Association Patterns of Endothelial Nitric Oxide ... - Wiley Online Library

Short Communication

doi: 10.1111/ahg.12192

Association Patterns of Endothelial Nitric Oxide Synthase Gene (NOS3) Variant Glu298Asp with Blood Pressure and Serum Lipid Levels in Subjects with Coronary Artery Disease from Pakistan and Abdul Rehman , Shabana † Saleem Ullah Shahid∗,† Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan

Summary Nitric oxide is an important antiatherosclerotic agent. The main determinant of nitric oxide levels is enzyme nitric oxide synthase encoded by the NOS3 gene, the common variants in this gene may be responsible for variations in plasma enzyme levels. The association of NOS3 variants with coronary artery disease (CAD) varies in different ethnicities. The current study aimed to determine the association of NOS3 Glu298Asp (rs1799983) with CAD and blood lipid levels in Pakistani subjects. Six hundred thirty-six samples (412 cases, 224 controls) were genotyped by TaqMan allelic discrimination assay and serum total cholesterol, and High Density Lipoprotein cholesterol (HDL-C)/Low Density Lipoprotein cholesterol (LDL-C) and triglycerides were measured. The genotype frequency was Glu/Glu = 64.6%, Glu/Asp = 30.1%, and Asp/Asp = 5.3% in cases, and Glu/Glu = 68.8%, Glu/Asp = 26.7%, and Asp/Asp = 4.5% in controls. The Asp298 (T) frequency was not significantly higher in cases than controls (20.4% vs 17.9%, P = 0.28) and risk allele was not associated with CAD (OR 1.15 (0.86–1.54), P = 0.33) and the tested lipid traits but had a strong association with blood pressure (for systolic and diastolic P = 1.9×10−–56 and 4×10−–40 , respectively). In conclusion, although Glu298Asp did not show association with CAD and lipid profile in the studied cohort, it may exert its effect through blood pressure; however, the mechanism of this effect needs to be explored in the future. Keywords: Nitric oxide, polymorphisms, coronary artery disease, TaqMan

Introduction Nitric oxide (NO) is a small molecule that is produced through L-arginine to L-citruline oxidation by the nitric oxide synthase (NOS) enzyme (Palmer et al., 1988). It is a potent vasodilator and an important antiatherosclerotic agent. The main contributor of the circulating NO is NOS (Griffith & Stuehr, 1995). The NOS family has three well-known isoforms: neuronal NOS (nNOS, NOS1, Gene ID: 4842, OMIM: 163731), inducible NOS (iNOS, NOS2, Gene ID: 4843, OMIM: 163730), and endothelial (eNOS, NOS3, Gene ID: 4846, OMIM: 163729) (Zhou et al., 2016). The NOS3 gene (OMIM 163729) is located on chromosome ∗

Corresponding author: SALEEM ULLAH SHAHID, Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan, 54590. Tel: 00923007592547; Fax: 00924235952855; E-mail: [email protected] † These authors contributed equally to this work.

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2017 John Wiley & Sons Ltd/University College London

7q36.1 (7:150,991,055-151,014,598 alternative name: ECNOS), consists of 26 exons ranging in size from 68 to 579 bp spanning 21 kb, and encodes 10 splice variants. The NOS3 gene has a TATA-less promoter and exhibits proximal promoter elements consistent with a constitutively expressed gene, namely, SP1 and GATA motifs. This gene is highly expressed in the endothelium (Nadaud et al., 1994). The NOS3 gene is the second most reported gene associated with coronary artery disease (CAD) and the fourth most myocardial infarction (MI)–associated gene (www.hugenavigator.net). Many polymorphisms have been reported in this gene to date. The nonsynonymous missense polymorphism Glu298Asp or E298D (rs1799983) is a G>T transversion located in the exon 7 of the gene. The substitution of the codon GAG to GAT in the 894 position (G894T) changes glutamate with aspartate at the 298 amino acid position of protein (Glu298Asp or E298D) (Hingorani et al., 1995). Hingorani et al. (1997) first reported the association of this polymorphism with CAD.

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Later, the association was also confirmed in other studies. This variant has been reported to be associated with CAD in Caucasians and Asians with contradictory results. In 2006, a metaanalysis by Casas et al. (2006) included 42 studies comprising of 13,876 cases and 13,042 controls and found the OR 1.17 (CI: 1.07–1.28), P = 0.001. However, the authors observed a statistical evidence of small study ( 0.05).

