Forward

2376 to 2626

Forward

2869 to 3119

2

Forward

18192 to 18442

Forward

6509 to 6759

3

Forward

22274 to 22524

Forward

16614 to 16864

4

Reverse

22746 to 22496

Forward

37132 to 37382

5

Reverse

18539 to 18289

Reverse

37784 to 37534

6

Reverse

11775 to 11525

Reverse

25471 to 25271

7

Reverse

9846 to 9596

Reverse

3137 to 6887

8

Reverse

2873 to 2623

 

 

Table 5: Shows results of promotor 2.0 Prediction Server of nNOS, INOS , and eNOS genes

Rs id nNOS

Position

Sift Score

Prediction

Poly2 Score

Predioction in PolyPhen

Stability

rs 56308341

R23H

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 76839820

TIo5M

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 78402290

R19c

0.01

DAMAGINE

1

Probably damaging

Decrease

rs80348085

R48S

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 78422671

R1129H

0.02

DAMAGINE

0.903

Probably damaging

Decrease

rs 55922940

L216P

0.01

DAMAGINE

0.814

Possible damaging

Decrease

rs id inos

Position

Sift Score

Prediction in Sift Score

Poly2 Score

Predioction in PolyPhen

Stability

rs 28944201

R1009C

0.02

DAMAGINE

0.984

Probably damaging

Decrease

rs 406104261

R506W

0.02

DAMAGINE

0.996

Probably damaging

Decrease

rs 112588673

R452Q

0.01

DAMAGINE

0.99

Probably damaging

Decrease

rs 143835443

L720F

0.05

DAMAGINE

0.97

Probably damaging

Decrease

rs 150704221

R750H

0.01

DAMAGINE

0.821

Possible damaging

Decrease

145383683

V1037l

0.02

DAMAGINE

0.456

Possible damaging

Decrease

rs id enos

Position

Sift Score

Prediction in Sift Score

Poly2 Score

Predioction in PolyPhen

Stability

rs 146141837

R1172C

0.01

DAMAGINE

1

Probably damaging

Decrease

rs 14178 7079

R530Q

0.01

DAMAGINE

0.984

Probably damaging

Decrease

rs 143324164

R128W

0.01

DAMAGINE

0.993

Probably damaging

Decrease

rs 145711802

F172L

0.03

DAMAGINE

0.937

Possible damaging

Decrease

rs 141456642

E156K

0.03

DAMAGINE

0.935

Possible damaging

Decrease

rs 145000830

Q411H

0.02

DAMAGINE

0.591

Possible damaging

Decrease

rs 3918232

V827M

0.04

DAMAGINE

0.476

Possible damaging

Decrease

Figure 2: shows Structure model (cartoon shape) of wild type amino acid Arginine (R)of iNOS in “stick” (magenta color) (left), and mutant type Glutamine (Q) in “stick” (grey color) at position 452  using chimera software

Figure 3: shows Structure model (cartoon shape) of wild type amino acid (R) in “stick” (Magenta color) (left), and mutant type (H) in “stick” (Grey color) of nNOS gene (right) at position 1129   using chimera software

Figure4: shows wild amino acid (R) and mutant one (w) in position128 in eNOS gene and Close-up of the mutation using project hope. The protein is colored grey, the side chains of both the wild-type and the mutant residue are shown and colored green and red respectively

  1.  DISCUSSION

Nitric oxide synthase is an enzyme, with three isoform (i.e. nNOS, iNOS, eNOS) synthesis Nitric oxide (NO), as messenger molecule and neurotransmitter; it plays an important role in regulation and modulation of a lot of processes in the nervous, immune, and cardiovascular systems. From the database of single nucleotide polymorphisms in a national center of biotechnology (NCBI), 5293, 1441, and 782 SNPs in nNOS, iNOS, ande NOS respectively, were obtained and categorized as shown in figure 1, table1. So an effort was done  to identify most deleterious  SNPs that can affect the structure, function and stability of the proteins that encode these genes . SNPs was submitted to the SIFT as well as to the PolyPhen server, their prediction are six damaging SNPs in both nNOS and INOS ,and 7 damaging SNPs in eNOS as shown in tables 2. It has been reported in two studies done in 2002 and 2010 in Japanese population,  SNP (C276T)  and SNP (rs41279104) in nNOS are significantly associated with schizophrenia, with p value of allelic frequency = 0.000007. and p value of genotype = 0.0013 , allelic p = ), respectively [34,35]. Also mentioned in two studies, one done by Manso, H. and his colleages , and other in Chinese population, SNPs in nNOS (rs2293050, rs2139733, rs7308402 and rs1483757) were significantly associated with susceptibility to stroke and ischemic stroke (rs7308402) respectively,[11,12]. rs1800780 in the eNOS  associated with susceptibility to essential hypertension as reported in B. Yang et. al, 2013[24].

