中国汉族人群急性肺损伤/急性呼吸窘迫综合征的遗传易感性研究

【作者】 宋振举；

【导师】 白春学；

【作者基本信息】 复旦大学 ， 内科学， 2011， 博士

【摘要】 第一部分TIRAP基因多态性与脓毒症致ALI/ARDS易感性关联研究[目的]TIRAP基因编码的衔接蛋白-Mal是Toll样受体(TLRs)信号传导通路的重要衔接蛋白,在信号传导通路中具有重要的作用。有研究发现TLRs信号传导通路介导的炎症介质过量生成参与了ALI/ARDS的发病。最近研究发现TIRAP基因变异与多种炎症相关性疾病的易感性相关。本研究探讨TIRAP基因变异是否与中国汉族人群脓毒症致ALI/ARDS的发病相关。[方法]临床上收集278例脓毒症致ALI/ARDS患者、288例单独脓毒症患者和298例健康对照个体,利用中国汉族人群Hapmap数据库选择TIRAP基因的3个tag SNPs (rs595209, rs3802813和rs8177375)和2个文献报道的与炎症相关性疾病易感性相关的SNPs (rs8177374和rs7932766)进行研究；运用PCR直接测序的方法进行SNPs分型,基因型频率和等位基因频率在各组之间的比较采用χ2检验,运用Bonferroni方法进行校正和Logistic回归分析去除混杂因素的影响。[结果]研究发现TIRAP基因的5个SNPs等位基因频率分布在研究样本中均符合Hardy-Weinberg平衡,5个SNPs之间的连锁不平衡程度较弱。位于基因3’端区域的rs8177375和内含子区域的rs595209与中国汉族人群ALI/ARDS的易感性有关。ALI/ARDS患者rs8177375等位基因G的频率明显高于单独脓毒症组(p=0.00079)和正常对照组(p=0.0001),基因型频率相比也具有显著性的统计学差异,携带等位基因G的个体更易患ALI/ARDS; ALI/ARDS患者rs595209A的等位基因频率也显著高于单独脓毒症组(p=0.0041)和正常对照组(p=0.0027),携带等位基因A的个体更易患ALI/ARDS,基因型频率也存在显著性差异。rs8177375(C/A)和rs595209(A/G)组成的单倍型也与ALI/ARDS的易感性有关,与单个SNP相比差异更显著。ALI/ARDS患者组单倍型AG(rs595209A/rs8177375G)频率高于单独脓毒症组(OR=2.24,95%CI 1.52-3.29,p=0.00003)和正常对照个体(OR=2.13,95%CI 1.46-3.09,p=0.00006)。携带单倍型CA的个体不易患ALl/ARDS。运用Bonferroni方法进行校正和Logistic回归分析去除混杂因素后以上差异仍具有统计学意义。[结论]TIRAP基因多态性与中国汉族人群脓毒症致ALI/ARDS的易感性显著相关。第二部分TLRs信号传导通路相关基因多态性与ALI/ARDS易感性关联研究[目的]TLRs信号通路的过度激活和负性调节因子的功能失调均可引起炎症介质的过量生成,导致组织和器官的损伤,在ALI/ARDS的发病机制中具有重要的作用。本部分研究选择TLRs信号传导通路及负性调节因子的部分基因IRAK1, TRAF6、SIGIRR和IRAK3进行研究,探讨以上基因SNPs与中国汉族人群ALI/ARDS遗传易感的关联性。[方法]临床上收集336例ALI/ARDS患者和384名对照患者(入住ICU,具有ALI/ARDS发病的高危因素,而未进展至ALI/ARDS)入选本部分研究。根据Hapmap数据库共选择IRAK、TRAF6、SIGIRR和IRAK3基因的17个tag SNPs,利用SNPstream技术平台对17个SNPs进行分型。基因型频率和等位基因频率在各组之间的比较采用χ2检验,运用Bonferroni方法进行校正检验和Logistic回归分析去除混杂因素的影响。[结果]4个基因的17个SNPs成功分型,且所有的SNPs等位基因分布在研究样本中均符合HWE。研究发现：①TRAF6基因rs4755453的等位基因频率和基因型频率在ALI/ARDS组和对照组之间存在显著的统计学差异(p= 0.0000187, p= 0.00015)。ALI/ARDS患者rs4755453C的频率(10.7%)明显低于对照组(18.8%),等位基因C对ALI/ARDS的患病具有保护作用(OR=0.52,95%CI0.38-0.70); Logistic回归分析去除混杂因素后等位基因频率(p=0.000062,OR=0.61,95% CI 0.46-0.78)和基因型频率分布(p=0.00046)仍有显著性的统计学差异。Bonferroni校正后等位基因和基因型频率在两组之间仍存在显著性的统计学差异；②IRAK3基因rs1370128和rs2289314等位基因频率和基因型频率在对照组和ALI/ARDS疾病组之间也存在显著性的统计学差异,但运用Logistic去除混杂因素和Bonferroni校正后等位基因和基因型频率在两组之间无显著性差异(p>0.05)。Haplotype分析发现IRAK3基因10个SNPs组成2个单倍域共包括7种单倍型,2个单倍域与ALI/ARDS发病无明显的相关性；7种单倍型的分布频率在两组之间无显著的统计学差异；③IRAK1基因的1个tag SNP和SIGIRR基因的3个tag SNPs的等位基因和基因型频率分布在两组之间无显著的统计学差异。[结论]TRAF6基因多态性与中国汉族人群ALI/ARDS的遗传易感性相关。第三部分TNF-a信号传导通路相关基因多态性与ALI/ARDS易感性关联研究[目的]TNF-α是一种多效应的细胞因子,具有极强的促炎性反应以及免疫调节功能,是炎症反应的核心环节,已有大量的研究证实TNF-α在ALI/ARDS发病机制中具有重要的作用。TNF-α主要通过细胞膜上的受体(TNF receptor, TNFR)发挥生物学效应。A20是TNF-α信号传导通路的负反馈调节因子,是防止体内炎症反应失控的重要调节蛋白。本研究选择TNF-α、TNFRSFIA、TNFRSF1B和TNFAIP3作为候选基因,探讨以上基因的SNPs与中国汉族人群ALI/ARDS遗传易感的相关性。[方法]临床上收集336例ALI/ARDS患者和384名对照患者(入住ICU,具有ALI/ARDS发病的高危因素,而未进展至ALI/ARDS)入选本部分研究。共选择TNF-a、TNFRSF1A、TNFRSF1B和TNFAIP3基因的14个SNPs进行研究,利用PCR-直接测序法和SNPstream技术平台对14个SNPs进行分型。