辽宁地区汉族人群TAP2基因4个SNPs位点遗传分布及法医学意义研究

发布时间：2018-03-10 12:48

本文选题：TAP2基因　切入点：单核苷酸多态性　出处：《中国医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 前言抗原处理相关转运体(transporter associated with antigen processing,TAP)蛋白是一种分布于内质网膜上的转运蛋白,功能是将胞浆内内源性抗原的降解产物转运至内质网腔。TAP基因于1990年分别由Trowsdale J等和Spies T等同时发现。1991年WHO的HLA命名委员会将这一新发现的基因统一命名为TAP基因。TAP基因位于6p21.3,MHCⅡ区的DQB1和DPB1之间,包括TAP1和TAP2两个基因。TAP2基因长16.9kb,含有12个外显子和11今内含子。 2008年,NCBI报道在TAP2基因中发现了167个SNPs位点,其中有19个位于外显子。目前,国内外对TAP2基因的研究主要涉及的是疾病发生发展机制和免疫相关性方面。而有关TAP2基因在中国辽宁汉族人群的分布及其法医学应用价值国内外尚无报道。本研究选择TAP2基因第12外显子中的4个SNPs位点,分别为651密码子[2073(?)GT(Arg)→(?)GT(Cys),rs4148876]、665密码子[2115(?)CA(Thr)→(?)CA(Ala),rs241447]、687密码子[2181(?)AG(stop)→(?)AG(Gln),rs241448]和697密码子[2213GT(?)(Val)→GT(?)(Val),rs241449],调查这4个SNPs位点在辽宁地区汉族人群的频率分布和单体型的构成情况,为人类遗传学、法医学和临床医学等学科提供有价值的参考数据。材料与方法 1、226例静脉抗凝血采自辽宁地区汉族无血缘关系的健康献血者。18例已知亲缘关系家系的DNA样品由中国医科大学法医血清学教研室提供。 2、应用双向等位基因特异性扩增(Bi-ASA)技术调查辽宁地区北方汉族人群TAP2基因4个SNPs位点的分布频率,并应用于亲子鉴定案例。 3、直接计数法计算TAP2基因4个SNPs位点的等位基因频率和基因型频率;Hardy-Weinberg平衡检验以及连锁不平衡分析;应用SPSS11.5统计软件进行x~2检验及Fisher确切概率法来比较不同人群间等位基因频率分布差异是否具有显著性;计算杂合度(H)、多态性信息量(PIC)、个人识别率(DP)和非父排除率(EPP)。结果 1、在辽宁地区汉族人群中,TAP2基因651位点检测出C和T两个等位基因,C等位基因的频率为0.9071,T等位基因的频率为0.0929;两个等位基因构成C/C、C/T和T/T三种基因型,频率分别为0.8086、0.1770和0.0044。 665位点检测出A和G两个等位基因,A等位基因的频率为0.6593,G等位基因的频率为0.3407;两个等位基因构成A/A、A/G和G/G三种基因型,频率分别为0.4513、0.4159和0.1327。 687位点检测出T和C两个等位基因,T等位基因的频率为0.6593,C等位基因的频率为0.3407;两个等位基因构成T/T、T/C和C/C三种基因型,频率分别为0.4513、0.4159和0.1327。 697位点检测出G和T两个等位基因,G等位基因的频率为0.6593,T等位基因的频率为0.3407;两个等位基因构成G/G、G/T和T/T三种基因型,频率分别为0.4513、0.4159和0.1327。 2、连锁不平衡分析结果表明,651分别与665、687和697位点处于连锁平衡状态;665、687和697这三个位点之间处于连锁不平衡状态。本研究检出这4个位点构成的4种单体型:C-A-T-G型、C-G-C-T型、T-A-T-G型和T-G-C-T型,频率分别为0.6018、0.3053、0.0575和0.0354。 3、建立了一种双SNPs位点复合扩增分型技术,并对665、687和697三个位点中任意两个位点的组合进行了同步基因分型研究。 4、在18个2代3口家系中,有2个家系的生物学父子关系在665、687和697位点被否定;18个家系在651位点均未否定生物学父子关系。讨论本研究应用双向等位基因特异性扩增技术检测ZAP2基因4个SNPs位点在辽宁地区汉族人群中的多态性分布。即在单管中加入两对引物进行PCR扩增,其中一对为公用引物,用于扩增含有SNP位点的片段;另两条为等位基因特异性引物,分别与相应的一条公用引物构成上、下游引物进行扩增反应。两条特异性引物的3′末端对应于SNP的变异点上,由引物的3′端控制引物的延伸反应,根据扩增片段的数目和长度确定该SNP位点的基因型。并通过在两条等位基因特异性引物的5′末端附加一段由10个G或C组成的“GC尾巴”,提高延伸反应的特异性。在单个SNP位点分型的基础上,本研究还探讨了一种进行两个SNPs位点复合扩增的方法。即在一个PCR反应体系中,加入两对等位基因特异性引物和一对公用引物,根据扩增片段在电泳图谱中出现的数目和位置不同对两个SNPs位点进行同步基因分型。这一方法提高了SNPs分析的检测效率,为后续三个或更多SNPs位点的同步检测奠定了基础。本研究将辽宁汉族人群TAP2基因651、665和687位点等位基因分布调查结果与其他人群的研究报道资料进行比较,发现在651位点,辽宁汉族人群与津巴布韦人群具有显著性差异(P＜0.05);在665位点,与台湾汉族、津巴布韦、高加索和荷兰人群具有显著性差异;在687位点,与维吾尔族、津巴布韦和西班牙人群具有显著性差异。