P2X_7基因多态性与中国汉族儿童结核病易感性的研究

Association of polymorphisms in the P2X_7 gene with tuberculosis in Chinese Han children

目的探讨中国汉族儿童P2X7基因多态性与结核病易感性相关关系,以探讨P2X7基因在儿童结核病发病中的作用。方法病例组为2005年1月至2008年9月首都医科大学附属北京儿童医院收治的汉族结核病患儿;对照组为同期在儿外科行手术前体检的患儿。按照年龄等与病例组3:1进行匹配。扩增基因组DNAP2X7基因,应用限制性酶切片段长度多态性(PCR-RFLP)分析方法和碱基特异性PCR方法,分别对P2X7基因1513和-762位点多态性与儿童结核病易感性进行相关分析。结果病例组纳入96例,平均年龄(5.5±4.5)岁;对照组纳入384例,平均年龄(5.9±4.0)岁。P2X7基因1513位点A/C和C/C基因型分布,病例组(38.5%和8.3%)较对照组(31.0%和11.5%)比率增高,但差异无统计学意义(χ2=2.306,P=0.316);1513C在病例组和对照组分布频率分别为27.6%和27.0%,差异无统计学意义(χ2=0.033,P=0.856)。-762位点C/C基因型在整体人群中的分布频率为56.5%,-762C在病例组和对照组中的分布频率分别为77.4%和71.7%,对照组和病例组各基因频率或基因型频率差异无统计学意义(χ2=4.742,P=0.093)。上述基因型和等位基因频率在肺结核亚组和肺外结核病亚组差异均无统计学意义。结论宿主P2X7基因1513位点A/C和-762位点T/C的转换可能与中国汉族儿童结核病易感性无相关性。因此,P2X7基因多态性与结核病发病以及是否为结核病易感性的影响因素有待进一步验证。

Objective To explore the association of P2X7 gene polymorphisms with the susceptibility to pediatric tuberculosis in Chinese Han. Methods Genomic DNA was extracted from peripheral blood by EDTA anticoagulation , using a standard salting-out procedure. The concentration and purity of DNA were estimated spectrophotometrically. The 1513A/C polymorphism was detected via RFLP, by using the restriction enzyme HaeII. The polymorphism at position -762 in the promoter of the P2X7 gene was studied by allele-specific PCR. Products were run on 1.5% agarose gel stained with ethidium bromide, whereupon it was visualized using an UV transilluminator. Statistical analyses were carried out using SPSS software, version 11.5 （SPSS Inc. , Chicago, IL, USA）. The Hardy-Weinberg equilibrium （HWE） , which indicated the absence of discrepancy between genotype and allele frequency, was determined for both control subjects and patients. Overall genotype frequencies in each patient group were compared using a 3 × 2 X2 test, and a 2× 2 X2 test was also used to detect any significant difference between the two groups in terms of allele frequency. Patient and clinical information was collected from patient files, and all participating subjects and parents gave written, informed consent. Results From Jan, 2005 to Sep, 2008, ninety-six cases of pediatric tuberculosis admitted to Beijing Children＇s Hospital were identified by clinical manifestation, culture and X-ray results, and by other means, including forty-one active pulmonary tuberculosis and fifty-five extrapulmonary tuberculosis. Three hundred and eighty four children, who were inpatients children for physical examination in surgery department, were collected as controls. The mean age was 5.5 years old （ SD ： 4.5 ; range ： three months-15 years old） for patients and 5.9 years old（ SD ： 4.0 ; range ： three months-16 years old） for control subjects. All controls were matched with tuberculosis patients by age, sex, and area. Members of both patient and control groups were of Chinese Han ancestry. The genotypic and allelic frequencies of two different SNPs of the P2X7 gene were analyzed. The frequencies of the alleles were found in HWE. The frequency of A/C and C/C genotype at the 1513 site was higher in tuberculosis patients （38.5% and 8.3% ） than in controls （31.0% and 11.5% ）, but no statistical difference was obtained （X2 =2. 306, P = 0. 316）. Similarly, the frequency of 1513C in tuberculosis patients and controls （27.6% and 27% ） had no statistical difference （X2 = 0. 033, P = 0. 856）. The frequency of C/C genotype at the - 762 site was high in all the subjects enrolled （56.4%）, while the frequencies of -762 C in tuberculosis patients and controls were 77.4% and 71.7% , no statistical difference was obtained （X2 = 22. 670 ,P = 0. 102）. There were no significant associations between each of the genotypie or allelic distributions and pulmonary or extrapulmonary TB. Conclusions The 1513 and -762 polymorphisms in human P2X7 gene had no association with pediatric tuberculosis in China. Although the mechanism of protection or pathogenicity by these genotypes had not been fully elucidated, we considered these apparently discrepant results to be of interest. Individual SNP within genes varied markedly among different racial groups, the number of subjects enrolled was different, the P2X7 receptor gene was not the major gene to produce a marked effect. Thus, further studies are needed to clarify whether the polymorphisms in P2X7 gene is associated with tuberculosis or suseeptive to tuberculosis.