Heme oxygenase-2 gene mutations and blood bilirubin level in Iranian patients with premature atherosclerosis

Heme oxygenase-2 (HO-2) is a critical antioxidative stress enzyme found in endothelial cells and adventitial nerves. This enzyme in conjunction with other HOs (1 and 3) metabolize heme molecule into ferrous iron, carbon monoxide (CO), and biliverdin which is further converted to bilirubin. Both biliverdin and bilirubin are potent antioxidants, reducing the risk of atherosclerosis. HO-2 also induces endothelial relaxation by synthesizing CO. This is the first study to evaluate the association of HO-2 gene mutation in patients affected with atherosclerosis. Blood samples from patients (n=137) and normal controls (n=100) were collected. Three pairs of primers were designed to amplify exons 2 to 4 related to human HO-2 gene. The PCR products were analyzed by SSCP and sequencing to find out mutations. Iron and bilirubins (Total, Direct and Indirect) levels were determined in patients and controls. Two nucleotide substitutions were found among 10% of patients, consisted of a newly reported transversion mutation, C to A substitution in codon A70D (GCC to GAC) (Ala to Asp) and a previously reported transition mutation, A to G substitution in codon K89E (AAG to GAG) (Leu to Glu). Significant associations were obtained between risk of atherosclerosis and A437G substitution in codon K89E of HO-2 gene (P< 0.006 and chi(2) >6.82) and reduced level of total (P< 0.016 and chi(2) >6.01), and indirect (P< 0.016 and chi(2) >5.99) bilirubins with no significant association with serum iron and direct bilirubin. No significant associations were observed among C381A substitution in codon (A70D, P< 0.11 and chi(2) >2.97), level of serum iron, bilirubin and risk of atherosclerosis. These findings indicate the importance of A437G substitution in the development of atherosclerosis. Further studies are required to study the association of HO-2 gene mutations with atherosclerosis in other populations.