I- mutant server’s result showed that, some deleterious SNPs decrease the stability of these proteins while the other increase it as shown in tables 2. To visualize the effect of these damaging SNPs, cph modeler sever, Chimera software and project hope has been used for probably damaging SNPs as shown in figure 2,3,4. Important point is that, only one 3D structure of nNOS (1129) , iNOS (452), and eNOS (128) were done from all other damaging SNPs, the problem is that, there is no pdb id for protein encoding these genes, in addition, there is no full structure done for these isoforms , then protein sequence were checked again for their 3D structural  using pBLAST analysis and  also protein data bank (pdb), they showed similarity is very weak association with proteins found in data bank http://www. rcsb.org/pdb/home/home.do, so, visualization of 3D structure of  deleterious SNPs that  found  just  in chain C -which extent from 755 to 1418- and chain A- from 83 to 505- and from 76 to 480 in nNOS, iNOS, and eNOS respectively has been appeared. Hap map project (http://hapmap. ncbi.nlm.nih.gov/) and 1000 genome project has been used to check the allelic frequency of these damaging SNPs that has been appeared in chains of the protein encoded genes,  SNP (rs78422671) in nNOS which is missense transition  mutation from G To A  that change the amino acid sequence from Arginine (R) – hydrophilic- to Histidine (H)- hydrophilic- not found in Hap Map project, in 1000 genome project (MAF = 0.0002/1). SNP (rs112588673) in iNOS ,which is missense transition  mutation from G to A, that convert the amino acid from Arginine (R)- hydrophilic - to Glutamine (Q)- hydrophilic-, not found neither in HapMap project, nor in 1000 genome project. The last SNP (rs143324164) in eNOS ,which is missense transition  mutation from C to T, that convert  he amino acid from Arginine (R)- hydrophilic - to t tryptophan (W)

- Aromatic , hydrophobic -, it was not found in HapMap project, but was found in 1000 genome project with MAF = 0.0004/2 . Furthermore, cis regulatory elements in three genes (nNOS, iNOS, and eNOS) were determined, as shown in tables 4,5,6. Numbers of promotor start sites by Proscan version 1.7 server is equal to that came out by TSSG prediction program , but  differ from the results when Promotor 2.0 prediction server was used , so this server less reliable than others two. Combination of clinical evidences, and in silico analyses strongly recommended to  increase our knowledge and to understand  the effect deleterious SNPs and their role in the pathogenesis of the NO related diseases. Hence, these most deleterious SNPs may constitute distinct genetic markers that may be used as powerful mutation-screening in disease epidemiological studies.

V.     CONCLUSION

Bioinformatics field is a powerful field nowadays worldwide and used in all fields of life sciences. Six SNPs in both (nNOS, iNOS) ,and seven SNPs in eNOS genes as damaging., these deleterious SNPs will affect the bioavailability of nitric oxide, and thus will contribute in the pathogenesis of human immune, neuro and cardiovascular diseases.

  1.  RECOMMENDATION

More researches are warranted to detect the deleterious SNPs that come out from this study. x-ray crystallography and NMR must be done for other chain of the Three isoforms of human NOS , in order to  be able to do 3d structure of them and  to visualize  the mutation that may occur on them

Disclosure statement.

The authors declare that, there is no conflict of interest regarding the publication of this paper.

