TLR通路基因多态性与变应性鼻炎的关联研究
Association of Toll-like receptor pathway gene polymorphisms with allergic rhinitis
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收稿日期: 2019-02-15 修回日期: 2019-05-06 网络出版日期: 2019-08-06
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Received: 2019-02-15 Revised: 2019-05-06 Online: 2019-08-06
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陈若希, 张清照, 陆美萍, 朱歆洁, 程雷.
CHEN Ruoxi, ZHANG Qingzhao, LU Meiping, ZHU Xinjie, CHENG Lei.
研究发现,Toll样受体(Toll-like receptors,TLR)及其通路与鼻部炎性疾病的发生发展相关[9,10]。TLR能通过调控多种与AR发生密切相关的细胞如树突状细胞、肥大细胞和调节性T细胞(regulatory T-cells, Treg),参与Th1/Th2免疫反应平衡的调控。研究显示[11,12,13,14,15,16,17,18]TLR2、TLR5、TLR7、TLR9对变应性炎症主要起负性调节作用,通过诱导Treg细胞扩增等机制,促使免疫系统向Th1分化,抑制或逆转Th2反应。TLR通路中的一些细胞因子比如MyD88[19]、TIRAP[20,21]、CD14[15,16,17,18,19,20,21,22]等在信号传导中发挥十分重要的作用。
1 材料与方法
1.1 研究对象 病例选择按照WHO“变应性鼻炎及其对哮喘的影响(ARIA 2008)”和中华医学会“变应性鼻炎诊断和治疗指南(2009年,武夷山)”的诊断依据。研究对象为2008年5月至2016年1月在南京医科大学第一附属医院(江苏省人民医院)就诊的持续性AR患者,病史和临床资料来源于门诊病史采集及面对面的问卷调查。所有病例均来自江苏和安徽的汉族人群,对于患者的年龄、性别无特殊限制,采血前4周内未使用过糖皮质激素,2周内未使用过抗组胺药、白三烯受体拮抗剂等抗过敏药物。
对照选自同时期来医院进行健康体检的人群,所选对照要求:①无AR及鼻部疾病症状和病史;②无哮喘等其他变态反应疾病症状和病史;③血清变应原特异性IgE阴性;④直系一代亲属内无AR或其他变态反应疾病史。病例和对照按照频数匹配的原则进行匹配,年龄范围为±5岁,男女性别按照病例中的比例进行匹配。
本研究得到研究机构伦理委员会的批准,严格遵循相关规定获得研究对象(监护人)的书面知情同意。所有病例和对照均为华东地区无血缘关系的汉族人。
1.2 标本收集和DNA提取 对病例及对照者进行问卷调查,问卷的主要内容包括一般情况、人口学特征、疾病的症状、体征、病史、家族史及伴随疾病。视觉模拟评分(VAS):从0 cm(无影响)至10 cm(影响最重)评价患者对鼻部症状的主观感觉,包括流涕、喷嚏、鼻塞、鼻眼痒及总的鼻部症状。VAS评分<5诊断为轻度鼻炎,VAS评分≥5为中重度鼻炎[34]。
经知情同意,研究对象抽取肘部静脉血2 mL,装入含惰性分离胶的促凝管中,在临床检验中心进行血常规检测。同时抽取静脉血5mL,分装入三个清洁EDTA抗凝管中,其中1管置于-20 ℃冰箱保存,剩余两管经1 000 rpm离心10 min,分离得到血浆,分装于试管,血浆置-70℃低温冰箱冷冻保存待测。使用QIAGEN公司生产的全血基因组DNA提取试剂盒,提取DNA最低浓度为100 ng/μL,纯度为1.8~2.0。同时采用1%琼脂糖凝胶电泳质检。
1.3 SNPs的基因分型 根据文献报道并结合生物信息网络数据库,优先选取位于启动子区(promoter)、基因编码区(coding gene region)、5′-非翻译区(5′-untranslated region,5-UTR)、3′-非翻译区(3′-untranslated region,3-UTR)及3′-侧翼区(3′-near gene)的碱基突变。选取的基因多态位点在中国人群中最小等位基因频率(minor allele frequency, MAF)大于0.05。经过筛选选取TLR2的rs7656411、rs76112010、rs7682814和CD14的rs2563298、rs2569190、rs2569191作为候选SNPs(表1),采用TaqMan基因分型系统进行检测。设计PCR引物,定制TaqMan荧光双标记探针,然后运用TaqMan荧光定量PCR技术对SNPs位点基因型进行分析,其结果用序列检测软件进行输出。另外,采取TaqMan技术随机抽取一定比例(不低于10%)的随机样本进行重复检测,以验证检测方法的可靠性。
