Endothelial nitric oxide synthase gene introne4 VNTR polymorphism in sickle cell disease and β-thalassemia major: Relation to vascular complications

Abstract

growing body of data suggests that thalassemia has many biologic and epidemiologic factors in common with SCD, including chronic hypoxia, long term effect of splenectomy, red cell membrane pathology, coagulation abnormalities, platelet activation, oxidative stress, iron overload and chronic hemolysis. Many mechanisms contribute to the complex pathophysiology of both thalassemia and SCD, with dysfunction of the vascular endothelium as a unifying theme. The constitutive endothelial NO synthase (eNOS), an enzyme that generates NO, is expressed in the endothelium. Among the reported polymorphisms of the eNOS gene, a polymorphism in intron 4 of the eNOS gene is a candidate gene in cardiovascular and renal diseases. In view of these data, this study aimed to assess the 27-base pair tandem repeat polymorphism in intron4 of eNOS gene in young patients with SCD and β-thalassemia major and its potential involvement in disease severity and various hemolysis-associated complications. The study included 55 patients with chronic hemolytic anemia; 25 patients with SCD and 30 patients with β-TM. Patients were compared with 25 age- and sex-matched healthy subjects served as controls. The mean age of SCD patients was 12.9 ± 4.5 years and that of β-TM patients was 14.4 ± 2.3 years while the controls had a mean age of 10.4 ± 3.6 years. All included patients were subjected to detailed medical history and thorough clinical examination with special emphasis on disease duration, anthropometric measures, blood pressure, evidence of renal, hepatic or cardiac disease, frequency of sickling crisis, history of splenectomy, transfusion history, chelation and/or hydroxyurea therapy. Laboratory investigations included CBC, hemoglobin analysis, liver and kidney function tests, markers of hemolysis (LDH and indirect bilirubin), mean serum ferritin calculated in the last year prior to the study and urinary albumin excretion. eNOS genotype was determined using polymerase chain reaction (PCR). In the current work, two alleles of the VNTR in the human eNOS gene were detected in the studied population; one allele contained four of 27bp repeats (eNOS4a allele) giving rise to a PCR product of 393 bp, whereas the other contained five of such repeats (eNOS4b allele), yielding a PCR product of 420 bp. The distribution of eNOS intron 4 alleles and genotypes was not significantly different between β-TM patients and healthy controls. The frequency of eNOS4a allele (aa and ab genotypes) was significantly higher in SCD patients with pulmonary hypertension, nephropathy (micro- or macro-albuminuria) or history of thrombosis. SCD patients with frequent sickling crisis ≥3 attacks/year in the last year prior to the study showed higher frequencies of the eNOS4a allele. SCD patients with eNOS4a allele had higher transfusion index, WBCs, LDH, serum ferritin and UACR with lower hemoglobin and HbF levels. In β-TM, the frequency of eNOS4a allele was significantly higher in patients with pulmonary hypertension and cardiomyopathy compared with bb genotype.