有关TAP2基因697位点频率分布的资料目前尚未见报道,因此不能进行不同人群之间基因分布的比较。在4个SNPs位点中,651位点的H、PIC、DP和EPP分别为0.1687、0.1450、0.3149和0.0772;665/687/697位点的H、PIC、DP和EPP分别为0.416、0.3484、0.6088和0.1744。在所检测的18个2代3口家系中,有2个家系在665/687/697位点排除父权;18个案例在651位点均未否定生物学父子关系。结论 1、本研究所建立的SNPs双位点复合扩增技术,可同时对两个SNPs位点进行基因分型,具有简单、快速、特异性好的特点。 2、TAP2基因651和665/687/697位点在辽宁汉族群体中具有遗传多态性,且符合Hardy-Weinberg平衡。651、665、687位点在不同种群间具有遗传差异。 3、651位点属于中等鉴别能力的遗传标记,665/687/697位点属于较高鉴别能力的遗传标记,在法医学亲子鉴定和个人识别中具有实际应用价值。
[Abstract]:Preface
Transporter associated antigen processing (transporter associated with antigen processing, TAP) is a protein located in endoplasmic reticulum transport protein function is a degradation product of transport to the endoplasmic reticulum cavity.TAP gene in the cytoplasm of endogenous antigen respectively in 1990 by Trowsdale J and Spies T also found that.1991 WHO HLA Nomenclature Committee this will be a new gene named as TAP gene.TAP gene at 6p21.3 between MHC II region of DQB1 and DPB1, including TAP1 and TAP2 two gene.TAP2 gene was 16.9kb long with 12 exons and 11 introns present.
In 2008, NCBI reported 167 SNPs loci were found in the TAP2 gene, including 19 in exon. At present, the domestic and foreign research on TAP2 gene is mainly involved in the development and mechanism of immune related diseases occurred. The TAP2 gene in Liaoning Han population Chinese distribution and medical application of the domestic law there is no report.
This study selected TAP2 gene twelfth exon 4 SNPs loci in the 651 codon, respectively [2073 (?) GT (Arg), (?) GT (Cys), rs4148876], [2115 codon 665 (?) CA (Thr), (?) CA (Ala), rs241447], password 687 [2181 (?) AG (stop), (?) AG (Gln), rs241448] and [2213GT codon 697 (?) (Val), GT (?) (Val, rs241449]), the composition of this survey the frequency distribution of 4 SNPs loci in Liaoning Han population and haplotype, for human genetics, which provides valuable reference data for forensic science and clinical medicine.
Materials and methods
1226 cases of venous anticoagulant were collected from.18 blood donors in Liaoning area. The DNA samples of known families were provided by the Department of forensic serology, China Medical University.
2, the bidirectional allele specific amplification (Bi-ASA) technique was applied to investigate the distribution frequency of 4 SNPs loci of TAP2 gene in northern Han population of Liaoning, and applied to paternity testing cases.