表1 TLR2和CD14等位基因的基本信息及基因频率分布
Table 1
| 序号 | SNPs | 染色体位置 | 碱基变化 | 最小等位基因(MAF) | HWE Pa | ||
|---|---|---|---|---|---|---|---|
| 数据库 | 病例组 | 对照组 | |||||
| 1 | rs7656411 | 3′near gene | G/T | 0.419 | 0.482 | 0.451 | 0.168 |
| 2 | rs76112010 | 5′near gene | A/G | 0.136 | 0.203 | 0.169 | 0.661 |
| 3 | rs7682814 | 5′near gene | A/G | 0.167 | 0.202 | 0.172 | 0.286 |
| 4 | rs2563298 | 3′UTR | A/C | 0.151 | 0.123 | 0.184 | 0.067 |
| 5 | rs2569190 | 5′UTR | A/T | 0.500 | 0.380 | 0.412 | 0.988 |
| 6 | rs2569191 | 5′near gene | C/T | 0.453 | 0.363 | 0.426 | 0.625 |
1.4 血清总IgE和特异性IgE检测 使用ImmunoCAP体外变应原检测系统检测血清总IgE和特异性IgE水平。本地区常见吸入物变应原包括屋尘螨、粉尘螨、猫毛、狗毛、德国小蠊、链格孢霉、艾蒿及豚草花粉等。血清总IgE检测范围为2~5 000 kU/L;特异性IgE检测范围为0.35~100 kUA/L,特异性IgE水平≥0.35 kUA/L为阳性。
1.5 统计学处理 采用Epidata软件对临床资料和实验室数据进行录入和管理。应用SPSS及SAS软件进行资料的统计学分析。分析方法包括计数资料的单因素
2 结 果
2.1 一般情况及资料分析 此次研究共纳入研究对象947例,其中病例为452例,对照495例,病例组和对照组的年龄及性别经检验无统计学差异。病例组的总IgE水平(264.0 [120.6~593.5] kU/L)和血清ECP水平(12.7 [5.2~28.7] μg/L) 明显高于(P<0.001)对照组 (总IgE:26.8 [11.1~52.2] kU/L,ECP:4.6 [3.1~7.5] μg/L)。在AR患者中,247例 (54.6%) 的症状为轻度(VAS<5),194例 (42.9%) 为中重度(VAS≥5),并且有118例 (26.1%) 伴随哮喘疾病,122例(27.0%)有过敏性疾病的家族史(表2)。
表2 病例组和对照组研究对象的临床特征
Table 2
| 变量 | 病例组 (n = 452) | 对照组 (n = 495) | P |
|---|---|---|---|
| 年龄 (岁, | 19.70 ± 12.60 | 20.10 ± 12.61 | 0.597a |
| 性别[n(%)] | |||
| 男 | 302(66.8) | 303(61.2) | 0.073a |
| 女 | 150(33.2) | 192(38.8) | |
| 鼻炎病程 (年, | 6.62 ± 5.87 | ||
| 鼻腔总症状 ( | 5.25 ± 2.39 | ||
| 血清总IgE [kU/L, M (IQR)] | 264.0 (120.6~593.5) | 26.8 (11.1~52.2) | < 0.001b |
| 特异性IgE [kUA/L, M (IQR)] | |||
| 屋尘螨 | 29.0(4.6~72.5) | — | |
| 粉尘螨 | 24.1 (5.0~69.9) | — | |
| ECP [μg/L, median(IQR)] | 12.7 (5.2~28.7) | 4.6 (3.1~7.5) | < 0.001b |
| 伴随哮喘 | |||
| 是 | 118(26.1) | ||
| 否 | 271(60.0) | ||
| 家族史 | |||
| 是 | 122(27.0) | ||
| 否 | 269(59.5) | ||
2.2 单个基因多态位点分析
