P-058: INFLUENCE OF HEME OXYGENASE-I GENE PROMOTER POLYMORPHISM ON THE VASO OCCLUSIVE CRISIS OF SICKLE CELL ANEMIA PATIENTS OF EASTERN INDIA

Abstract

Purpose: Intravascular hemolysis in sickle cell anemia (SCA) leads to release of heme into the circulation and oxidative stress predisposing to vaso occlusive crisis (VOC). Heme is metabolized by the Heme Oxygenase (HO) enzyme resulting in decrease in oxidative stress. The HO-1 expression is altered by HMOX-1 gene promoter polymorphism. We investigated the role of three HMOX-1 gene polymorphisms [two single nucleotide polymorphisms (SNP), 19(G>C)(rs2071747) and -413A>T(rs2071746), and one dinucleotide (GT)n length polymorphisms (rs3074372)] on VOC in SCA patients of eastern India. Materials and methods: A total of 170 SCA cases (80 mild and 90 severe phenotypes) and 101 healthy controls were considered for this study. Those without incidence of a single pain episode or VOC episode were considered as ‘mild phenotype’ of SCA while those who had three or more acute pain episodes in the last twelve months were considered as ‘severe phenotype’. Genotyping of HMOX-1 (19 G>C) polymorphism was done by ARMS-PCR. Whereas, -413A>T polymorphism and (GT)n repeats were done by DNA sequencing. In HMOX-1 gene promoter, the (GT)n repeats were categorized into three types: first, (GT)n≤27 is noted as ‘Small (S)’ repeats; second, (GT)n=28-32 as ‘Medium (M)’ repeats and third, (GT)n≥33 as ‘Large (L)’ repeats. We categorized repeats into two groups: group-I contains S/S and S/M; whereas, group-II contains L/L, L/M, M/M, and S/L. Genotypes and allele frequency were calculated and compared between cases and controls using SNPstarts. Fisher’s exact test, odds ratio (ORs) and 95% confidence intervals (95% CIs) were calculated by using GraphPad Prism v5.0. The p-value of <0.05 was considered statistically significant. Results: On analysis of HMOX-1 (19G>C) polymorphism (rs2071747) between the mild and severe phenotype of VOC, no significant (p>0.05) variation was found in the allele and genotype distribution. Whereas, in HMOX-1 (-413 A>T) polymorphism, a significantly higher mutant allele (T) (p=0.0185) and genotype were found [AA vs TT (p=0.011) and AA vs AT+TT (p=0.02)] in severe phenotype. However, there is no difference among AA vs AT (p=0.56). On (GT)n repeat analysis, it was found that small repeats were associated with the mild phenotype and large repeats with that of severe phenotype in SCA {Gp-I vs Gp-II [p=0.0014; OR(95%CI)- 3.143 (1.538-6.422)]}. The difference was significant when comparison of severe phenotype SCA was done with controls for (GT)n repeats with larger repeats being commoner in severe phenotype. Conclusion: No difference was noted between mild and severe SCA phenotypes HMOX-1 (19G>C) polymorphism. The prevalence of the small repeat (GT)n was found to be significantly higher in the mild SCA phenotypes and controls. Higher incidence of mutant allele and genotype of HMOX-1(-413T) polymorphism and larger (GT)n repeats in the promoter region of the HMOX-1 gene were associated with the severe phenotypes. Both have a negative effect on the HO-1 enzyme activity leading to increased VOC in severe SCA- phenotype. The capacity of HO-1 enzyme activity appears to be overwhelmed by heme-induced oxidative stress in severe phenotypes and the affected patients suffer more VOC compared to mild SCA phenotypes and controls. The authors do not declare any conflict of interest