维吾尔族饲鸽者肺患者外周血HLA—DQA1基因甲基化分析

Methylation analysis of HLA-DQA1 gene in peripheral blood of Uygur patients with pigeon breeder＇s disease

目的通过检测维吾尔族饲鸽者肺患者外周血HLA-DQA1基因甲基化程度探讨其临床意义。方法以20例确诊维吾尔族饲鸽者肺患者为病例组，20名维吾尔族饲养鸽子未发病者为阴性对照组，20名维吾尔族未饲养鸽子健康体检者为正常对照组。收集资料并抽取外周血标本，常规提取DNA、亚硫酸盐转化、PCR扩增、体外转录和RNaseA特异性酶切，并行基质辅助激光解吸电离飞行时间质谱分析技术检测HLA-DQAI基因甲基化情况。比较3组研究对象HLA—DQA1基因甲基化率是否存在差异，然后3组间两两比较HLA—DQAI基因甲基化率是否存在差异。结果检测HLA-DQA1片段的CpG位点总数4个（CpG-I、CpG-2、CpG-3、CpG-4），实际检测到的CpG位点数3个（CpG-Z、CpG-3、CpG-4）。3组样本中CpG-2、CpG-4甲基化率差异无统计学意义（Х^2值分别为1．062、1．867，P值均〉9．05）。3组样本中CpG-3甲基化率差异有统计学意义（Х^2=11．978，P〈0.05）。病例组、阴性对照组CpG-3甲基化率高于正常对照组（Z值分别为-3．116、-2．781，P值均〈0．017），但病例组与阴性对照组CpG-3甲基化率差异无统计学意义（Z=-0．576，P〉0．017）。结论接触鸽子会影响HLA—DQA1基因甲基化。但HLA-DQAI基因甲基化对维吾尔族饲鸽者肺的发病有无影响尚需进一步研究。

Objective To detect the HLA-DQA1 methylation status in peripheral blood of Uygur patients with pigeon breeder ＇s disease and explore its clinical significance. Methods 20 cases of Uygur patients with pigeon breeder＇ s disease were diagnosed as the case group, 20 cases of healthy Uygur persons who fed pigeons were selected as negative control group, another 20 cases of healthy Uygur persons who did not raise pigeons were selected as normal control group. All data were collected and all blood samples were extracted. The conventional DNA extraction, bisulfite conversion, polymerase chain reaction amplification, transcription in vitro and RNase A specific enzyme cut, matrix assisted laser desorption ionization time of flight mass spectrometry were used to detect HLA-DQA1 gene methylation status. Then SPSS 20.0 software was used for statistical analysis. HLA-DQA1 methylation rate was compared among the three groups and between every two groups. Results In detection of HLA-DQA1 fragment of CpG loci in total four （CPG-1, CPG-2, CpCr-3, CpG-4）, three sites were actually detected （CPG-2, CpG-3, CpG-4）. There was no statistical significance in CPG-2 and CpCr-4 methylation distribution among three groups （ Х^2= 1. 062,1. 867, both P 〉0.05）. There was statistical significance in CpG-3 methylation distribution among three groups （Х^2= 11. 978, P 〈0.05）. The methylation rate of CpG-3 in case group and negative control group was higher than that in normal control group （ Z =-3. 116, -2. 781, both P 〈0. 017）, but there was no difference in the methylation rate of CpG-3 between case group and negative control group （ Z = -0. 576, P 〉0. 017）. Conclusions Pigeon exposure can affect the methylation of HLA-DQA1 gene. However, it still needs to further study whether HLA-DQA1 gene methylation can impact on the incidence of Uygur pigeon breederrs disease.