Discussion The results of current study showed that although the risk allele was high in frequency in the CAD group compared to the controls, the difference was not statistically significant. The Asp298 allele was not associated with CAD in


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all the three genetic models (additive, dominant, recessive) tested. The findings are consistent with other studies where the polymorphism was not found to be associated with CAD in Asian-Indians (Saini et al., 2012; Narne et al., 2013). Contrary to this, the polymorphism was found to be associated with CAD in Asian-Indians (Angeline et al., 2010; Saini et al., 2011). A meta-analysis of 39 published studies consisting of 7489 cases and 7051 controls also identified a significant association of the 298Asp with CAD in all the genetic models tested (Tian et al., 2013). It is striking to note that Glu298Asp has been consistently reported to be associated with CAD in meta-analysis studies since long ago, but in individual studies, the association remained controversial. Casas et al. (2004) reported a meta-analysis of 14 studies comprising of 6036 cases and 6106 controls. Of these studies, individually, only 3 reported a significant risk associated with Asp298, whereas 13 studies did not report the association, but in their combined metaanalysis, the risk turned out to be statistically significant (OR, 1.31; 95% CI, 1.13 to 1.51, P = 0.0003) (Casas et al., 2004). Contradictory results regarding the association of this variant with CAD have already been reported in the Pakistani people. In a study by Taqddus et al. (2014) including 280 subjects (160 cases, 120 controls), Asp298 allele was totally absent in cases as well as controls. In another study by Nawaz et al. consisting of 376 subjects (198 cases, 178 controls), the Asp298 allele was significantly higher in cases than controls (0.73 vs. 0.3) and associated with CAD risk. However, the genotyping techniques in both of these studies (PCR followed by RFLP and allele specific PCR) was different than our technique (TaqMan technique). A small group of Punjabi people (n = 96) from Lahore, Pakistan (PJL) (http://asia. ensembl.org/Homo_sapiens/Variation/Population?db=core; r=7:150998523-150999523;v=rs1799983;vdb=variation; vf=102374312#373533_tablePanel) were genotyped in the 1000 Genomes Project phase III, and the Asp298 allele frequency was found to be 0.151% (Handsaker et al., 2012).We compared our Asp298 frequency in controls with PJL genotypes, and both were found to be comparable (0.179 vs. 0.151, P = 0.4). However, when the genotypes in controls by Nawaz et al. (2015) were compared with the PJL, a significant difference between the allele frequencies (0.297 vs. 0.151, P = 0.0002) was seen. Both of the studies (Taqddus et al., 2014; Nawaz et al., 2015) also deviated from HWE in cases as well as controls. The variant did not appear to be associated with serum lipid levels, including TC, LDL-C, HDL-C, and TG levels. However, it was significantly associated with SBP and DBP. This finding is in contrast with the results reported in Caucasians (Karvonen et al., 2002). The lack of association with lipid profile is in agreement with the results reported by Rossi et al. (2003) for NOS3 polymorphism but the authors also

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did not find any relation with blood pressure. Another study reported an association of the variant with both lipid traits and blood pressure and proposed that the effect on blood pressure is modulated through changing lipid traits in a Brazilian population (Pereira et al., 2006). Our results, however, indicated that the effect of the NOS3 variant on blood pressure is not modulated by the lipid profile and may involve some other mechanism that needs to be explored further in the future studies. The molecular effects of the Glu298Asp polymorphism on enzyme function are still not completely understood. The kinetics of NO synthesis does not differ between NOS3 Glu298 and Asp298. However, the polymorphism might cause an increased susceptibility of the polypeptide to degrade due to a tight run of alpha helix (Kelm, 2006). The peptide with Asp298 was found to be more susceptible to proteolytic cleavage in the endoplasmic reticulum than with a peptide having Glu298 at this locus, that is, an amino terminal 35 kDa and carboxy terminal 100 kDa fragments. The cleaved eNOS is expected to be deficient in NOS activity, and this would lead to decreased NO in subjects homozygous for Asp298 (Tesauro et al., 2000). It has also been reported that this polymorphism may affect protein–protein interaction and interaction of the protein with chaperon (Monti et al., 2003). The steady-state level of the NOS enzyme in subjects with the 298Asp variant might, therefore, be lower, with a resultant reduction in capacity for NO production. NO levels in 298Asp carriers may be sufficient to maintain vascular homeostasis in the absence of other risk factors. The study had some limitations, including the power issue and small sample size. The power of this sample size as calculated came out to be 85% for this outcome. For a power to be 100%, approximately 500 cases and 500 controls were required. Secondly, the cases and controls were not age matched; it looked as if controls were younger than cases, but a careful examination revealed that there was a broader age range in cases (28–95 years) than in controls (35–86 years). A greater number of control subjects were in the middle age range than cases. The cases had more subjects than controls, which fell in the younger as well as elderly age groups.

Conclusion In conclusion, in our studied subjects, the polymorphism Glu298Asp cannot be attributed as an independent genetic risk factor for CAD development. We found different allele frequencies and association with CAD than previous studies in Pakistan. Hence, it is suggested that further studies with a larger sample size from this ethnicity should be conducted. However, if this polymorphism appears in GWAS of CAD association in the future, this will provide very valuable information.

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NOS3 Glu298Asp and Coronary Artery Disease in Pakistani subjects

Acknowledgements The Higher Education Commission (HEC) of Pakistan is acknowledged for providing financial support for the study under the Indigenous 5000 Programme (grant number Bm6131) and International Research Support Initiative Programme (IRSIP) under grant IRSIP 24 BMS 41.

Conflict of Interest None declared.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Supplementary table 1. Primers and probes used for TaqMan amplification.

Supplementary table 2. Sequence of primers used for sequencing Supplementary table 3. Power Calculation Supplementary table 4. Adjusted logistic regression Received: 31 October 2016 Accepted: 16 March 2017

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