河北汉族人群线粒体DNA遗传多态性研究及其法医学应用

发布时间：2018-08-03 18:26

【摘要】： 目的：线粒体DNA（mitochondrial DNA,mtDNA）因具有拷贝数多、母系遗传、进化速度快等特点而被应用于法医学个人识别和母系亲子鉴定中，尤其对于微量、降解、无核生物检材提供了一种有效的检测途径。mtDNA由编码区和非编码区构成。非编码区也叫控制区（control region,CR）或D-loop(displacement loop)区，包括三个高变区：高变区1（hypervariable region,HV1）、高变区2（hypervariable region ,HV2）和高变区3（hypervariable region ,HV3）。其序列多态性区域主要集中在HV 1和HV 2，对世界上很多人群的调查结果显示mtDNA HV1、HV2区具有较高的遗传多态性，而编码区具有高度保守性。以往认为在健康人群中此区域没有基因突变，但近来国外有报道编码区8389-8865nt和Cyb基因在人群中也具有遗传多态性。目前国内已有对中国人群的此类报道，但尚未见编码区人群多态性的报道，更未见对河北汉族人群mtDNA多态性的研究。本研究选取了mtDNA HV1、HV2重叠片段和编码区8430-8673nt对100－175名河北汉族无关个体进行遗传多态性调查，同时对10个2代已确定父母子血缘关系的家系进行分析。毛干是现场常见的生物检材，只含有mtDNA，我们对20例毛干进行HV3 WP=4 区（CA）n重复子长度多态性分析。通过以上研究，首次获得河北汉族人群mtDNA的遗传学数据，为mtDNA数据库的建立及法医学应用奠定了基础。方法：采用盐析法提取新鲜血及腐败血样DNA，分别对mtDNA HV1、HV2重叠片段进行PCR扩增，6%非变性聚丙烯酰胺凝胶垂直电泳进行SSCP分型，硝酸银染色。对毛干用Chelex法提取DNA，PCR特异扩增，8%非变性聚丙烯酰胺凝胶垂直电泳分型，硝酸银染色。对样品逐一分型后，观察基因型分布，计算各基因型频率，选取SSCP带型比较接近，目测判型困难的基因型样本测序。用excell进行统计学处理，计算遗传变异度和人群偶合概率。将各基因型频率分布与国内外其他人群进行比较。测序结果用NCBI BLAST与Anderson序列比对，各序列之间用DNASTAR软件比对。结果：在159名河北汉族无关个体中，HV1A共检出39个基因型，基因频率在0.006289-0.09433之间，偶合率P为0.0381，遗传变异度H为0.9680。在104名河北汉族无关个体中，HV1B共检出25个基因型，基因频率在0.009615-0.1442之间，偶合率P为0.0793，遗传变异度H为0.9296。在100名河北汉族无关个体中，HV2A共检出22个基因型，基因频率在0.01-0.18之间，偶合率P为0.0914，遗传变异度H为0.9178。在101名河北汉族无关个体中，，HV2B共检出19个基因型，基因频率在0.009901-0.1881之间，偶合率P为0.0873，遗传变异度H为0.9218。在175名河北汉族无关个体中，编码 WP=5 区8430-8673nt共检出29个基因型，基因频率在0.0057-0.1657之间，偶合率P为0.0936，遗传变异度H为0.9116。在河北汉族人群100名健康无关个体中线粒体DNA 5个片段共分出91种单倍型。联合HV1、HV2和编码区8430－8673基因片段遗传变异度H为0.9977,偶合概率为0.013297。以上5个位点所选基因型样本经测序证实各序列之间均有差异。HV1、HV2区各序列与Anderson序列比较，共检测出65个变异位点，44个与MITOMAP收录的基因突变相同。其中16223t，16362C，73g，263g, 8563g,8584a等与以往报道的中国汉族其他地区（北京、山西、哈尔滨、广州、台湾）的结果相符。在98、197、204、316、16168、16184、16188、16195、16266等位置发现的基因突变与MITOMAP所收录不同。87、253、317、389、411、16013、16061、16370、8410、8459、8604、8650等位置在MITOMAP中未见收录。应用MITOMAP Mitoanalyzer Tool (NIST) 分析这些突变位置，编码区除8410c-t、8604t-c为同义突变，其它均为错义突变。以上5个位点通过10个家系2代人30名个体的分析发现孩子的基因型与母亲基因型相同，而与父亲的不同，表明mtDNA是通过母亲传递给后代，为母系遗传。在人群及家系调查中均未发现异质现象。HV3区（CA）n重复子调查结果：对20名河北汉族无关个体提供的毛发进行分析，共发现3个基因型：4、5、6，重复次数在4-6次之间，片段大小为88-92bp，基因型频率分别为0.3、0.25、0.45，遗传变异度H为0.678。用Fisher WP=6 确切概率法将河北汉族人与其他人群比较，发现基因型频率分布与中国汉族及其他种族间存在显著性差异。结论：HV1、HV2区重叠片段包含mtDNA D-loop50%以上的点突变，且相同的多态位点在两个重叠片段中分别位于不同的位置，通过SSCP分析得到两种不同的带型，提高了SSCP检测的灵敏度。HV1、HV2区及编码区8430－8673区段片段各长250bp左右，适合SSCP方法检测。尽管有文献报道PCR-SSCP方法可检出90%以上的点突变，但由于SSCP方法电泳影响因素很多（如电压、凝胶浓度、温度、缓冲液等），与序列测定相比，SSCP方法检出的基因型类型很少，这说明SSCP会漏检一些突变，但测序方法费时、费用昂贵且对实验室条件有一定要求，SSCP技术操作相对简单、快速、成本低、容易掌握，适于大样本筛查，所以PCR-SSCP技术可在基层法医检案中应用，作为一种初步筛选方法，以节约时间，降低消耗。本研究所采用的实验方法对含量约0.5-1ng/μl的DNA、降解DNA和毛干DNA进
