High prevalence of polymorphisms of angiotensin-converting enzyme (I/D) and endothelial nitric oxide synthase (Glu298Asp) in patients with systemic sclerosis.

PURPOSE: Systemic sclerosis is characterized by progressive microvascular occlusion and fibrosis and by an imbalance in the fibrinolytic system. In vivo and in vitro studies suggest that the renin-angiotensin system partly regulates vascular fibrinolytic balance. Angiotensin II increases the production and secretion of plasminogen activator inhibitor-1, while angiotensin-converting enzyme (ACE) contributes to reduced production of tissue plasminogen activator and endothelial nitric oxide synthesis by bradykinin degradation. The aim of our study was to investigate the effects of ACE insertion/deletion (I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894-->T) and T-786-->C polymorphisms in patients with systemic sclerosis. SUBJECTS AND METHODS: We studied 73 consecutive patients (47 with limited and 26 with diffuse cutaneous systemic sclerosis) and 112 control subjects. ACE I/D and eNOS polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The ACE I/D and the eNOS G894-->T polymorphisms were more common in patients than in controls (for the ACE D allele: odds ratio [OR] = 3.4; 95% confidence interval [CI]: 1.5 to 7.9; P = 0.003; for the eNOS T allele: OR = 1.9; 95% CI: 1.0 to 3.4; P = 0.04). There was no association between the eNOS T-786-->C polymorphism and systemic sclerosis. CONCLUSIONS: Our findings of an increased risk of systemic sclerosis in ACE D and eNOS 894T allele carriers suggest that these polymorphisms may contribute to the pathogenesis of the disease.

High-speed detection of the G894T polymorphism in exon 7 of the eNOS gene by real-time fluorescence PCR with the Light-Cycler.

In endothelial cells nitric oxide (NO) is synthesized by endothelial-nitric oxide synthase (e-NOS), constitutively expressed and encoded by a 26-exon gene, located on chromosome 7q35-36. The prevalence of the T rare variant of the G894T polymorphism in exon 7 of the e-NOS gene (Glu-->Asp amino acid substitution) has been reported to be significantly higher in patients with coronary spasm and coronary artery disease. To date G894T polymorphism detection is performed by PCR-RFLP assay. In order to establish a high-speed genotyping method, we have taken advantage of the Light Cycler instrument, a thermal cycler that combines rapid-cycle DNA amplification with a real-time fluorescence monitoring. This technology is based on hybridization of the adjacent fluorescently labeled probes with PCR products. This methodology is considered more accurate and less time-consuming than conventional PCR-RFLP assay. To validate this technique we genotyped 270 healthy subjects. The results were consistent with those obtained from PCR-RFLP assay.