3, the allele frequency and genotype frequency direct counting method to calculate the TAP2 gene of 4 SNPs loci; Hardy-Weinberg balance test and linkage disequilibrium analysis; application SPSS11.5 statistical software for x~2 test and Fisher exact probability method to whether differences in allele frequency distribution between groups compared different significantly; calculation of heterozygosity (H), polymorphism information content (PIC), personal identification rate (DP) and the probability of exclusion (EPP).
Result
1, in Liaoning Han population, two alleles of C and T were detected at 651 loci of TAP2 gene. The allele frequency of C was 0.9071, the frequency of T allele was 0.0929, and two alleles constituted three genotypes of C/C, C/T and T/T, and the frequencies were 0.8086,0.1770 and 0.0044. respectively.
665 loci detected two alleles of A and G, the frequency of A allele was 0.6593, the allele frequency of G was 0.3407, two alleles constituted three genotypes of A/A, A/G and G/G, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
687 loci detected two alleles of T and C, the frequency of T allele was 0.6593, the allele frequency of C was 0.3407, two alleles constituted three genotypes of T/T, T/C and C/C, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
697 loci detected two alleles of G and T, the frequency of G allele was 0.6593, the allele frequency of T was 0.3407, two alleles constituted three genotypes of G/G, G/T and T/T, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
2, the linkage disequilibrium analysis showed that 651 and 665687 respectively and 697 loci in linkage equilibrium; between 665687 and 697 of the three loci in linkage disequilibrium. The detection of these 4 loci of 4 haplotypes: C-A-T-G type, C-G-C-T type, T-A-T-G type and T-G-C-T type, frequency respectively. 0.6018,0.3053,0.0575 and 0.0354.
3, a double SNPs loci composite amplification typing technology was established, and the synchronous genotyping of 665687 or 697 three loci with any two loci was studied.
4, in the 18 2 generation and 3 families, 2 families were denied the biological father son relationship at 665687 and 697 sites; 18 families did not deny biological father son relationship at 651 sites.
discuss
The research and application of bidirectional allele specific amplification technology to detect the ZAP2 gene of 4 SNPs loci in Liaoning Han population polymorphism distribution. Adding two pairs of primers in a single tube PCR amplification, one of the common primers containing SNP sites for amplification of fragments; another two for allele specific primers were formed with a corresponding common primers, amplification primers. The 3 'end of the corresponding two specific primers for SNP point mutation, by primer extension reaction control primer 3' end, according to the number and length of the amplified fragment to determine the genotype SNP loci. By adding a section consisting of 10 G or C "GC tail" in the 5 'end of the two allele specific primers, increase specific extension reaction.
Based on single SNP locus typing, this study also discusses one of the two SNPs loci multiplex PCR method. In a PCR reaction system, adding two allele specific primers and a pair of common primers according to the amplified fragment appeared in electrophoresis in the number and position of different of the two SNPs loci were simultaneous genotyping. This method improves the detection efficiency of SNPs analysis, which laid the foundation for the subsequent simultaneous detection of three or more SNPs sites.
The distribution of TAP2 gene in Liaoning Han population and 651665 alleles in 687 loci survey results and other population studies reported data were compared, found in 651 loci in Liaoning Han population and Zimbabwe population have significant difference (P < 0.05); in 665 sites, and the Han nationality in Taiwan, Zimbabwe, the Caucasus and Holland people have significant differences; in 687 sites, and Uygur, Zimbabwe and Spain, the crowd has significant difference. The TAP2 697 gene frequency distribution of the data has not been reported, so the comparison of gene distribution between different groups.
In 4 SNPs loci, 651 loci H, PIC, DP and EPP were 0.1687,0.1450,0.3149 and 0.0772 respectively; 665/687/697 loci H, PIC, DP and EPP were 0.416,0.3484,0.6088 and 0.1744. in the 18 generation 2 3 families detected in families with 2 excluding paternity at 665/687/697 loci; 18 A case in 651 sites allthe18caseswerenotexcludedfromthepaternity.
conclusion
1, the SNPs double site multiplex amplification technology established in this study can genotyping two SNPs loci simultaneously, which has the characteristics of simple, rapid, and good specificity.
2, the 651 and 665/687/697 loci of TAP2 gene have genetic polymorphism in Liaoning Han population, and Hardy-Weinberg balance.651665687 loci have genetic differences among different populations.
The 3651 locus is a genetic marker with moderate discriminating ability. The 665/687/697 locus is a genetic marker with high discriminating ability. It has practical application value in forensic paternity testing and personal identification.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R394;D919

【共引文献】