Impact of Polymorphisms of TNF-α, CTLA-4 and IL-10 Genes on Inhibitor Development In Chinese Patients with Hemophilia A.
Impact of Polymorphisms of TNF-α, CTLA-4 and IL-10 Genes on Inhibitor Development In Chinese Patients with Hemophilia A.
复制标题
TNF-α、CTLA-4 和 IL-10 基因多态性对中国甲型血友病患者抑制剂开发的影响。
DOI：
10.1182/blood.v116.21.3665.3665
复制
发表时间：
2010
期刊：
影响因子：
20.3
通讯作者：
C. Ruan
中科院分区：
文献类型：
--
作者：
Lu;Ziqiang Yu;Wei Zhang;Lijuan Cao;C. Ruan
摘要3665引言：血友病A是替代治疗过程中的严重并发症，三分之一到四分之一的患者会产生因子VIII抑制物。虽然因子VIII基因突变是主要决定因素，但HLA等位基因和细胞因子（如TNF-α、CTLA-4和IL-10）的基因多态性也与抑制剂的产生相关。为探讨TNF-α-308 G>A、CTLA-4-318 C>T和IL-10基因CA重复序列微卫星多态性在抑制剂形成中的作用，对140例经血浆来源的F-10浓缩物治疗的中国血友病A患者和108例正常对照进行了分析。方法：从所有患者和正常对照的柠檬酸盐保存的血液中提取基因组DNA。PCR扩增TNF-α和CTLA-4的启动子区，分别用Nco I和Mse I酶切TNF-α和CTLA-4的PCR产物，然后进行2%琼脂糖凝胶电泳。PCR产物直接测序鉴定基因型。采用PCR扩增IL-10启动子区的短串联重复序列，并进行毛细管电泳分析。同时用改良Nijmegen法测定血友病A患者血浆中凝血因子VIII抑制物的活性。结果如下：血友病A组-308G和A等位基因频率分别为90.7%和9.3%，正常对照组分别为90.3%和9.7%。血友病A组-318C和T等位基因频率分别为87.9%和12.1%，正常对照组分别为86.6%和13.4%。血友病A患者134 bp等位基因阳性率为27.9%，134 bp等位基因阴性率为72.1%，而正常对照组分别为29.6%和71.4%。两组间-308G/A等位基因频率、-318C/T等位基因频率和134 bp等位基因频率差异无统计学意义。在34/140（24.3%）例患者中鉴定出F β [抑制剂活性。在有抑制物的患者中，13/34例（38.2%）携带-308A等位基因，其G和A等位基因频率分别为75%和25%。携带-308 A等位基因的血友病A患者发生抑制物的风险高（OR，7.519; 95%CI，3.168 '17.844），尤其是重型血友病A患者（OR，8.163; 95%CI，2.521'26.434）。在有抑制物的患者中，10/34例（29.4%）携带-318T等位基因，C等位基因频率为83.8%，T等位基因频率为16.2%。同时携带C等位基因和T等位基因的患者发生抑制物的风险差异无统计学意义（OR，1.586，95%CI，0.729 '3.450）。在抑制物患者中，等位基因134 bp阳性和阴性的频率分别为44%和56%，等位基因134 bp阳性和阴性患者发生抑制物的风险差异无统计学意义（OR，1.769，95%CI，0.676 '4.627）。结论：TNF-α-308G>A基因多态性与中国血友病A患者凝血因子VIII抑制物的发生显著相关。TNF-α基因可能是血友病A患者替代治疗免疫应答的一个有用的标志物和潜在的调节因子。披露：没有相关的利益冲突。
Abstract 3665 Introduction: 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. Methods: 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 Nco I or Mse I, 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. Results: 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). Conclusion: 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. Disclosures: No relevant conflicts of interest to declare.