侵袭性曲霉病患者Toll样受体4基因变异的初步研究

A preliminary study on the mutation of TLR4 gene in patients with invasive aspergillosis

目的 Toll样受体(Toll-like receptors,TLRs)家族是最重要的病原识别受体,动物实验发现TLR4-/-小鼠更容易感染曲霉。文中探讨我国汉族人群TLR4基因编码区变异情况以及该基因变异与侵袭性曲霉病(invasive aspergillosis,IA)易感性的关系。方法回顾性收集2011年6月至2012年12月南京军区南京总医院确诊为IA的25例住院患者设为IA组,其中男性17例,平均年龄(52.4±12.3)岁,有基础疾病12例、无基础疾病的13例;健康体检者103例设为对照组,其中男性70例,平均年龄(56.0±17.2)岁。所有研究对象均来源于汉族人群。采集研究对象外周静脉血,提取外周血中DNA,采用聚合酶链式反应(PCR)扩增TLR4基因编码区序列后测序,测序结果与NCBI Gen Bank中的TLR4编码序列进行比对,并分析基因突变引起的氨基酸改变以及对蛋白质功能的影响。比较IA组与对照组TLR4基因突变率的差异。结果研究共发现2个突变点,分别为219 C>G和1420 C>T,均为错义突变。蛋白结构预测结果显示219 C>G突变导致TLR4识别病原体的功能区改变,TLR4 1420 C>T未引起结构域改变。IA组TLR4的突变频率为8%(2/25),2例TLR4基因突变的患者均无基础疾病;对照组未发现突变,2组间比较差异有统计学意义(P=0.037)。结论 IA患者存在TLR4基因编码区错义突变(219C>G与1420C>T),该基因的突变可能增加机体对IA的易感性。

Objective Toll-like receptor （TLR） gene family is the most important pathogen recognition receptor and animal experiment have found TLR4 mice is inclined to infect aspergiUosis （IA）. The study was to investigate the variation of TLR4 gene in Chinese Han nationality and its relation with the susceptibility of IA. Methods 25 patients diagnosed with proven IA from June 2011 to December 2012 in our hospital were enrolled, among which 17 were males. Their average age was 52, 4 ± 12.3, and 12 patients had underlying diseases, the others had no underlying diseases. The control group consisted of 103 normal persons, among which 70 were males. Their average age was 56.0 ± 17.2. All of the subjects were Chinese Han population. DNA was extracted from peripheral blood. Polymerase chain reaction （PCR） was applied to amplify the coding sequence of TLR4 gene followed by sequencing. The sequencing result was compared with TLR4 coding sequence in NCBI GenBank along with the analysis on amino acid change caused by genetic mutation and its effect on protein function. Comparison analysis was made on genetic mutation rate between IA group and control group. Results Two missense mutations,TLR4 219 C 〉 G and 1420 C 〉T, were identified in TLR4. The prediction result of protein structure showed 219 C 〉 G resulted in the change of functional area for TLR4 to recognize pathogen and 1420 C 〉 T caused no change in domains. The variation rate of TLR4 in IA group was 8% （2/25） and both the patients had no underlying diseases. No mutation has been founded in control group and the difference between two groups was of statistical significance （ P = 0. 037 ）. Conclusion Two missense mutations （219 C 〉 G and 1420 C 〉 T） have been detected in encoding area of TLR4 gene of IA patients. Variation in the coding region of TLR4 gene may increase the susceptibility to IA.