CD40-1C/T基因多态性与儿童哮喘的相关性分析

引用本文

杜建钢, 徐红星, 竭琼菲, 钟桥, 吴元建, 徐吉成. CD40-1C/T基因多态性与儿童哮喘的相关性分析. 2013, 28(1): 17-20
DU Jiangang, XU Hongxing, JIE Qiongfei, ZHONG Qiao, WU Yuanjian, XU Jicheng. Analysis on the correlation of CD40-1C/T gene polymorphism with children asthma. Labratory Medicine, 2013, 28(1): 17-20 复制到剪切板

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CD40-1C/T基因多态性与儿童哮喘的相关性分析

杜建钢¹, 徐红星¹, 竭琼菲¹, 钟桥¹, 吴元建¹, 徐吉成²

1.南京医科大学附属苏州市立医院本部检验科,江苏苏州 215002

2. 南京医科大学附属苏州市立医院本部儿科,江苏苏州 215002

作者简介：杜建钢,男,1977年生,主管技师,主要从事免疫检验工作。

通讯作者：徐红星,联系电话：051262362318。

摘要

目的

研究CD40基因5'非翻译区-1位点C/T多态性在本地区哮喘儿童人群中的分布,探讨其与哮喘发病风险之间的关系。

方法

以134例哮喘患者作为研究对象,儿童门诊健康体检者113名作为对照组。应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测哮喘组和对照组CD40-1C/T位点的基因型及等位基因频率;采用免疫比浊法测定血清总IgE水平。分析该基因多态性与儿童哮喘发病风险及IgE水平之间的关系。

结果

哮喘组和对照组之间C与T等位基因频率分布差异无统计学意义(P>0.05);CC基因型与CT+TT基因型之间的分布差异有统计学意义(P<0.05),CC基因型的发病风险是CT+TT基因型的1.75倍[95%可信区间(CI):1.013.04]。哮喘组IgE水平在CC与CT、TT基因型间差异有统计学意义(P<0.05)。

结论

CD40基因5'非翻译区-1位点CC基因型可能与本地区儿童哮喘的发生有关。

关键词: CD40; 哮喘; 基因多态性

中图分类号:Q503 文献标志码:A 文章编号:1673-8640(2013)01-0017-04

Analysis on the correlation of CD40-1C/T gene polymorphism with children asthma

DU Jiangang¹, XU Hongxing¹, JIE Qiongfei¹, ZHONG Qiao¹, WU Yuanjian¹, XU Jicheng²

1.Department of Clinical Laboratory,Suzhou Municipal Hospital, Nanjing Medical University,Jiangsu Suzhou 215002, China

2.Department of Pediatrics,Suzhou Municipal Hospital,Nanjing Medical University,Jiangsu Suzhou 215002, China

Abstract

Objective

To investigate the distribution of CD40 gene 5' untranslated region-1 site C/T gene polymorphism,and to study its relationship with the risk of asthma in local asthma children.

Methods

A total of 134 asthma children were enrolled, and 113 healthy children from healthy examination subjects were as control group. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotype and the allele frequencies of CD40-1C/T gene in asthma and control groups. The serum total level of IgE was determined by immunonephelometry. The relationship of CD40-1C/T gene polymorphism with the risk of asthma and IgE level was analyzed.

Results

The distributions of the C and T allele frequencies between asthma and control groups had no statistical significance (P>0.05). The distribution difference of CC and CT+TT genotype frequencies was statistically significant (P<0.05). The risk of CC genotype was 1.75 times higher than that of CT+TT genotype [95%confidence interval (CI): 1.01-3.04].There were significant differences in the level of IgE between CC genotype and CT or TT genotype in asthma group (P<0.05).

Conclusions

CC genotype of CD40 gene 5' untranslated region -1 site might be associated with the development of asthma in local asthma children.

