The Impact of Diabetes Mellitus on the Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms (4a/4b, G894T, and T786C) with Clopidogrel Resistance in Coronary Artery Disease Patients Undergoing Percutaneous Coronary Interventions

Abstract

Ali A. R. Aldallal, Bassim I Mohammad, Ahmed N. Rgeeb, Salam Jasim mohammed, Khalid Amber.

Background: Compared with the general population, patients with diabetes mellitus (DM) usually show higher platelet reactivity, which increases their risk of atherothrombotic events, such as acute coronary syndrome, during the percutaneous coronary intervention (PCI). Endothelial nitric oxide synthase (eNOS) gene polymorphisms have been associated with many diseases, but their role as risk factors of coronary artery disease (CAD) in DM is yet unknown. Although acetylsalicylic acid and clopidogrel are prescribed individually or in combination for high-cardiovascular risk patients with overall improvements in outcomes, DM patients are still at greater risk of recurrent ischemic events.
Objective: This study was conducted to determine the effect of DM on the association of e-NOS polymorphisms with clopidogrel resistance in patients with CAD undergoing PCI.
Methods: Three-hundred twenty-four of CAD patients were recruited in this case-control study. All patients were subjected to platelets function test within at least two hours before PCI techniques to determine the clopidogrel resistance first then classified accordingly to clopidogrel resistance group (case group n=111) and non-clopidogrel resistance group (control group n=213). Each group was subdivided into a diabetic patient’s group and non-diabetic patients’ group. Blood samples were taken for genotyping analysis and phenotyping determination by measuring the level of nitric oxide in the plasma via colorimetric method. The genotype analysis was carried using allele-specific polymerase chain reaction based on the banding pattern on gel electrophoresis(GEP) to evaluate whether genetic e-NOS polymorphisms (T786C, exon 7 G894T and intron 4 (4a4b) affect NO formation in both groups. Odds ratios for these polymorphisms were then estimated for the clopidogrel and non-clopidogrel resistance groups.
Results: Approximately 34.25% of the study population showed clopidogrel resistance. No significant difference in serum NO levels between the case and control groups (26.1 ± 1.52 µM vs. 25.41 ± 1.18 µM; P = 0.727) was observed.
The effect of diabetes on clopidogrel resistance was significant in the case group (20.06 ± 1.82 µM) in diabetes patients when compared with 26.52 ± 2.40 µM in non- diabetes patients (P = 0.03), but within a control group, the effect diabetes on clopidogrel resistance was not significant (P = 0.07), while the effect of diabetes was not significant in diabetes patient (P = 0.09) and non- diabetes (P = 0.4) when the comparison between case and control. The study found that e-NOS intron 4 (4a4b) polymorphisms genotypes were significantly associated in the diabetic group as compared with non –diabetic group in patients' resistant to clopidogrel [ab genotype: OR = 2.4 (1.1–5.69), P=0.02; aa genotype: OR = 2.8 (0.89–8.7), P=0.03], while there was a negative association between diabetic group and non–diabetic group in patients' sensitive to clopidogrel. However, e-NOS polymorphisms of the T786C and exon 7 G894T genotypes showed no association between diabetic and non-diabetic patients of both resistant or sensitive patients except mutant homozygous CC of T786C showed a significant association between diabetic and non-diabetic patients in case and control groups (P = 0.02, 0.04) respectively.
Conclusions: Our study indicates that e-NOS gene polymorphisms, specifically the 4a allele (4a/ab) and mutant homozygous CC (T786C), maybe determinants of clopidogrel resistance in diabetic CAD patients.