| Background and purpose:Pulmonary tuberculosis(PTB) is caused by mycobacterium tuberculosis(MTB), and the infection rate and fatality are both very high. It’s a serious threat to human’s health, and brings human a great pressure financially and mentally. According to statistics, there are about 2 billion people who are infected with mycobacterium tuberculosis in the word now, and about 1/10 of the people above developed into active tuberculosis. According the statistics coming from WHO, there are 22 high-burden countries of PTB around the world, including China, whose mortality of PTB ranks ninth among all kinds of diseases.The pathogenesis of pulmonary tuberculosis is very complicated,it is not only related with the pathogen virulence and environmental factors, but also with the host genes. The genetic susceptibility of tuberculosis is determined by many genes, and it is different in some races and regions, so we should make a plan for prevention, diagnosis and treatment, according to different clinical group. Therefore, it will provide some theoretical basis and reference data to develop targeted prevention, diagnosis and treatment that screen susceptibility genes of tuberculosis from different regions and populations, and explor the molecular mechanism of the disease.With the development of human genomics and proteomics, many genes who are associated with the susceptibility of TB have been found in recent years.In 2002, Lagali, P.S and others found the FAM46 A gene for the first time, and the gene located on cromosome 6q14.1 encoded FAM46 A protein. There is a VNTR sequence in the coding sequence of exon 2 in the FAM46 A gene. In 2014, Godfrey Essien Etokebe and others found that the FAM46 A gene was associated with the susceptibility of pulmonary tuberculosis, and they believe that VNTR can participate in the regulation of many biological processes, for example, morphological development and gene transcription, besides, it includes protein function and so on, but we didn‘t find any reports in the rest of the populations.The EBF1 gene is located on cromosome 5q34 and is the specific factor of T cell lineage. In 2012, Eileen Png and others found the rs10515787 in EBF1 gene was was associated with the susceptibility of PTB in Indonesians. Because the body’s immune response to mycobacterium tuberculosis is mainly related to T lymphocytes, so the author concluded that the gene was not only involved in B cell immune but was associated with NOTCH signaling who plays a very important role in T cell differentiation, and affected the host immune response to the intracellular bacteria infection ultimately. But this argument lacks more experimental support, Eileen Png’s results is race genetic differences or common phenomenon? It is the pathogenesis in Chinese population? It needs to be studied further.Several studies have shown that protein tyrosine phosphatase non-receptor type 22(PTPN22) gene is associated with a variety of immune disease. PTPN22 gene located on chromosome lpl3.l-lp13.3, and encodes lymphoid tyrosine phosphatase(LYP) who could Inhibit T cell activation, not infavor of T cell signaling, and causing related disease. In recent years, some scholars found that the rs2488457 loci in PTPN22 gene was associated with autoimmune diseases,such as type I diabetes, idiopathic urticaria. In 2009, Lamsyah and others found the rs33996649 loci in PTPN22 gene was associated with PTB in Moroccans. Is the rs2488457 loci in PTPN22 gene associated with susceptibility to tuberculosis? Is the rs33996649 loci PTPN22 gene related to the occurrence of tuberculosis in Chinese Han population? It remains to be further research.So in our study, we selected the VNTR loci in FAM46 A gene, the rs10515787 loci in EBF1 gene, and the rs2488457 loci and rs333996649 loci in PTPN22 gene to analyze the relation between their polymorphisms and the susceptibility of tuberculosis in Henan Han population. It will provide certain reference and theoretical basis for exploring tuberculosis molecular genetic mechanism of Henan han nationality population, and making a effective prevention, diagnosis and treatment plan of PTB.Research object:Case groups: To collect 200 cases new tuberculosis patients’ blood samples from the infectious disease hospitals of Anyang and Xinxiang Henan for over two years. Their ages are from 20 to 56(the average age: 38.9±17.6) years old, including 146 male subjects, 54 female subjects. Tuberculosis diagnosis standard was accord with WS 288-2008 diagnosis standards promulgated by the ministry of health in China in 2008, becides, we should rule out other immune disease,such as diabetes, HIV and so on, including some patients who have used immunosuppressant.Control group: To selecte 200 cases healthy individuals in the same period with patients randomly whose eges are from 20 to 52(the average age: 36.0±15.7) years old, including 178 male subjects, 22 female subjects. Exclusion criteria is the same as the case group.All the research object of case group and the control group come from Henan region, and there is no genetic relationship between the objects of study, getting the informed consent of objects themselves or guardians.Research methods:To take 2 ml research object of peripheral blood and extract genomic DNA using the phenol-chloroform method. First we amplificate VNTR by STR-PCR,and then detect the polymorphism of VNTR loci in the FAM46 A gene through capillary electrophoresis(CE) for amplification products, and the alleles are named after their repetitions. For rs10515787 loci in the EBF1 gene, and rs2488457 and rs33996649 loci in the PTPN22 gene, we first amplificate the purpose fragment by PCR, then the amplification products are digested by TasⅠ, SacⅠ and MspⅠ restriction enzymes respectively, and detect their genotypes through 2%, 3.5% and 2% of agarose gel electrophoresis respectively after digestion, and then analyze and observe the result by ultraviolet gel imaging system, and record finally.Statistical disposal:We use the SPSS17.0 statistical software to analyze all the datas(the inspection level: α=0.05), and then calculate the OR value and the 95% confidence interval to estimate the influence of the risk of tuberculosis of genetic mutations.. In order to estimating the representation of detas selected, we use the HWE 1.20 software to do Hardy-Weinberg(HWE) tests for control groups. Using the MDR Software package(version 0.6.1) to analyse the interactions between genes.Results:1. The frequencies of genotypes of four locus detedced above in control group were all accorded with Hardy-Weinberg balance(P>0.05) in Henan han population, which showed they were good representatives of groups.2. We detected four kinds of alleles of VNTR loci in the FAM46 A gene they are 1,2,3 and 4 respectively, and nine kinds of genotypes they are 1/1, 1/2, 2/2, 1/3, 2/3, 3/3, 2/4, 3/4 and 4/4 respectively. The relative risk estimates showed 2/2 genotype was positively correlated with the susceptibility of tuberculosis, and 2/3 genotype was negatively correlated with the susceptibility of tuberculosis in Henan Han population.3. The differences of allele and genotype frequency of rs10515787 loci in the EBF1 gene had statistical significance(P<0.05)after comparison for them between patients and control group, and AA genotype and A allele is the risk factor of tuberculosis.4. The difference of CG genotype frequency of rs2488457 in the PTPN22 gene had statistically significant(P<0.05)after comparison for them between two groups, and the relative risk estimates showed it’s negatively correlated with susceptibility of tuberculosis in Henan Han population.5. W e didn’t found rs33996649 loci in the PTPN22 gene polymorphism in Henan Han population, and the results of testing all subjects individuals were all CC genotype.6. There were interactions between rs10515787 in the EBF1 gene and rs2488457 and rs33996649 in the PTPN22 gene.Conclusion:1. The VNTR loci in FAM46 A gene, rs10515787 loci G/A in EBF1 gene, and rs2488457 loci G/C in PTPN22 gene polymorphisms were associated with the susceptibility of tuberculosis.2. There were interactions between rs10515787 in the EBF1 gene and rs2488457 and rs33996649 in the PTPN22 gene. |
PDF Full Text Request