Keyword: CD40; Asthma; Gene polymorphism

Show Figures

引言

CD40属肿瘤坏死因子受体(TNF-R)超家族成员,主要表达在B细胞、胸腺上皮细胞、活化的单核巨噬细胞、树突状细胞、造血细胞、上皮细胞、内皮细胞及一些肿瘤细胞等^{[ 1]}。CD40和CD40L是T细胞与抗原提呈细胞(APC)表面一对重要的共刺激分子,是免疫细胞间重要的信号转导途径。CD40基因含1个831 bp的开放阅读框架,其基因由8个内含子和9个外显子构成,定位于20号染色体的q11-13,CD40基因5'UTR-1位点位于CD40基因翻译起始密码子前一位,该位点存在T→C多态性。国外有报道,CD40基因多态性与某些疾病如甲状腺疾病^{[ 2]}、系统性红斑狼疮^{[ 3]}等相关。目前,关于CD40基因多态性与哮喘的关系报道较少。鉴于哮喘发生时,变应原与致敏靶细胞表面IgE结合,激活多种炎症细胞,释放多种生物活性介质,发生免疫反应。我们拟分析CD40基因5'UTR-1C/T多态性在本地区儿童中的分布,并探讨其与哮喘的相关性。

材料和方法

一、研究对象

选取2010年5月至2011年5月南京医科大学附属苏州市立医院本部儿科门诊及住院哮喘患儿134例,均符合儿童哮喘防治规范诊断标准^{[ 4]}。其中男童74例,女童60例,平均年龄6岁。另选择113名同期南京医科大学附属苏州市立医院本部健康体检儿童作为对照组,其中男童61名,女童52名,平均年龄5岁。

二、方法

1.基因组DNA提取

乙二胺四乙酸(EDTA)抗凝静脉血200 μL,应用TianGen生物技术有限公司提供的基因组DNA提取试剂盒提取DNA。

2.引物设计与合成

参照文献[5]设计合成引物,由上海捷瑞生物技术公司合成。

3.PCR扩增

PCR反应体系为25 μL,其中含模板DNA 2 μL(100 ng/μL),P1、P2引物各0.5 μL(20 μmol/L),dNTP预混液12.5 μL(2.5 mmol/L),其余用灭菌双蒸水补足。PCR反应条件:94 ℃预变性5 min后进入循环,94 ℃变性30 s,58 ℃退火35 s,72 ℃延伸45 s,循环40次;再72 ℃延伸10 min。取PCR产物10 μL,经2%琼脂糖凝胶电泳,紫外灯下观察扩增结果。

4.扩增产物的限制性酶切鉴定

取PCR扩增产物10 μL,用限制性内切酶 NcoI(Fermentas公司)水解PCR产物,水解片段进行2%琼脂糖凝胶电泳,通过BIORAD凝胶成像系统判断结果。

5.DNA测序

将3种基因型的扩增产物进行测序,由苏州工业园区金唯智生物技术有限公司完成。

6.血清总IgE水平测定

采用BNⅡ特定蛋白仪(免疫比浊法)测定血清总IgE水平。

三、统计学方法

采用SPSS 11.0统计软件进行统计分析。以Hardy-Weinberg平衡检验确认研究样本的群体代表性,基因型频率和等位基因频率比较采用χ²检验,以比数比(odds ratio, OR)及95%可信区间(confidence interval, CI)表示各基因型与发病风险的相关性。总IgE水平呈偏态分布,以中位数(四分位间距)表示,两样本比较采用Wilcoxon秩和检验。

一、CD40-1C/T基因多态性酶切结果及测序结果

PCR扩增片段为320 bp,CD40-1C/T位点C等位基因有限制性内切酶 NcoI酶切位点。经 NcoI酶切及琼脂糖凝胶电泳后,CD40基因-1C/T位点各基因型酶切结果见图1:CC基因型为184、136 bp 2条条带,CT基因型为320、184、136 bp 3条条带,TT基因型为320bp 1条条带。3种基因型的测序结果见图2。