基因型频率和等位基因频率在各组之间的比较采用χ2检验,运用Bonferroni方法进行校正和Logistic回归分析去除混杂因素影响。[结果]14个SNPs的等位基因分布在研究样本中均符合HWE。研究发现：①TNFAIP3基因rs661561等位基因频率在ALI/ARDS组和对照组之间存在着显著的统计学差异(p=0.0002),等位基因T频率在ALI/ARDS组为7.5%,明显低于对照组的频率13.6%,等位基因T对ALI/ARDS的发病具有保护作用(OR=0.52,95%CI0.36-0.74)。rs661561基因型GG、GT和TT的频率在两组之间也存在着显著的统计学差异(p=0.001)。Logistic回归分析去除混杂因素后rs661561等位基因频率(p=0.00052,OR=0.56,95%CI0.46-0.81)和基因型频率分布(p=0.0016)在两组之间的差异仍有显著性的统计学意义。Bonferroni校正后等位基因和基因型频率在两组之间仍存在显著性的统计学差异。Haplotype分析发现由2个SNPs(rs661561和rs610604)组成的单倍域与ALI/ARDS的发病明显相关,单倍型GT的频率在两组之间存在着显著的统计学差异(p=0.00039,OR=1.87,95% CI 1.32-2.65), ALI/ARDS患者单倍型GT的频率(92.2%)明显高于对照组(86.3%),携带GT的个体易患ALI/ARDS;单倍型TG的频率在两组之间也存在显著的统计学差异(p=0.029,OR=0.63,95%CI0.42-0.96),携带单倍型TG个体不易患ALI/ARDS, Logistic回归分析后仍具有显著性的统计学差异；②TNF-α基因的4个SNPs、TNFRSF1A基因的2个SNPs以及TNFRSF1B基因的2个SNPs等位基因频率和基因型频率两组相比无显著的统计学差异。[结论]编码TNF-a信号传导通路的负性调节因子A20蛋白的-TNFAIP3基因变异与中国汉族人群ALI/ARDS的遗传易感性相关。更多还原

【Abstract】 PART I Genetic variants in the TIRAP gene are associated with increased risk of sepsis-associated acute lung injuryBackground:Toll like receptors (TLRs) signaling pathways, including the adaptor protein Mal encoded by the TIRAP gene, play a central role in the development of acute lung injury (ALI). Recently, the TIRAP variants have been described association with susceptibility to inflammatory diseases. The aim of this study was to investigate whether genetic variants in TIRAP are associated with the development of ALI.Methods:A case-control collection from Han Chinese of 298 healthy subjects,278 sepsis-associated ALI and 288 sepsis alone patients were included. Three tag single nucleotide polymorphisms (SNPs) of the TIRAP gene and two additional SNPs that have previously showed association with susceptibility to other inflammatory diseases were genotyped by direct sequencing. The differences of allele, genotype and haplotype frequencies were evaluated between three groups.Results:The minor allele frequencies of both rs595209 and rs8177375 were significantly increased in ALI patients compared with both healthy subjects (odds ratio (OR)= 1.47,95% confidence interval (CI) 1.15-1.88, p= 0.0027 and OR= 1.97, 95% CI 1.38-2.80, p= 0.0001, respectively) and sepsis alone patients (OR= 1.44, 95% CI 1.12-1.85, p= 0.0041 and OR= 1.82,95% CI 1.28-2.57, p= 0.00079, respectively). Haplotype consisting of these two associated SNPs strengthened the association with ALI susceptibility. The frequency of haplotype AG (rs595209A, rs8177375G) in the ALI samples was significantly higher than that in the healthy subjects (OR= 2.13,95% CI 1.46-3.09, p= 0.00006) and the sepsis alone group (OR = 2.24,95% CI 1.52-3.29, p= 0.00003). Carriers of the haplotype CA (rs595209C, rs8177375A) had a lower risk for ALI compared