利用外显子组测序技术探寻5q31-33区中儿童支气管哮喘易感基因

发布时间：2018-04-25 16:03

本文选题：支气管哮喘 + 全外显子组测序　；参考：《皖南医学院》2017年硕士论文

【摘要】：目的:采用全外显子组测序技术探寻5q31-33区中儿童支气管哮喘的易感基因。方法:从皖南医学院第一附属医院等多家医疗单位收集到支气管哮喘核心家系18例,从中选取1例母亲和儿子均为哮喘患者,而父亲正常的家系进行全外显子组测序;另从120例散发哮喘儿童中选取20例重度患者进行全外显子组测序,并针对5q31-33区进行重点分析,以期找出该区域内与哮喘相关联的SNPs位点。结果:1、本研究对一例母亲和儿子均为哮喘患者,而父亲正常的哮喘核心家系进行全外显子组测序,通过对测序结果的多步骤筛选分析,最终,在该家系中发现一个母亲和儿子共同拥有的错义突变位点p.Ala514Thr,并且父亲不存在该突变位点,该位点位于PCDH 1基因的外显子2区。2、通过对20例重度哮喘患儿的全外显子组测序结果在5q31-33区内进行重点分析,我们共发现了124个在哮喘患者和正常对照者之间存在差异的突变位点。结论:1、PCDH1基因可能是支气管哮喘的易感基因之一。2、全外显子组测序是探寻复杂疾病易感基因的有效方法之一,尤其对于罕见突变位点的发掘能力较高,相比其他方法而言,具有样本量较少、测序通量更高、测序成本更低等优势。目的:通过对PCDH 1基因的启动子和五个外显子区域进行序列测定,从而寻找PCDH 1基因启动子和编码区的多态性位点。方法:针对PCDH 1基因的启动子和五个外显子设计引物,对所收集的138例哮喘儿童和150例健康对照者进行PCR扩增,并对扩增产物进行sanger测序,将测序结果与NCBI及Hap Map数据库进行对比,以此确定PCDH 1基因多态性的分布特征。采用Pearsonχ2检验比较各位点基因型和等位基因频率在哮喘组和对照组的分布。结果:1.本实验共在PCDH1基因中发现了五个SNPs位点,其中有4个为新发现的位点。2.本实验所检出的五个SNPs的分布及频率在哮喘组和对照组之间均未见明显的统计学差异(P0.05)。结论:本实验尚未检出PCDH1基因中与哮喘相关联的SNPs位点,但由于本实验样本量较少和可能存在的人群因素的影响,所以还需进行进一步的验证。
[Abstract]:Objective: to explore the susceptibility gene of bronchial asthma in children in 5q31-33 region by using total exon sequence analysis. Methods: 18 cases of bronchial asthma nuclear family were collected from the first affiliated Hospital of Southern Anhui Medical College. One case of mother and son were selected as asthma patients, while the father's normal family was sequenced. In addition, 20 patients with severe asthma were selected from 120 children with sporadic asthma to sequence the whole exon group, and to analyze the 5q31-33 region in order to find out the SNPs site associated with asthma in this region. Results: 1. In this study, we sequenced the whole exon group of an asthmatic nuclear family whose mother and son were both asthmatic, and finally analyzed the results by multistep screening. A missense mutation site, p.Ala514Thr, shared by mother and son, was found in the family and did not exist in the father. The locus was located in exon 2 of PCDH 1 gene. The results of total exon group sequencing in 20 children with severe asthma were analyzed in the 5q31-33 region. We found a total of 124 mutation sites with differences between asthmatic patients and normal controls. Conclusion: 1 / 1 PCDH1 gene may be one of the susceptible genes of bronchial asthma. Total exon sequencing is one of the effective methods to explore the susceptible genes of complex diseases, especially for rare mutation sites, compared with other methods. It has the advantages of small sample size, higher sequencing flux and lower sequencing cost. Objective: to search for the polymorphic sites of PCDH 1 gene promoter and coding region by sequencing the promoter and five exon regions of PCDH 1 gene. Methods: primers were designed for the promoter and five exons of PCDH 1 gene. PCR amplification was performed in 138 asthmatic children and 150 healthy controls, and the amplified products were sequenced by sanger. The results of sequencing were compared with NCBI and Hap Map databases to determine the distribution characteristics of PCDH 1 gene polymorphism. Pearson 蠂 2 test was used to compare the distribution of genotype and allele frequencies in asthma group and control group. The result is 1: 1. In this study, five SNPs loci were found in the PCDH1 gene, four of which were newly discovered. There was no significant difference in the distribution and frequency of five SNPs between the asthma group and the control group (P 0.05). Conclusion: the SNPs loci associated with asthma in the PCDH1 gene have not been detected in this study, but due to the small sample size and the influence of possible population factors, further verification is needed.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.6

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