中国北方汉族群体HLA-DRA基因座3个SNPs位点遗传多态性及法医学应用研究

发布时间：2018-05-13 09:25

本文选题：单核苷酸多态性(SNPs) + PCR-序列特异性引物(PCR-SSP)　；参考：《中国医科大学》2004年硕士论文

【摘要】： 前言人类白细胞抗原(human leukocyte antigen，HLA)编码基因位于人类第6号染色体短臂(6p21)上。1958年，由Dausset确立了第一个人类白细胞抗原Mac，它标志着HLA研究的开始。HLA系统之所以引起法医学的重视在于它的高度多态性。HLA每个基因座上均有众多的复等位基因，而且共显性遗传，各个基因座的基因随机组合，形成HLA血型系统高度的遗传多态性。由于HLA多态性是由单个碱基差异造成的序列多态性(即单核苷酸多态性)，各个位点碱基组合又是随机的，故人群中HLA基因型可达10~8种之多，这为法医学个人识别和亲子鉴定提供了理想的遗传标记。所谓单核苷酸多态性(single nucleotide polymorphisms，SNPs)是指基因组内特定核苷酸位置上存在着两种不同的碱基，其中最少的一种在群体中的频率不少于1％。而HLA上SNPs则要高的多。据估测，人类整个基因组内核苷酸的多态性分布频率为0.08％-0.2％，而位于HLA-Ⅰ类基因的核苷酸多态性分布频率竟高达8.6％。所以HLA基因单核苷酸多态性的研究成为目前法医学研究新的热点。多个SNPs位点的同时检测，即SNPs位点的复合扩增，是PCR技术上的一大进步，一次复合扩增同时获得多个SNPs位点的信息，提高了个人识别能力，对微量检材鉴定特别有价值，尤其适用于法医学检案。IMS-JST011107、IMS-JST011109、IMS-JST011110是位于JSNP数据库内的HLA-DRA基因座上的3个SNPs位点。HLA-DRA基因是HLA-Ⅱ类抗原重链基因之一，有3个等位基因，由4个外显子组成。这三个SNPs位点位于第三和第四外显子之间。2000年日本学者利用基因组扫描结合序列测定的方法，首次获得了HLA-DRA基因座3个SNPs位点的等位基因频率分布数据。国内尚无有关这三个SNPs位点的群体遗传学数据。本研究利用PCR-SSP技术复合扩增HLA-DRA基因座的3个SNPs位点，调查了它们在中国北方汉族群体中等位基因分布频率，，为它们在法医学个人识别和亲子鉴定中的实际应用提供了必需的基础数据，并探讨在法医学领域应用的价值。材料与方法本研究应用PCR一SSP技术复合扩增HLA一DRA基因座上3个SNPs位点IMS一JSIDlllO7、IMS一JS功11109和IMS一JS，ID 11110，以聚丙烯酞胺凝胶电泳分离扩增产物结合银染显带的方法，对170例无血缘关系的中国北方汉族个体进行多态性分析。摸索IMS一JS功1一107、IMs一Js功一1 209和IMs- Js功11110位点的复合扩增的最适条件，并在亲子鉴定案例中应用。结果在退火温度为58‘’C，两个PCR复合扩增体系在xMS一JS功2 1 1 07、xMS- JS功川09和IMS一JS功1 1 1 10位点引物浓度比分别为0.4/0.6/1 .0(林mol/ L)和0.8/0.8/0.8(林m0FL)时，复合扩增产物的电泳谱型清晰，达到了基因分型的目的。 IMS一JS功1 1 107位点在170例中国北方汉族个体共检出由两个等位基因构成的三种基因型:C/T、C/C、T/T.其中T/T为巧例，频率为0 .0882;C/ T为73例，频率为0.4249;C/C为82例，频率为0.4823;等位基因C频率为 O‘6971，等位基因T频率为0.3029。IMS一JS功1 1 109位点检出由两个等位基因构成的三种基因型:C/T、C/C、T/T.其中T/T为12例，频率为0.0706; C/T为84例，频率为0.4941;C/C为74例，频率为0.4353;等位基因C频率为0.6823，等位基因T频率为0.3176。IMS一JS功11110位点检出由两个等位基因的构成三种基因型:C/A、A/A、C/C。其中灯A为n例，频率为 0.0647;C/A为83例，频率为0.4882。C/C为76例，频率为0.4470:等位基因C频率为0.6912，等位基因A频率为0.3088(表3)。此外，由于本研究采用单侧的引物，3个SNPs位点均未检测到新的变异。复合扩增3个SNPs位点检测5例亲子鉴定案例，有4例双亲均可以提供给子相应的等位基因，1例父亲不能提供给子相应的等位基因。案例1 在IMS一JS，ID 1 1 107位点父、子、母的基因型分别为C/C、T/T、T/T。父不能提供给子等位基因T。其中案例1、2的复合扩增图谱如(图8)所示，案例1- 5的基因型检测结果见(表4)。讨论本研究成功地摸索了IMS一JS，ID 1 1 107、IMS一JS，ID 1 1 109和IMS一JS功1 1 1 10 三个位点复合扩增的条件，且效果良好，提高了个人识别能力，具有快速、鉴别率高、节省检材等优点，可以用于个人识别和亲子鉴定案例。 HLA一DRA基因座3个SNPs位点在中国北方汉族群体中的DP值分别为0.5674、0.5664、0.5577;EPP值分别为0.1666、0.1704、0.1679;PIC值分别为0.4223、0.4335、0.4269;H值分别为0.4294、0.4941、0.4882。CDP为 0.9165，eEPP为0.4247，累积Pxe为0.8124(表5)。这三个位点都是具有高鉴别能力的遗传标记系统，在法医学个人识别和亲子鉴定中具有很高的应用价值。本研究获得的中国北方汉族群体IMS一JsTDI 1 107、IMS一JS功1 1109和 IMS一JS功11110三个SNPS位点的基因频率分布结果与国外报道的日本人群的频率分布进行x，检验，发现两群体之间无显著性差异(P0.05)(表 6)。这说明了IMS一JS功1 1 107、IMS一JS功11109和IMS一JS功11110位点的等位基因在中国汉族和日本人群中分布具有同源性，这一现象可以用来解释中日两群体具有共
[Abstract]:Preface
The human leukocyte antigen (human leukocyte antigen, HLA) coding gene is located on the human chromosome sixth on the short arm (6p21).1958, and the first human leukocyte antigen Mac is established by Dausset, which marks the beginning of the HLA study of the.HLA system that causes forensic medicine to focus on its high polymorphism.HLA on each of the loci. Multiple alleles, and co dominant inheritance, and a random combination of genes in each loci, form a genetic polymorphism of the HLA