本文選題：支氣管哮喘 + 全外顯子組測(cè)序�。� 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:采用全外顯子組測(cè)序技術(shù)探尋5q31-33區(qū)中兒童支氣管哮喘的易感基因。方法:從皖南醫(yī)學(xué)院第一附屬醫(yī)院等多家醫(yī)療單位收集到支氣管哮喘核心家系18例,從中選取1例母親和兒子均為哮喘患者,而父親正常的家系進(jìn)行全外顯子組測(cè)序;另從120例散發(fā)哮喘兒童中選取20例重度患者進(jìn)行全外顯子組測(cè)序,并針對(duì)5q31-33區(qū)進(jìn)行重點(diǎn)分析,以期找出該區(qū)域內(nèi)與哮喘相關(guān)聯(lián)的SNPs位點(diǎn)。結(jié)果:1、本研究對(duì)一例母親和兒子均為哮喘患者,而父親正常的哮喘核心家系進(jìn)行全外顯子組測(cè)序,通過對(duì)測(cè)序結(jié)果的多步驟篩選分析,最終,在該家系中發(fā)現(xiàn)一個(gè)母親和兒子共同擁有的錯(cuò)義突變位點(diǎn)p.Ala514Thr,并且父親不存在該突變位點(diǎn),該位點(diǎn)位于PCDH 1基因的外顯子2區(qū)。2、通過對(duì)20例重度哮喘患兒的全外顯子組測(cè)序結(jié)果在5q31-33區(qū)內(nèi)進(jìn)行重點(diǎn)分析,我們共發(fā)現(xiàn)了124個(gè)在哮喘患者和正常對(duì)照者之間存在差異的突變位點(diǎn)。結(jié)論:1、PCDH1基因可能是支氣管哮喘的易感基因之一。2、全外顯子組測(cè)序是探尋復(fù)雜疾病易感基因的有效方法之一,尤其對(duì)于罕見突變位點(diǎn)的發(fā)掘能力較高,相比其他方法而言,具有樣本量較少、測(cè)序通量更高、測(cè)序成本更低等優(yōu)勢(shì)。目的:通過對(duì)PCDH 1基因的啟動(dòng)子和五個(gè)外顯子區(qū)域進(jìn)行序列測(cè)定,從而尋找PCDH 1基因啟動(dòng)子和編碼區(qū)的多態(tài)性位點(diǎn)。方法:針對(duì)PCDH 1基因的啟動(dòng)子和五個(gè)外顯子設(shè)計(jì)引物,對(duì)所收集的138例哮喘兒童和150例健康對(duì)照者進(jìn)行PCR擴(kuò)增,并對(duì)擴(kuò)增產(chǎn)物進(jìn)行sanger測(cè)序,將測(cè)序結(jié)果與NCBI及Hap Map數(shù)據(jù)庫進(jìn)行對(duì)比,以此確定PCDH 1基因多態(tài)性的分布特征。采用Pearsonχ2檢驗(yàn)比較各位點(diǎn)基因型和等位基因頻率在哮喘組和對(duì)照組的分布。結(jié)果:1.本實(shí)驗(yàn)共在PCDH1基因中發(fā)現(xiàn)了五個(gè)SNPs位點(diǎn),其中有4個(gè)為新發(fā)現(xiàn)的位點(diǎn)。2.本實(shí)驗(yàn)所檢出的五個(gè)SNPs的分布及頻率在哮喘組和對(duì)照組之間均未見明顯的統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:本實(shí)驗(yàn)尚未檢出PCDH1基因中與哮喘相關(guān)聯(lián)的SNPs位點(diǎn),但由于本實(shí)驗(yàn)樣本量較少和可能存在的人群因素的影響,所以還需進(jìn)行進(jìn)一步的驗(yàn)證。
[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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.6

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