 REFERENCES

  1. Wang, D. G., J. B. Fan, et al. 1998. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science. 15,280(5366) (May 1998), 1077-1082.DOI=https://doi.org/10.1126/science. 280.5366.1077.
  2. Sachidanandam, R., D. Weissman, et al. 2001. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature. 409, 6822 (Feb. 2001), 928-933.DOI=https://doi.org/10.1038/350 57149
  3.  O’Dell TJ, Hawkins RD, Kandel ER, Arancio O. 1991. Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc Natl Acad Sci USA. 88,24 (Dec.1991), 11285–11289.
  4. Schuman E. M., Madison D. V. 1991. A requirement for the intercellular messenger nitric oxide in long-term potentiation. Science. 6, 254 (Dec.1991), 1503–1506.
  5. Ignarro L.J. 1990.  Nitric Oxide. A Novel Signal Transduction Mechanism For Transcellular Communication. Hypertension. 16,5 (Nov.1990), 477-83.DOI=https://doi.org            /10.1161/01.HYP.16. 5. 477.
  6. Zhou L., Zhu D.Y. 2009. Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide 20,4 (Jun.2009), 223–230. DOI=http:// dx.doi.org/10.1016/j.niox.2009.03.001.
  7. Fo¨r stermann U., Closs E. I., Pollock J. S., Nakane M., Schwarz P., Gath I., Kleinert H. 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension. 23, 6 Pt 2 (Jun 1994), 1121–1131. DOI=https:// doi.org/10.1161/01.HYP.23.6.1121.
  8. Nakane M., Schmidt H. H., Pollock J. S., Fo¨r stermann U., Murad F. 1993. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 316, 2 (Jan 1993), 175–180.DOI=https://doi.org/ 10.1016 /0014- 5793(93)81210-Q.
  9. Steinert J.R., Chernova T., Forsythe I.D. 2010. Nitric oxide signaling in brain function, dysfunction, and dementia. Neuroscientist. 16, 4 (Aug. 2010), 435–452. DOI=https:// doi.org/ 10.1177/1073858410366481.
  10. F. Freudenberg†, A. Alttoa‡,§ and A. Reif†,∗. 2015. Neuronal nitric oxide synthase (NOS1) and its adaptor,NOS1AP, as a genetic risk factors for psychiatric disorders.Genes, Brain and Behavior 14 (2015): 46–63 doi: 1 http://doi.org/ 10.1111/gbb.12193
  11. Manso H., Krug T., Sobral J., Albergaria I., Gaspar G., Ferro J. M., Oliveira S. A., Vicente A. M. 2012. Variants within the nitric oxide synthase 1 gene are associated with stroke susceptibility. Atherosclerosis 220, 2,443-448. DOI=http:// doi.org/10.1016/j.atherosclerosis.2011.11.011.
  12. Dai, Y., He Z., Sui R., Jiang  Z.,  Ma S. 2013. Association of nNOS gene polymorphism with ischemic stroke in Han Chinese of North China. Scientific World Journal 2013, 891581. DOI= http://dx.doi.org/10.1155/2013/891581.
  13. Kro¨ncke K. D., Kolb-Bachofen V., Berschick B., Burkart V., Kolb H. 1991. Activated macrophages kill pancreatic syngeneic islet cells via arginine-dependent nitric oxide generation. Biochem Biophys Res Commun. 175, 3 (Mar. 1991), 752–758. DOI= http:// doi.org/10.1016/ 0006-291X(91)91630-U.
  14. Li L. M., Kilbourn R. G., Adams J., Fidler I. J. 1991. Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells. Cancer Res. 51, 10 (May 1991), 2531–2535.
  15. Green S. J., Mellouk S., Hoffman S. L., Meltzer M. S., Nacy C. A. 1990. Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes. Immunol Lett. 25, 1-3 (Aug.1990), 15–19. DOI= http:// doi.org/10.1016/0165-2478(90)90083-3.
  16. Langrehr J. M., Hoffman R. A., Billiar T. R., Lee K. K., Schraut W. H., Simmons R. L. 1991. Nitric oxide synthesis in the in vivo allograft response: a possible regulatory mechanism. Surgery. 110, 2 (Aug. 1991), 335–342.
  17.  Fo¨rstermann U. 2000. Regulation of nitric oxide synthase expression and activity In: Mayer B, ed. Handbook of Experimental Pharmacology—Nitric Oxide. Berlin: Springer. 71–91.
  18. MacMicking J. D., Nathan C., Hom G., Chartrain N., Fletcher D. S., Trumbauer M., Stevens K., Xie Q. W., Sokol K., Hutchinson N et al. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell .81, 641–650.