	Figure Option View Download New Window
	图1 CD40基因PCR扩增产物 NcoI酶切结果注:M为DNA marker;1、4为CT基因型;2、5为CC基因型;3为TT基因型;6:PCR扩增产物

二、对照组与哮喘组CD40-1C/T位点各基因型分布的Hardy-Weinberg平衡检验

用χ²检验各基因型观察值与预期值之间差异是否具有统计学意义,以确定等位基因频率和基因型频率分布是否符合Hardy-Weinberg平衡。其中,对照组χ²值为2.126, P>0.05;哮喘组χ²值为0.742, P>0.05,均符合遗传平衡定律。

	Figure Option View Download New Window
	图2 CT、CC、TT 3种基因型的测序结果

三、哮喘组CD40基因-1C/T位点多态性分析

哮喘组和对照组等位基因C、T频率比较差异无统计学意义( P>0.05)。哮喘组患者CC基因型的频率(36.6%)明显高于对照组(24.8%;χ²=3.97, P=0.043)。见表1。

表1 哮喘组和对照组CD40-1C/T多态性比较[例(%)]

四、哮喘组和对照组的分类统计分析

鉴于哮喘患者CC基因型频率明显高于对照组,故将CT与TT基因型合并为CT+TT组。结果显示,CC基因型和CT+TT组的组间分布差异有统计学意义( P<0.05), OR值>1,并且95% CI的下限值也>1,可推断CC对于哮喘的发生可能是个危险因素。结果见表2。

表2 哮喘组和对照组CD40-1C/T多态性的分类统计分析[例(%)]

五、CD40-1C/T位点各基因型血清总IgE水平比较

对照组CD40-1C/T位点各基因型间IgE水平差异无统计学意义( P>0.05)。哮喘组CC基因型总IgE水平明显高于CT、TT基因型( P<0.05),而CT与TT基因型之间差异无统计学意义( P>0.05),见表3。

表3 CD40-1C/T位点各基因型血清总IgE水平[中位数(四分位间距)]

讨论

哮喘是由多种细胞(如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞、气道上皮细胞等)和细胞因子参与的气道慢性炎症性疾病,其发病重要环节是高水平IgE的表达使得机体免疫耐受破坏产生变态反应。哮喘的相关基因尚未完全明确。IgE的水平与T、B淋巴细胞的激活状态有直接联系,而CD40和CD40L是T淋巴细胞与APC表面一对重要的共刺激分子,是促使T淋巴细胞完全活化的第二信号。CD40主要表达在B细胞、单核细胞和树突状细胞表面,属TNF-R超家族,在B细胞活化、抗体产生和类别转换、记忆B细胞分化等方面发挥关键作用。CD40L属TNF超家族,主要表达在活化的CD4⁺ T细胞和肥大细胞表面。因此推断,CD40/CD40L对于哮喘的发生可能有一定的作用。

已有研究表明哮喘患者T、B淋巴细胞表达CD40/CD40L水平明显增高;急性发作哮喘患儿的B细胞表面CD40表达率高于对照组,CD4⁺T细胞表面CD40L表达率亦比对照组高^{[ 6, 7]}。国外有文献报道,Grave's病患者CD40表达水平与CD40-1C/T基因多态性中CC基因型相关^{[ 8]};CD40基因多态性与急性冠状动脉综合症相关^{[ 9]}。有关CD40基因多态性与儿童哮喘的关系,国内未见有这方面的报道,本研究就其相关性进行了分析。