blood type system. Because HLA polymorphism is a sequence polymorphism (single nucleotide polymorphism) caused by a single base difference (i.e., single nucleotide polymorphisms), and the combination of each site base is random, so the HLA genotype can reach 10~8 species in the population. It provides an ideal genetic marker for forensic personal identification and parent-child identification. Single nucleotide polymorphisms (SNPs) refers to the existence of two different bases on the specific nucleotide sites in the genome, the least of which is less than 1%. in the group and higher in SNPs on HLA. It is estimated that the polymorphism of nucleotide polymorphisms in the whole human genome is 0.08%-0.2%, and the frequency of nucleotide polymorphisms in the HLA- I gene is up to 8.6%., so the study of the single nucleotide polymorphism of the HLA gene has become a new hot spot in forensic research.
The simultaneous detection of multiple SNPs loci, that is, multiplex amplification of the SNPs site, is a major advance in PCR technology. A composite amplification is used to obtain information of multiple SNPs sites at the same time. It improves the individual recognition ability and is of particular value for the identification of trace materials, especially for the forensic case.IMS-JST011107, IMS-JST011109, and IMS-JST011110 is located in JSNP. The 3 SNPs loci.HLA-DRA gene in the HLA-DRA loci in the database is one of the HLA- class II antigen heavy chain genes, with 3 alleles and 4 exons. The three SNPs loci are located between the third and the fourth exon of the Japanese Japanese scholars for the first time using the method of genomic scanning and sequencing. The HLA-DRA gene was obtained for the first time. There is no data on the allele frequency distribution of the 3 SNPs loci. There is no population genetic data about these three SNPs loci. This study used PCR-SSP technology to amplify the 3 SNPs loci of the HLA-DRA loci and investigated the frequency of their secondary gene distribution in the Han population in northern China, for their personal identification and parent children in forensic medicine. The practical application of identification provides the necessary basic data and discusses the value of application in forensic medicine.
Materials and methods
In this study, PCR SSP technique was used to amplify 3 SNPs sites on HLA DRA locus.
Point IMS one JSIDlllO7, IMS one JS work 11109 and IMS one JS, ID 11110, polypropylene phthalamine gel gel
170 cases of unrelated northern Han Chinese were analyzed by the method of combining silver staining and banding.
The individuals were analyzed for polymorphism. IMS 1 JS 107 IMs Js Js and IMs-
The optimal conditions for the multiplex amplification of Js power 11110 loci were applied in paternity testing cases.
Result
At the annealing temperature of 58 '' C, the two PCR multiplex amplification system worked at xMS JS 21107, xMS-
JS, Chuan Chuan 09 and IMS one JS work 11110 site primer concentration ratios were 0.4/0.6/1.0 (Lin mol/)