6个SNPs中,CD14的rs2563298(C>A)和rs2569191(C>T)基因位点在与AR疾病存在阳性相关(rs2563298:P = 0.003, rs2569191:P = 0.008)。rs2563298位点相比野生CC基因型,携带CA基因型 (OR =0.65; 95%CI = 0.48~0.89)、AA基因型(OR =0.40; 95%CI = 0.18~0.89)和CA/AA联合基因型(OR =0.62; 95%CI = 0.46~0.83)的个体可以明显降低AR的发病风险。rs2569191位点的CT(OR =0.65; 95%CI = 0.48~0.87)、TT(OR =0.65; 95%CI = 0.44~0.95)和联合基因型CT/TT(OR =0.65; 95%CI = 0.49~0.85),相比野生型CC,明显降低了AR的发病风险。虽然TLR2的rs7656411位点总效应为阴性结果,但对比野生型GG,联合基因型GT/TT明显增加了AR的发病风险(OR =0.65; 95%CI = 0.49~0.85, P=0.035)(表3)。
表3 TLR2和CD14多态性基因分型及在病例与对照组中的频率分布
Table 3
| SNPs | 基因型 | 病例组 | 对照组 | Crude OR(95%CI) | Adjusted OR (95%CI)a | Pb | |||
|---|---|---|---|---|---|---|---|---|---|
| n | % | n | % | ||||||
| TLR2 | |||||||||
| rs7656411 | n = 441 | n =493 | |||||||
| GG | 112 | 25.4 | 156 | 31.6 | 1.00 (reference) | 1.00 (reference) | 0.079 | ||
| GT | 233 | 52.8 | 229 | 46.5 | 1.42 (1.05~1.92) | 1.41 (1.04~1.91) | |||
| TT | 96 | 21.8 | 108 | 21.9 | 1.23 (0.86~1.79) | 1.22 (0.84~1.76) | |||
| GT/TT | 329 | 74.6 | 337 | 68.4 | 1.36 (1.02~1.81) | 1.35 (1.01~1.79) | 0.035 | ||
| T allele | 0.451 | 0.186 | |||||||
| rs76112010 | n = 446 | n = 485 | |||||||
| GG | 287 | 64.4 | 331 | 68.3 | 1.00 (reference) | 1.00 (reference) | 0.144 | ||
| GA | 137 | 30.7 | 141 | 29.1 | 1.12 (0.84~1.49) | 1.15 (0.86~1.53) | |||
| AA | 22 | 4.96 | 13 | 2.7 | 1.95 (0.97~3.95) | 1.98 (0.98~4.02) | |||
| GA/AA | 159 | 35.7 | 154 | 31.2 | 1.19 (0.91~1.53) | 1.22 (0.93~1.60) | 0.209 | ||
| A allele | 0.203 | 0.089 | |||||||
| rs7682814 | n = 441 | n = 486 | |||||||
| GG | 285 | 64.6 | 330 | 67.9 | 1.00 (reference) | 1.00 (reference) | 0.074 | ||
| GA | 134 | 30.4 | 145 | 29.8 | 1.07 (0.81~1.42) | 1.09 (0.82~1.45) | |||
| AA | 22 | 5.00 | 11 | 2.26 | 2.32 (1.10~4.86) | 2.29 (1.09~4.81) | |||
| GA/AA | 42 | 15.9 | 38 | 13.9 | 1.15 (0.88~1.52) | 1.17 (0.90~1.55) | 0.292 | ||
| A allele | 0.202 | 0.097 | |||||||
| CD14 | |||||||||
| rs2563298 | n = 434 | n =479 | |||||||
| CC | 336 | 77.4 | 326 | 68.1 | 1.00 (reference) | 1.00 (reference) | 0.003 | ||
| CA | 89 | 20.5 | 131 | 27.3 | 0.66 (0.48~0.90) | 0.65 (0.48~0.89) | |||