本研究哮喘组和对照组C与T等位基因频率分布差异无统计学意义( P>0.05)。哮喘组CC基因型频率明显高于对照组( P<0.05);合并CT与TT基因型的个体,比较CC和CT+TT基因型之间的分布,则两者间差异有统计学意义( P<0.05)。CC基因型个体的发病风险是CT+TT基因型个体的1.75倍。同时,哮喘组CC基因型总IgE水平明显高于CT和TT基因型。因此,CC基因型可能与本地区儿童哮喘的发生有关。有研究报道,CD40-1C/T基因多态性与急性冠状动脉综合症等患者CD40表达水平相关^{[ 10]}。故本研究推断哮喘患者CC基因型可能通过上调CD40基因转录和翻译效率,使哮喘患者也产生较高水平的CD40,促使T、B淋巴细胞活化,进而对哮喘发生产生作用。

总之,CD40-1C/T基因多态性与儿童哮喘的发生有一定的关联,携带CC基因型的儿童有更高罹患哮喘的风险,应对这部分人群予以关注。同时,单个CD40-1C/T基因多态性并不能全面评估CD40基因对儿童哮喘的影响。因此,有必要将进一步筛查CD40基因其他位点的多态性,进行多位点的连锁分析,综合评价CD40基因对支气管哮喘疾病的作用。

The authors have declared that no competing interests exist.

参考文献

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[1]	Cagnoni F, Oddera S, Giron-Michel J, et al. CD40 on adult human airway epithelial cells: expression and proinflammatory effects[J]. J Immunol, 2004, 172(5): 3205-3214. [本文引用:1] [JCR: 5.52]
[2]	Tomer Y, Concepcion E, Greenberg DA, et al. A C/T single-nucleotide polymorphism in the region of the CD40 gene is associated with Graves' disease[J]. Thyroid, 2002, 12(12): 1129-1135. [本文引用:1] [JCR: 3.544]
[3]	Chadha S, Miller K, Farwell L, et al. Haplotype structure of TNFRSF5-TNFSF5 (CD40-CD4OL) and association analysis in systemic lupus erythematosus[J]. Eur J Hum Genet, 2005, 13(5): 669-676. [本文引用:1] [JCR: 4.319]
[4]	全国儿科哮喘防治协作组. 儿童哮喘防治规范(试行)[J]. 中华儿科杂志, 1998, 49(12): 747. [本文引用:1]
[5]	Kim TY, Park YJ, Hwang JK, et al. A C /T polymor-phism in the 5'-untranslated region of the CD40 gene is associated with Graves' disease in Koreans[J]. Thyroid, 2003, 13(10): 919-925. [本文引用:1] [JCR: 3.544]
[6]	祝戎飞, 黄爱霞. 协同刺激分子CD28/B7及CD40/CD40L在哮喘特异性免疫治疗中的作用[J]. 华中医学杂志, 2006, 30(4): 278-280. [本文引用:1]
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[9]	Tian C, Qin W, Li L, et al. A common polymorphism in CD40 Kozak sequence (-1C/T) is associated with acute coronary syndrome[J]. Biomed Pharmacother, 2010, 64(3): 191-194. [本文引用:1] [JCR: 2.068]
[10]	Yan J, Wang C, Du R, et al. Association analysis of CD40 gene polymorphism with acute coronary syndrome[J]. Clin Exp Med, 2010, 10(4): 253-258. [本文引用:1] [JCR: 2.397]

2004

5.52

0.0

... 引言CD40属肿瘤坏死因子受体(TNF-R)超家族成员,主要表达在B细胞、胸腺上皮细胞、活化的单核巨噬细胞、树突状细胞、造血细胞、上皮细胞、内皮细胞及一些肿瘤细胞等^[1] ...

2002

3.544

0.0

... 国外有报道,CD40基因多态性与某些疾病如甲状腺疾病^[2]、系统性红斑狼疮^[3]等相关 ...

2005

4.319

0.0

. 2005, 13(5):669-676

Haplotype structure of TNFRSF5-TNFSF5 (CD40|[ndash]|CD40L) and association analysis in systemic lupus erythematosus

The European Journal of Human Genetics is the official Journal of the European Society of Human Genetics, publishing high-quality, original research papers, short reports, News and Commentary articles and reviews in the rapidly expanding field of human genetics and genomics.

