Is the endothelial nitric oxide synthase (eNOS) gene a susceptibility gene for coronary artery disease, hypertension and type 2 diabetes among North Indian populations?

Coronary artery disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) are all global health problems. This is particularly evident amongst South Asian population groups. The conventional risk factors do not fully explain the higher prevalence of these diseases among South Asians. The endothelial Nitric Oxide Synthase (eNOS) gene is responsible for the production of Nitric Oxide (NO), which may contribute to the physiology of all three disease states. Endothelial dysfunction (which is characterised by a reduction in basal NO) has been shown to be present in, or prior to all three diseases. Numerous variations exist within the eNOS gene, of these variations three have been shown to have a possible functional effect. The first is the Glu298Asp polymorphism within the exon region of the gene, resulting in an amino acid substitution of Glutamate (Glu) to Aspartate (Asp). The second, known as the T-786C polymorphism, is a thymine to cytosine mutation at position -786 in the promoter region. Finally a VNTR polymorphism in Intron 4 causes either a 4 27bp repeat or a 5 27bp repeat. It is hypothesised that these variations could have an effect on the ability of eNOS to produce NO and thus may increase the risk or contribute to the development of the diseases. Previous studies on these variants have shown conflicting results and further studies are warranted to understand and confirm the role of eNOS gene polymorphisms in cardio-metabolic diseases. There is very limited research into the distributions of these genetic variants and their interaction in diseases processes in North Indian populations. Objectives: 1. To analyse through a case control study three different polymorphisms of the eNOS gene for possible association with Coronary Artery Disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) in North Indian population groups. 2. To statistically evaluate descriptive statistics including; age, gender, smoking, dietary behaviours and lipid parameters for possible influence on disease and potential interaction with genetic polymorphisms. 3. To evaluate linkage disequilibrium between the three eNOS variants and carryout haplotype analysis to work out haplotype risk in different diseases. 4. To analyse through a case control study the deletion variant of the Angiotensin-converting enzyme (ACE) gene for possible association with Coronary Artery Disease (CAD), Hypertension (Ht) and Type 2 Diabetes Mellitus (T2DM) in North Indian population groups. 5. To determine a possible interactive effect of the eNOS polymorphisms with the ACE polymorphism. Subjects and Methods: The Glu298Asp and Intron 4 variants were genotyped using a PCR-RFLP technique, the T-786C variant was genotyped using a real time-PCR technique. The ACE deletion variant was also genotyped using a standard PCR technique. The genotyping was undertaken in a total of 457 CAD patients and 220 matched controls from Lucknow, Uttar Pradesh in North India, 319 T2DM patients and 307 matched controls from Punjab, North India and 210 Ht and 162 matched controls, also from Punjab, North India. Results: CAD: The Glu298Asp was significantly associated with CAD among smokers (TT+GT vs. GG OR=2.84 (CI: 1.61-5.0), p<0.001). The Intron 4 variant was also significantly associated with CAD in a smoking dependent manner (4aa+4ab vs. 4bb OR=0.56 (CI: 0.33-0.96). The T-786C variant showed no overall influence on CAD risk. There was also evidence for both synergistic and haplotypic effects of the eNOS gene on CAD status (haplotype G-C-4b OR=4.76 (CI: 1.43-15.78), p<0.001). The ACE genetic variant was confirmed to be a strong independent risk factor for CAD under a dominant model (OR=2.18 (CI: 1.46-3.25), p<0.001). There was no evidence for an interactive effect between the ACE deletion and any of the three eNOS variants incorporated in the current study. Ht: The Glu298Asp variant was not shown to increase Ht risk, with a reduced risk association found under a recessive model (OR=0.316 (CI:0.089-1.116), p=0.061). The T-786C variant s role in disease remained unclear with the findings showing a non significant increased risk. The Intron 4 variant was also shown to increase Ht risk, in a non significant manner. Sufficiently powered studies would be required to clarify these possible associations. The combined analysis, using logistic regression and haplotype analysis revealed no significant associations, but there was a possible protective effect of the T-C-4b haplotype (OR=0.46 (CI: 0.21-1.01), p=0.054). The ACE gene variant was confirmed to be a strong independent risk factor for Ht under a recessive model (OR=1.81 (CI: 1.20-2.74), p=0.01). Again there was no evidence for an interactive effect between the ACE deletion and any of the three eNOS variants in hypertension. T2DM: The Glu298Asp variant was found to be associated with T2DM under a dominant model, the protective effect remained significant following adjustment for conventional risk factors and other gene variants (OR=0.407 (CI: 0.231-0.717), p=0.002). The T-786C variant showed no overall influence on T2DM risk. The Intron 4 variant also found no overall influence. Haplotype analysis found the T-T-4b was found to be significantly protective for T2DM (OR=0.41 (CI: 0.26-0.65), p=0.0002). Finally the ACE gene variant was confirmed to be a risk factor for T2DM under a dominant model (OR=2.62 (CI: 1.51-4.54), p=0.001). Overall Conclusions: To conclude, this study successfully identified the frequency of three eNOS gene variants and the ACE deletion variant in three complex diseases within north Indian populations. There is a clear role of the eNOS gene in all three diseases and consequently the genetic variants have susceptible/protective associations. The association with disease was found to be present at an individual level, in association with risk factors and at a haplotypic level. These findings warrant further studies to confirm and untangle the genetics of complex diseases and genetic risk profiles calculations which will contribute to the field of medical genomics/personalised medicare and interventions among North Indian populations.