Impact of Polymorphisms of TNF-α, CTLA-4 and IL-10 Genes on Inhibitor Development In Chinese Patients with Hemophilia A.

The inhibitor against factor VIII would develop in one-third to one-fourth patients with hemophilia A, which is the severe complication during replacement therapy. Although mutation in factor VIII gene is the main determinant, HLA alleles and gene polymorphism of cytokines, such as TNF-α, CTLA-4 and IL-10, are also associated with inhibitor development. The polymorphisms of TNF-α-308G>A, CTLA-4 -318C>T and one of the CA repeat microsatellites in IL-10 gene were analyzed in 140 Chinese patients with hemophilia A treated with plasma-derived F‡[ concentrates and in 108 normal controls, in order to evaluate their contribution to inhibitor development.

Genomic DNA was extracted from citrate-preserved blood from all patients and normal controls. The promoter region of TNF-α and CTLA-4 were amplified by PCR, then the PCR products of TNF-α and CTLA-4 were digested by NcoI or MseI, respectively, then subjected to 2% agarose gel electrophoresis. PCR products were also directly sequenced to identify the genotype. The short tandem repeat in the promotor region of IL-10 was amplified by PCR, then analyzed by capillary electrophoresis. The inhibitor activities against factor VIII in plasma of patients with hemophilia A were measured by modified-Nijmegen assay simultaneously.

The frequencies of -308 G allele and A allele in patients with hemophilia A were 90.7% and 9.3% respectively, compared with 90.3% and 9.7% in normal controls. The frequencies of -318 C allele and T allele in patients with hemophilia A were 87.9% and 12.1% respectively, compared with 86.6% and 13.4% in normal controls. The allele 134bp positive and allele 134bp negative in patients with hemophilia A were 27.9% and 72.1% respectively, compared with 29.6 % and 71.4% in normal controls. There were not significant difference of -308G/A allele frequencies ƒ A-318C/T allele frequencies and 134bp allele frequencies between two groups. The F‡[ inhibitor activity was identified in 34/140 (24.3%) patients. In the patients with inhibitor, 13/34(38.2%) patients carried the -308 A allele, the frequencies of -308 G allele and A allele in these patients were 75% and 25% respectively. The patients with hemophilia A who carried -308 A allele had a high risk of inhibitor development (OR,7.519; 95%CI,3.168 ‘17.844), especially for the severe patients with hemophilia A (OR,8.163; 95%CI,2.521 ‘26.434). Also in the patients with inhibitor, 10/34(29.4%) patients carried the -318 T allele, the frequencies of -318 C allele and T allele in these patients were 83.8% and 16.2% respectively. There is no statistical significance regarding the risk of inhibitor development between the patients who were carrier of C allele and T allele (OR,1.586,95%CI,0.729 ‘3.450). The frequencies of allele 134bp positive and allele 134bp negative in patients with inhibitor were 44% and 56% respectively, there was no statistical significance regarding the risk of inhibitor development between the patients who were the allele 134bp positive and those who were allele 134bp negative (OR,1.769,95%CI,0.676 ‘4.627).

TNF-α-308G>A polymorphism is significantly associated with factor VIII inhibitor development in Chinese patients with hemophilia A. TNF-αgene may be a useful marker and potential modulator of the immune response to replacement therapy in patients with hemophilia A.

No relevant conflicts of interest to declare.