with healthy control group (OR= 0.69,95% CI 0.54-0.88, p= 0.0003) and sepsis alone group (OR= 0.71,95% CI 0.55-0.91, p= 0.0006). These associations remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons.Conclusions:These results indicated that genetic variants in the TIRAP gene might be associated with susceptibility to sepsis-associated ALI in the Han Chinese population. However, the association needs to be replicated in independent studies. PARTⅡAn association study of TLRs signaling pathway genetic variants and susceptibility to ALI/ARDS in the Chinese Han populationBackground:Deregulated or excessive host immune responses contribute to the pathogenesis of ALI/ARDS. TLRs signaling pathways and their negative regulators play a pivotal role in the modulation of host immune responses and the development of ALI/ARDS. The objective of this study was to investigate the association of variants in the TLR signaling pathway genes and their negative regulator genes with susceptibility to ALI/ARDS in the Chinese Han population.Methods:720 critically ill patients with risk factors for ALI/ARDS admitted to the ICUs from 2006 to 2010. Cases were 336 patients who developed ALI/ARDS and controls were 384 subjects who did not developed ALI/ARDS. Four genes, namely IRAKI, TRAF6, SIGIRR and IRAK3, were investigated for their association with ALI/ARDS susceptibility by a tag SNP strategy. Seventeen tag SNPs were selected based on the data of Chinese Han in Beijing from the HapMap project and genotyped by the SNPstream system. The differences of allele and genotype distributions between two groups were compared with the chi-square test or Fisher’s exact test when appropriate. Multivariate logistic regression was used to adjust for potential confounding factors and Bonferroni method was used to correct for multiple comparisons.Results:All of the 17 tag SNPs were genotyped successfully and all of the genotype distributions were consistent with Hardy-Weinberg equilibrium. Single SNP analyzing showed rs4755453 in TRAF6 is associated with susceptibility to ALI/ARDS. The allele and genotype distributions of rs4755453 in TRAF6 were significantly different between ALI/ARDS and control groups (p=0.0000187, p=0.00015). The allele frequency of rs4755453C was associated with a decreased risk of ALI/ARDS (OR= 0.52,95%CI 0.38-0.70). These associations remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons. The allele and genotype distributions of rsl370128 and rs2289314 in 1RAK3 were also significantly different between ALI/ARDS and control groups. But this difference disappeared after adjustment for covariates in multiple logistic regression analysis and after Bonferroni correction. Two haplotype blocks were indentified in the