[Abstract]:Objective: the mitochondrial DNA (mitochondrial DNA (mtDNA)) has been applied to forensic personal identification and maternal parent-child identification because of its multiple copy number, maternal inheritance and rapid evolution, especially for micro, degradation, and non nuclear biomaterials..mtDNA is composed of the coded region and non coding region. The non coding region is a non coding region. Also called the control area (control region, CR) or D-loop (displacement loop) area, including three high variable zones: high variable zone 1 (hypervariable region, HV1), high variable region 2 (hypervariable region, HV2) and high variable region 3. MtDNA HV1, HV2 region has high genetic polymorphism, and the coding region is highly conserved. It was considered that there was no genetic mutation in this area in healthy people, but recently there have been reports of genetic polymorphism of 8389-8865nt and Cyb genes in the coded regions abroad. The mtDNA polymorphism of the Hebei Han population was not studied. The genetic polymorphism of 100-175 unrelated Hebei Han individuals was investigated by mtDNA HV1, HV2 overlapping fragments and coding region 8430-8673nt. The common biological samples at the scene contain only mtDNA. We performed HV3 on 20 cases of hair shaft.
WP=4
CA n repeats polymorphism analysis. Through the above study, the genetic data of mtDNA in Hebei Han population was obtained for the first time, which laid the foundation for the establishment of mtDNA database and the application of forensic medicine.
Methods: fresh blood and DNA were extracted by salting out. MtDNA HV1, HV2 overlapped fragments were amplified by PCR respectively. 6% non denatured polyacrylamide gel electrophoresis was used for SSCP typing and silver nitrate staining. DNA was extracted by Chelex method, PCR specific amplification, 8% non denatured polyacrylamide gel electrophoresis and silver nitrate staining. After the sample was divided, the genotype distribution was observed, the genotype frequency was calculated, and the SSCP band type was selected to be close, and the genotype samples were sequenced. The genetic variation and the population coupling probability were calculated by excell. The genetic frequency distribution was compared with the other population at home and abroad. The sequencing results were NC BI BLAST and Anderson sequence alignment, each sequence was compared with DNASTAR software.
