Genetic risk factors for coronary artery spasm:: Significance of endothelial nitric oxide synthase gene T-786→C and missense Glu298Asp variants

Background: We recently identified two endothelial nitric oxide synthase (eNOS) gem polymorphisms, Glu298Asp and T-786-->C, which are independently associated with coronary spasm. eNOS gene intron 4b/a polymorphism is also reported to be involved in smoking-dependent coronary artery disease. The genetic linkage among these polymorphisms remains unknown. Also, it is unclear which variant is most responsible for coronary spasm. In the present study, we first examined the genetic linkage among these three variants. Next, we studied the risk factors of coronary spasm by using all significant genetic and conventional risk factors in a large-scale study. Methods: The genotype and allele frequencies for the T-786-->C, intron 4b/a, and Glu298Asp variants were assessed in 423 randomly selected DNA samples to examine their genetic linkages. The relative capacities of all risk factors to predict coronary spasm were then analyzed using multiple logistic regression in 201 patients with coronary spasm and 345 volunteers. Results: Comparison of allele frequencies revealed that the eNOS intron 4a allele was significantly linked to the T-786-->C mutation (P<0.00001), whereas there was not a linkage between the intron 4a allele and the Glu298Asp variant (P=0.1437) or between the Glu298Asp variant and the T-786-->C mutation (P=0.1996). Multiple logistic regression revealed that the most predictive independent risk factor for coronary spasm was the T-786-->C mutation (P<0.001), followed by cigarette smoking (P<0.001), hypertension (P=0.004), and the Glu298Asp variant (P=0.028). Conclusions: We found that the T-786-->C mutation and the intron 4a allele are in linkage disequilibrium. We previously showed that the T-786-->C mutation reduced eNOS gene promoter activity. In that context, our results strongly suggest that the T-786-->C mutation underlies the functional characteristics of the intron 4a allele. Further, multiple logistic regression analysis revealed that the T-786-->C mutation is the most predictive risk factor for coronary spasm, followed by cigarette smoking. Given that those effects are potentially additive, patients carrying the eNOS gene variants should be strongly cautioned against smoking.