The electrophoretic pattern of the amplified products was clear and reached the base when L and 0.8/0.8/0.8 (Lin m0FL).
For the purpose of classification.
IMS a JS work 11107 loci detected two alleles in 170 northern Han Chinese individuals.
Because of the three genotypes: C/T, C/C, T/T., T/T is a good example, with a frequency of 0.0882; C/
T was 73 cases, frequency was 0.4249, C/C was 82, frequency was 0.4823, allele C frequency was
O '6971, allele T frequency 0.3029.IMS, JS JS 11109 loci detected by two alleles.
There are three genotypes of genes: C/T, C/C, T/T., of which T/T is 12 and the frequency is 0.0706.
C/T was 84 cases, frequency 0.4941, C/C 74 cases, frequency 0.4353, allele C frequency.
The rate of allele T was 0.6823, and the frequency of 0.3176.IMS allele was 11110. The detection of 11110 loci was two.
Alleles consist of three genotypes: C/A, A/A, C/C., where the lamp A is n, and the frequency is
0.0647, C/A for 83 cases, frequency 0.4882.C/C for 76 cases, frequency of 0.4470: alleles.
The frequency of the gene C was 0.6912, and the allele A frequency was 0.3088 (Table 3).
Unilateral primers were used and no new mutations were detected in 3 SNPs loci.
5 cases of paternity testing were detected by multiplex amplification of 3 SNPs loci, and 4 parents were able to mention it.
The corresponding alleles were supplied to the offspring, and 1 fathers could not provide the corresponding alleles to the offspring. Case 1
At IMS JS, ID 11107 loci, the parent genotype is C/C, T/T, T/T. the father can not mention.
Supply sub allele T., in which case 1,2 composite amplification map, as shown in Figure 8, case 1-
The results of 5 genotypes were found (Table 4).
discuss
This study has successfully explored IMS one JS, ID 11107, IMS JS, ID 11109 and IMS JS 11110.
The three loci amplify the conditions, and the effect is good, which improves the ability of personal identification.
It can be used for personal identification and paternity testing cases.
The DP values of 3 SNPs loci of HLA DRA locus in northern Han population of China are respectively
The values are 0.5674,0.5664,0.5577; EPP values are 0.1666,0.1704,0.1679 and PIC respectively.
Don't be 0.4223,0.4335,0.4269; H value is 0.4294,0.4941,0.4882.CDP respectively.
0.9165, eEPP is 0.4247, and cumulative Pxe is 0.8124 (Table 5). These three loci are all
The highly discriminating genetic marker system is very high in forensic personal identification and paternity testing.
Applied value.
In this study, IMS JsTDI 1107, IMS JS and 11109 JS were obtained from the Han population in northern China.
The gene frequency distribution of IMS 11110 JS three SNPS loci results are compared with those reported in Japan.
There was no significant difference between the two groups in the frequency distribution of X (P0.05).
6). This shows that IMS one JS work 11107, IMS one JS work 11109 and IMS one JS 11110 loci.
The distribution of alleles in Chinese Han and Japanese populations is homologous. This phenomenon can be used to explain.
The two groups of China and Japan have the same

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2004
【分类号】：D919

【参考文献】