| AA | 9 | 2.1 | 22 | 4.60 | 0.40 (0.18~0.88) | 0.40 (0.18~0.89) | |||
| CA/AA | 98 | 68.5 | 153 | 31.9 | 0.62 (0.46~0.84) | 0.62 (0.46~0.83) | 0.002 | ||
| A allele | 0.123 | <0.001 | |||||||
| rs2569190 | n = 450 | n = 481 | |||||||
| AA | 174 | 38.7 | 166 | 34.5 | 1.00 (reference) | 1.00 (reference) | 0.351 | ||
| AG | 210 | 46.7 | 233 | 48.4 | 0.86 (0.65~1.14) | 0.86 (0.64~1.15) | |||
| GG | 66 | 14.7 | 82 | 17.1 | 0.78 (0.88~1.92) | 0.76 (0.52~1.94) | |||
| AG/GG | 276 | 61.3 | 315 | 65.5 | 0.84 (0.52~1.31) | 0.84 (0.64~1.09) | 0.188 | ||
| G allele | 0.380 | 0.150 | |||||||
| rs2569191 | n = 441 | n = 472 | |||||||
| CC | 187 | 42.4 | 153 | 32.4 | 1.00 (reference) | 1.00 (reference) | 0.008 | ||
| CT | 188 | 42.6 | 236 | 50.0 | 0.65 (0.49~0.87) | 0.65 (0.48~0.87) | |||
| TT | 66 | 15.0 | 83 | 17.6 | 0.65 (0.44~0.96) | 0.64 (0.44~0.95) | |||
| CT/TT | 254 | 57.6 | 319 | 67.6 | 0.65 (0.50~0.85) | 0.65 (0.49~0.85) | 0.002 | ||
| T allele | 0.363 | 0.006 | |||||||
2.3 基因多态位点与AR临床表型的相关性研究 由于病例组和对照组过敏状态的差异,所以分开进行总IgE、特异性IgE与各位点之间的相关性统计。在病例和对照组中,6个多态性位点均未发现与总IgE水平明显相关(P>0.05)。此外,其他AR相关临床指标包括症状严重度、特异性IgE水平及ECP水平均未发现与单个多态性位点有明显相关(P>0.05)。
分层分析中(表4),相比对照组,携带杂合/突变纯合性(MW/MM)基因型的rs7656411 (GT/TT)在男性组(OR = 1.62; 95%CI = 1.12~2.30)、有哮喘组(OR = 1.61; 95%CI = 1.13~2.28) 、有家族史组(OR =1.64; 95%CI = 1.16~2.33)的患者中提升了AR的患病风险。rs2563298的CA/AA基因型在年龄<18岁组、女性组、有/无哮喘组、有/无家族史组、高/低VAS组、低IgE组均相比对照组降低AR的发病风险。rs256191的CT/TT只有在无家族史组为阴性结果,在其他组别,如年龄组、性别组、有/无哮喘组、有家族史组、高/低VAS组、高/低IgE组均相比对照组降低了AR的发病风险。
表4 TLR2和CD14基因分型(MW/MM)与变应性鼻炎易感因素的分层分析
Table 4
| 变量 | 分组 | rs7656411 病例/对照 | rs2563298 病例/对照 | rs2569191 病例/对照 | |||
|---|---|---|---|---|---|---|---|
| 例数 | Adjusted OR (95%CI)b | 例数 | Adjusted OR (95%CI)b | 例数 | Adjusted OR (95%CI)b | ||
| 年龄(岁) | <18 | 247/239 | 1.33(0.88~1.99) | 244/239 | 0.54(0.36~0.80) | 247/236 | 0.65(0.44~0.94) |
| ≥18 | 194/254 | 1.36(0.91~2.06) | 191/240 | 0.73(0.47~1.13) | 194/236 | 0.66(0.44~0.98) | |
| 性别 | 男 | 295/303 | 1.60(1.12~2.30) | 289/296 | 0.75(0.52~1.07) | 293/289 | 0.66(0.47~0.93) |
