Correlation Study With Expression Of B Cell Activating Factor Of The TNF Family And Polymorphism Of BAFF In Patient With Immune Thrombocytopenic Purpura

Objective To test the distribution of polymorphism of promoter gene-871C/T of B cell activating factor of the TNF family (BAFF) in patients with immune thrombocytopenic purpura (ITP) and analyze the correlation between the polymorphism of the gene and ITP. Discuss the relationship between the level of BAFF antigen and polymorphism of this gene to research the pathogenesis of ITP.Methods1. The collection of samples. Patients, both outpatients and inpatients, are from the Departments of Hematology in the Second Hospital of Shanxi Medical University from May 2008 to November 2009. Among these patients, there are 147 cases of ITP with 60 males and 87 females, average age is 38.5±16.45; samples of the control group are the healthy medical examines in the medical examination center of our hospital healthy. There are 117 cases in total with 45 males and 72 females and average age is 32.5±17.31.2. The extraction of genomic DNA. Genomic DNA in the single nuclear cell was extracted from all samples with the method of column centrifugation.3. Amplification of PCR and analysis of the product. Polymorphism of BAFF promoter-871C/T gene was tested by the means of allele specific polymerase chain reaction (AS-PCR) and agarose gel electrophoresis. Genotypes of objects were distinguished according to strip electrophoresis and the frequencies of the genotype were counted.4. Gene sequencing. Genetype of C/C, T/T and C/T samples were sequenced to verify the existence of two alleles.5. Antigen of plasma BAFF were measured using ELISA in 72 cases of ITP and 45 controls, analyze the correlation of BAFF antigen among the different genotypes.6. A software of SPSS 13.0 was used for the statistical analysis and gene counting method for calculating genotype frequency and allele frequency of each group,x2 test to test the count data. M was used to indicate the measurement data, Mann-Whitney U non-parametric test to comparing the two groups of antigen and antigen of the three genotypes in each group were compared with the Kruskal-Wallis H non-parametric test and there was statistical significance when p<0.05.Results1. The mutation of the promoter gene-871C/T of BAFF is the mutation of C→T, with three genotypesC/C, C/T and T/T. Genotypes include C/C type 55.6%,C/T type 33.3%,T/T type 11.1% in the control group, C/C 32.7%, C/T 40.8%, T/T 26.5% in ITP group. The C and T allele frequency were72.6% and 27.4% respectively in the control group,53.1% and 46.9% in ITP group with p< 0.01.2. BAFF antigen in primary ITP,treated patients and health controls were 875.86 (416.89-8331) pg/mL,502.58 (226.9-792.23) pg/mL,736.88 (353.13-1725.93) pg/mL respectively, There was significant difference in the three groups p<0.01; In the comparision of primary ITP and health controls, z=2.028,p<0.05; the comparision of treated patients and health controls, z=3.497,p<0.01; the comparision of primary ITP and treated patients,z=5.162,p<0.01。3. BAFF antigen levels of three genotypes, C/C, C/T and T/T,753.45 (599.95-1335.16) pg/mL,820.03 (416.89-1137.17) pg/mL and 1256.37 (518.74-8331) pg/mL respectively in primary ITP,599.95(353.13-1045.44)pg/mL,795.19(448.94-1725.93)pg/mL and 946.22 (820.02-1137.17) pg/mL in the control group. The BAFF antigene of the C/C, C/T and T/T genotype in primary ITP Group x 2=4.894,p=0.083; in the control group x2=5.39, P=0.068。Conclusions1. There is significant correlation between gene polymorphism of promotor-871 C/T of BAFF and ITP, the gene may play an important role in the pathogenesis of ITP.2. The Gene polymorphism of promoter-871 C/T of BAFF may not affect the expression of BAFF antigen.3. The overexpression of BAFF antigen may be one of risk factors inducing ITP.4. It would be necessary to further study the function(or significance) of gene polymorphism with BAFF.