  19. Maria de Jesus Trovoada, Madalena Martins, Riadh Ben Mansour, Maria do Rosário Sambo, Ana B. Fernandes,Lígia Antunes Gonçalves, Artur Borja, Roni Moya, Paulo Almeida, João Costa, Isabel Marques, M. Paula Macedo,António Coutinho, David L. Narum, Carlos Penha-Gonçalves ,(2014) NOS2 Variants Reveal a Dual Genetic Control of Nitric Oxide Levels,Susceptibility to Plasmodium Infection, and Cerebral Malaria.iai.asm.org. 82,3(March 2014):1287– 1295.DOi=http://dx.doi.org/10.1128IAI.010 70-13.
  20. Garg U. C., Hassid A. 1989. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 83, 1774–1777. DOI= http://doi.org/10.1038/nmeth0410-248
  21. Nakaki T., Nakayama M., Kato R. 1990. Inhibition by nitric oxide and nitric oxide producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol. 189, 347–353.
  22. Murohara T., Asahara T., Silver M., Bauters C., Masuda H., Kalka C., Kearney M., Chen D., Symes J. F., Fishman M. C., Huang P. L., Isner J. M. 1998. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 101, 2567–2578.
  23. Rai H., Parveen F., Kumar S., Kapoor A., Sinha N. 2014. Association of endothelial Notric Oxide Synthase Gene Polymorphisms with Coronary Artery Disease: An Updated Meta- Analysis and Systematic Review. PloS ONE. 9, 11 (Jan. 2014). e113363. DOI=http://dx.doi.org/10.1371/ journal. Pone.0113363.
  24. Yang B., Xu J. R., Liu X. M., Yang Y., Na X. F., Li M., Wang Y. J. 2013. Polymorphisms of rs1799983 (G>T) and gene associated with susceptibility to essential hypertension in the Chinese Hui ethnic population .Genetics and Molecular Research Journal .12.3821-3829.
  25. Goldstein D. B. 2003. Pharmacogenetics in the laboratory and the clinic. N Engl J Med. 348(Feb.2003).553-556.DOI= http://doi.org/10.1056/NEJMe020173.
  26. Hassan M. M., Dowd A. A., Mohamed A.  H., Mahalah S. M.O.S., Kaheel H. H., Mohamed S. N., Hassan M. A. 2014. Computational analysis of deleterious nsSNPs within HLA-DRB1 and HLA-DQB1 genes responsible for Allograft rejection. Int J Comput Bioinfo In Silico Model. 3,6.562-577.
  27. PC. Ng, S. Henikoff. 2003. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res.31, 13 (Jul. 2003), 3812-3814. DOI= http://doi.org/ 10.1093/ nar/gkg509
  28. Kumar P., Henikoff S., Pauline C. Ng. 2009. Predicting the effects of coding non- synonymous variants on protein function using the SIFT algorithm. Nature Protocols. 4 (June, 2009), 1073-1081. DOI= http:// doi.org/10.1038/nprot.2009.86.
  29. Ramensky V., Bark P., Sunyaev S.  2002. Human non-synonymous SNPs: server and survey. Nucleic Acids Res. 30, 17 (Sep. 2002), 3894-3900.
  30. Adzhubei I. A. , Schmidt S., Peshkin L. , Ramensky V. E., Gerasimova A., Bork P., Kondrashov A. S., Sunyaev S. R. 2010. A method and server for predicting damaging missense mutations. Nat Methods. 7 (April 2010), 248-249.
  31. Solovyev V. V., Shahmuradov I. A., Salamov A. A. 2010. Identification of promoter regions and regulatory sites. Methods Mol Biol. 674, 57-83.DOI=http://doi.org/10.1007/978-1- 60761-854-6_5.
  32. Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E. 2004.UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 25, 13 (Oct.2004),1605-1612. DOI=http:// doi.org/ 10.1002/jcc.20084.
  33. Venselaar H. AH teBeek T., Kuipers R. K. P., Hekkelman M. L., Vriend  G. 2010. Protein structure analysis of mutations causing inheritable diseases, an e-science approach with life scientist friendly interfaces. BMC Bioinformatics. 11:548. DOI= http://doi.org/10.1186/1471-2105 -11-548.
  34. Shinkai, T., Ohmori O., Hori H. Nakamura J. 2002. Allelic association of the neuronal nitric oxide synthase (NOS1) gene with schizophrenia. Mol Psychiatry. 7,6. : 560-563.DOI= http://doi.org/10.1038/sj. mp.4001041.
  35. Cui, H., Nishiguchi N., Yanagi M., Fukutake M., Mouri K., Kitamura N., Hashimoto T., Shirakawa O., Hishimoto A. 2010. A putative cis-acting polymorp

 



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