| 女 | 146/190 | 0.97(0.60~1.57) | 146/183 | 0.42(0.25~0.72) | 148/183 | 0.62(0.40~0.98) | |
| 哮喘 | 无 | 263/493 | 1.28(0.81~2.03) | 261/479 | 0.45(0.27~0.75) | 262/472 | 0.53(0.35~0.81) |
| 有 | 116/493 | 1.61(1.13~2.28) | 115/479 | 0.60(0.42~0.85) | 116/472 | 0.70(0.51~0.96) | |
| 家族史 | 无 | 120/493 | 1.20(0.76~1.89) | 118/479 | 0.56(0.35~0.92) | 120/472 | 0.85(0.56~1.31) |
| 有 | 260/493 | 1.64(1.16~2.33) | 260/479 | 0.55(0.38~0.79) | 260/472 | 0.57(0.42~0.78) | |
| VAS | <5 | 247/493 | 1.25(0.88~1.77) | 241/479 | 0.65(0.45~0.92) | 246/472 | 0.72(0.52~0.99) |
| ≥5 | 194/493 | 1.45(0.99~2.12) | 193/479 | 0.60(0.40~0.88) | 195/472 | 0.57(0.41~0.80) | |
| IgE a | 低 | 135/493 | 1.41(0.99~2.00) | 234/479 | 0.45(0.31~0.67) | 237/472 | 0.61(0.44~0.84) |
| 高 | 206/493 | 1.31(0.90~1.89) | 200/479 | 0.82(0.57~1.19) | 204/472 | 0.71(0.50~0.99) | |
b运用Logist回归将年龄、性别作为调整因素。
2.4 多态性位点的基因-基因交互作用
表5 多因子降维模型分析基因-基因交互作用
Table 5
| 模型a | 训练样本准确度 | 检测样本准确度 | 交叉验证一致 性 | P |
|---|---|---|---|---|
| A6 | 0.5491 | 0.5264 | 8/10 | 0.0036 |
| A3 A6 | 0.5712 | 0.5196 | 8/10 | < 0.0001 |
| A1 A4 A6 | 0.5953 | 0.5487 | 8/10 | < 0.0001 |
图1
图1
最佳模型树状图。距离越近的位点,交互作用越强。连线颜色为红色,位点间的交互作用越接近协同作用,若连线颜色越浅,则表示交互作用较弱
Fig.1
Tree diagram of the best genotype models. The distance between SNPs indicates the intensity of the interactions. The colors indicate the type of interactions. Red, orange, and green denotes a high degree, lesser degree, and weak interaction, respectively
3 讨 论
TLR属于I型跨膜蛋白,是一类重要的模式识别受体,TLR对其配体的识别,不但启动了天然免疫系统,也激活了特异性免疫系统。TLR能通过调控多种与AR发生密切相关的细胞如树突状细胞、肥大细胞和调节性T细胞(regulatory T-cells, Treg),参与Th1/Th2免疫反应平衡的调控。我们从TLR通路中筛选了来自TLR2和CD14的6个SNP并进行了中国汉族人群的病例与对照研究。最终结果显示CD14的rs2563298、rs2569191位点与AR发病明显相关,TLR2的rs7656411(GT/TT基因型)与AR发病率相关。
TLR通过多种识别分子与其配体结合,经多种衔接分子将信号转导至细胞内,如IL-1受体相关激酶(IL-1 receptor-associated kinase,IRAK)、肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor,TRAF6)等,激活NF-κB或丝裂原活化蛋白激酶等,诱导免疫相关基因的表达[35]。TLR通路中的细胞因子在信号传导中发挥重要作用。细胞内TLR2/4信号传导起始的关键分子机制是TLR2/4、TIRAP和MyD88三者的TIR结构域的特异性组装[19]。CD14与TLR2、TLR4通过相互间的协同作用发挥其生物学功能[15,22]。