Results: among 159 unrelated individuals of Hebei Han, 39 genotypes were detected in HV1A, the frequency of gene frequency was between 0.006289-0.09433, the coincidence rate P was 0.0381, the genetic variability H was 0.9680. in 104 unrelated individuals of the Han nationality, and 25 genotypes were detected by HV1B. The gene frequency was between 0.009615-0.1442, the coincidence rate P was 0.0793, and the genetic variation H was H Among 100 unrelated individuals of Hebei Han, HV2A detected 22 genotypes with a genetic frequency of 0.01-0.18, the coincidence rate of P was 0.0914, the genetic variability H was 0.9178. in 101 unrelated individuals in the Han nationality, and 19 genotypes were detected by HV2B, the frequency of the gene frequency was 0.009901-0.1881, the coincidence rate P was 0.0873, and the genetic variability H was 0.9218.. In 175 unrelated individuals from Hebei Han, the code is coded.
WP=5
A total of 29 genotypes were detected in area 8430-8673nt, the frequency of gene frequency was between 0.0057-0.1657, the coincidence rate P was 0.0936, and the genetic variability H was 0.9116. in 100 unrelated individuals of the Hebei Han population, the mitochondrial DNA 5 fragments were divided into 91 haplotypes. The genetic variability of the combined HV1, HV2 and coding region 8430-8673 was 0.9977. The genotype samples of the 5 loci above 0.013297. showed that there were.HV1 differences between the sequences, and the sequence of HV2 region was compared with the Anderson sequence, and 65 mutation sites were detected, 44 were the same as those included in MITOMAP. Among them, 16223t, 16362C, 73g, 263G, 8563g, 8584a, etc. The results of Beijing, Shanxi, Harbin, Guangzhou and Taiwan were consistent. The gene mutations found in 981972043161616816184161881619516266 and other locations, such as the different.872533173894111601316061163708410845986048650 included in MITOMAP, were not included in the MITOMAP. MITOMAP Mitoanalyzer Tool (NIST) was used to analyze these The mutation location, the coding region is 8410c-t, 8604t-c is synonymous mutation, and the others are missense mutations. The above 5 loci through the analysis of 10 families and 2 generations of 30 individuals found that the child's genotypes are the same as the mother genotypes, but different from the father, it shows that mtDNA is passed through the mother to the offspring and is a maternal inheritance. In the population and family survey The results of the.HV3 region (CA) n repeats were not found. The hair of 20 independent Hebei Han individuals was analyzed, and 3 genotypes were found: 4,5,6, the number of repetitions was 4-6 times, the size of the fragment was 88-92bp, the genotype frequency was 0.3,0.25,0.45, and the genetic variability H was 0.678. using Fisher.
WP=6
The exact probability method was used to compare the genotype frequencies of the Han people in Hebei Province with those of other ethnic groups.
Conclusion: the overlap fragment of HV1, HV2 region contains point mutations above mtDNA D-loop50%, and the same polymorphic loci are located at different positions in two overlapped segments, and two different bands are obtained by SSCP analysis. The sensitivity.HV1 of SSCP detection is improved, HV2 area and 8430-8673 segment segments of the coding region are long 250bp, suitable for SSCP. Method detection. Although there are reports that PCR-SSCP method can detect more than 90% point mutations, but because of many factors affecting SSCP electrophoresis (such as voltage, gel concentration, temperature, buffer and so on), the SSCP method has few genotypes compared with sequence determination, which indicates that SSCP will leak some mutations, but the sequencing method is time-consuming and expensive and expensive. SSCP technology is relatively simple, fast, low cost, easy to master and suitable for large sample screening. So PCR-SSCP technology can be used in the primary forensic examination case, as a preliminary screening method, to save time and reduce consumption.
The experimental method used in this study is to degrade DNA and DNA into 0.5-1ng/ with DNA content of about L.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2003
【分类号】：D919

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