IRAK3 region. But no association was found between these two blocks and the susceptibility to ALI/ARDS.Conclusions:These results indicated that genetic variants in the TRAF6 gene might be associated with susceptibility to ALI/ARDS in the Han Chinese population. However, the association needs to be replicated in independent studies. PART IIIAn association study of TNF-a signaling pathway genetic variants and susceptibility to ALI/ARDS in the Chinese Han populationBackground:The pleiotropic cytokine tumour necrosis factor-a (TNF-a) is an important pro-inflammatory cytokine involved in the pathogenesis and development of ALI/ARDS. TNF-a binds directly to tumor necrosis factor receptors (TNFR) and induces the inflammatory response. A20, the product of the TNFAIP3 gene, is a key negative regulator of NF-κB activity downstream of TNF-a. The objective of this study was to investigate the association of variants in the TNF-a signaling pathway genes with susceptibility to ALI/ARDS in the Chinese Han population.Methods:TNF-a, TNFRSF1A, TNFRSF1B and TNFAIP3 were investigated for their association with ALI/ARDS susceptibility. Fourteen SNPs were genotyped in 336 ALI/ARDS patients and 384 controls. The differences of allele and genotype distributions between two groups were compared with the chi-square test or Fisher’s exact test when appropriate. Multivariate logistic regression was used to adjust for potential confounding factors and Bonferroni method was used to correct for multiple comparisons.Results:All of the 14 SNPs were genotyped successfully and all of the genotype distributions were consistent with Hardy-Weinberg equilibrium. Single SNP analyzing showed rs661561 in TNFAIP3 is associated with susceptibility to ALI/ARDS. There was significant difference of the allele and genotype distributions in ALI/ARDS patients compared with those of control subjects (p= 0.0002, p= 0.001). The minor allele frequency of rs661561T was associated with a decreased risk of ALI/ARDS (OR= 0.52,95% CI 0.36-0.74). A 2-SNP haplotype block harboring rs661561 and rs610604 also displayed strong association with ALI/ARDS risk. Haplotype GT and TG frequencies in ALI/ARDS patients were significantly different from that control subjects (p= 0.00039, OR= 1.87,95% CI 1.32-2.65; p= 0.029, OR = 0.63,95% CI 0.42-0.96, respectively). These associations remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons. No association was found between TNF-a, TNFRSF1A, TNFRSF1B variants and the susceptibility to ALI/ARDS.Conclusions:These results indicated that genetic variants in the TNFAIP3 gene might be associated with susceptibility to ALI/ARDS in the Han Chinese population. However, the association needs to be replicated in independent studies.更多还原