TLR2在单核-巨噬细胞、内皮细胞、上皮细胞、成纤维细胞等有表达。TLR2识别谱较广, 能够识别细菌肽聚糖、脂蛋白、病毒包膜蛋白等微生物成分并激活信号转导途径,最终导致获得性免疫的发生。大量的研究表明, TLR2对变应性反应有抑制作用[11,12,13,36],但也有报道TLR2对变应性反应有促进作用[37]。本研究中rs7656411位点 T等位基因与AR发病存在相关(P=0.035),该位点位于3′侧翼区,可能参与mRNA的转录从而影响基因的表达。目前尚未有rs7656411位点与AR相关性文献报道,但该位点被证实与中国人群哮喘发病相关[24]。TLR2的rs4696480位点在加拿大人群中与AR发病无相关[38],597 T/C和1350 T/C 位点在韩国人群中与AR发病无相关,仅单倍型(597C-1350C)有阳性相关[30]。TLR2的多态性位点在哮喘病例对照研究中较多,rs13150331、rs2289318、rs3804099位点在法国人群中与哮喘发病相关[39],也有阴性报道[40,41,42,43]。最近一篇Meta分析统计TLR2的4个位点rs5743708、 rs3804099、rs3804100和rs4696480对哮喘易感性,发现仅rs4696480位点与哮喘发病相关[44]。
CD14是脂多糖(lipopolysaccharide,LPS)的高亲和力受体,还能够介导其他G+及G-细菌成分与细胞的反应。LPS通过TLR4和cDl4介导刺激IL-12、IFN-γ分泌增加, 可以促使免疫系统向Th1转变。本研究中CD14的rs2563298和rs2569191位点与AR疾病存在阳性相关。尚未有此两位点与AR相关的文献报道,来自埃及[45]和巴基斯坦人群[46]的研究均证实了rs2569191位点与哮喘的相关性,一项爱尔兰的研究[47]证实rs2569191位点与荨麻疹及特异性疾病有关。在本研究中rs2569190位点与AR发病无明显相关,在日本人群研究[48]中结果为阴性,2018年一篇Meta分析[49]同样证实了本观点,但有研究报道不同结果[46,50]。一项来自中国北方人群研究[49]发现rs2569190位点TT基因型与AR发病相关,出现矛盾的结果可能由于那项研究的小样本量(病例92例,对照72例)可能导致结果的可信度较低,且中国南北地域差异引起的环境不同。
在研究TLR2和CD14基因多态性位点与AR相关临床指标的相关性中,我们发现TLR2的rs7656411、CD14的rs2563298和 rs2569191均与AR伴随哮喘的患者有明显相关,证实了诸多TLR2与CD14与哮喘相关的报道[39,44,45,46]。在本研究中各多态性位点与总IgE水平无明显相关。在日本[48]、中国儿童[51]、美国[52]以及德国人群[53]研究中同样证实了患者中的血清总IgE与CD14基因多态性无明显相关。然而一些不同人群的研究报道了阳性结果,比如俄罗斯过敏人群[54](TLR2 rs5743708)和美国哮喘人群[55](CD14 rs2569190),这也说明了种族在基因多态性中存在的差异。
本研究采用MDR模型分析发现TLR2与CD14可能存在基因交互作用。已有多项研究发现TLR2与CD14的协同作用,TLR2是LPS的信号转导分子,它的活化有赖于LPS结合蛋白(LPS binding protein,LBP)和CD14的存在。LPS能够诱导肥大细胞释放 TNF-α、IL-5、IL-10和IL-13,激活TLR2或TLR4信号转导通路可以促进Th2型免疫反应,同时也依赖CD14的存在[56]。此外有研究显示过敏性疾病中CD14与环境之间同样有交互作用,皮肤点刺阳性患者中携带CD14 rs2569190 CC基因型且在农村环境可降低哮喘发病率[39]。通过MDR模型研究,在菲律宾过敏人群[57](CD14与IL4、FCER1B、 IL4RA、 ADRB2)和韩国过敏儿童人群[58](CD14与IL-4Rα、 IL-13、IL-13Rα1、CTLA4)均发现了CD14与过敏相关基因的交互作用。过敏性疾病本身即是基因与环境共同作用,因此基因与基因、基因与环境交互作用将会更全面阐释过敏疾病发生与发展。
综上所述,本研究证实TLR通路中TLR2和CD14基因多态性在汉族人群AR的发病过程中起到重要作用,需要更多功能性的研究去证实其分子机制,以及搜集更多环境因素资料以体现在AR中的基因-环境交互作用。
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