... 国外有报道,CD40基因多态性与某些疾病如甲状腺疾病^[2]、系统性红斑狼疮^[3]等相关 ...

1998

0.0

... 材料和方法一、研究对象选取2010年5月至2011年5月南京医科大学附属苏州市立医院本部儿科门诊及住院哮喘患儿134例,均符合儿童哮喘防治规范诊断标准^[4] ...

2003

3.544

0.0

2006

0.0

... 急性发作哮喘患儿的B细胞表面CD40表达率高于对照组,CD4⁺T细胞表面CD40L表达率亦比对照组高^[6,7] ...

2006

0.0

... 急性发作哮喘患儿的B细胞表面CD40表达率高于对照组,CD4⁺T细胞表面CD40L表达率亦比对照组高^[6,7] ...

2005

4.717

0.0

... 国外有文献报道,Grave's病患者CD40表达水平与CD40-1C/T基因多态性中CC基因型相关^[8] ...

2010

2.068

0.0

. 2010, 64(3):191-194 DOI:10.1016/j.biopha.2009.09.021

A common polymorphism in CD40 Kozak sequence (-1C/T) is associated with acute coronary syndrome

Abstract Objective Evidence suggests the CD40-CD40L pathway as a key process in the development, progression, and outcome of acute coronary syndrome (ACS). We hypothesized that the -1C/T polymorphism of the CD40 gene would be associated with ACS and influence the CD40 expression. Methods The genotype distribution and allele frequency of CD40-1C/T polymorphism in 248 ACS patients and 206 controls were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Monocytes from 90 healthy volunteers were incubated with IFN-γ. CD40 expression was detected by flow cytometry. Result Patients with ACS showed a significant increase of CD40 expression compared with controls ( P P P P P Conclusions These results suggest that the -1C allele of the CD40 (-1C/T) gene polymorphism is a genetic factor that may determine an individual's susceptibility by ACS in Chinese. The CD40 -1C/T polymorphism is a novel regulator of CD40 expression.

... CD40基因多态性与急性冠状动脉综合症相关^[9] ...

2010

2.397

0.0

. 2010, 10(4):253-258 DOI:10.1007/s10238-010-0092-7

Association analysis of CD40 gene polymorphism with acute coronary syndrome

1.Division of Cardiology, Department of Medicine, The Affiliated Hospital of Jiangsu University, 438 Jiefang Road, 212001 Zhenjiang, Jiangsu Province People’s Republic of China

Current evidence shows that the CD40–CD40 ligand (CD40–CD40L) system plays a crucial role in the development, progression and outcome of acute coronary syndrome (ACS). The aim of this study is to investigate whether a CD40 gene (−1C/T) single nucleotide polymorphism (SNP) is associated with ACS and CD40 expression. We included controls ( n = 163) and patients with either ACS ( n = 210) or stable angina (SA, n = 189) in the study. The gene polymorphism was measured using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and identified by sequencing. CD40 expression on platelets was detected by flow cytometry. The results showed that the frequency of the CC genotype of CD40 gene in the ACS group was significantly higher than in the SA and control groups ( P P P < 0.001). The C allele increased the risk of ACS when compared with the SA group (OR = 1.841, 95% CI: 1.390–2.437) and the control groups (OR = 1.877, 95% CI: 1.402–2.515). No significant differences in genotypes or allele frequencies were found between the SA and control groups. CD40 expression on platelets in patients with C-allele carriers was significant higher than in the T-allele carriers in each group. In conclusion, our results suggested that the CD40 gene (−1C/T) polymorphism was associated with ACS in Chinese people. The −1C-allele gene carriers had a higher expression of CD40 and an increased risk of suffering from ACS.

... 有研究报道,CD40-1C/T基因多态性与急性冠状动脉综合症等患者CD40表达水平相关^[10] ...