中国南方汉族人群6个Y-STR基因座 遗传多态性及法医学应用

为研究中国南方汉族人群DYS393等6个Y-STR基因座的遗传多态性并用于法医学鉴定,通过采用PCR复合扩增和基因测序仪荧光检测方法,检查204个无关男性个体,调查南方汉族的6个Y-STR基因座的单倍型频率,并对93对真父子和38对非父子的亲子鉴定样本进行检测。结果DYS393基因座检出5个等位基因,DYS19基因座检出6个等位基因,DYS389Ⅱ基因座检出8个等位基因,DYS390基因座检出6个等位基因,DYS391基因座检出4个等位基因,DYS385 基因座检出44个等位基因,共检出176种单倍型。93对真父子中,观察到2例分别有1个基因座突变。检测38对非父子,有1个或2个Y-STR基因座排除的案例各有1例（2.6%）;有3 个和3个以上的Y-STR基因座可以排除父子关系的案例为35例（92.1%）;6个Y-STR基因座不能排除父子关系的为1例。结果表明6个Y-STR基因座具有丰富的遗传多态性,可用于法医学个体识别和亲子鉴定。Abstract: To study the genetic polymorphisms of six Y-chromosome specific STR loci in the southern Chinese Han population and apply it in forensic science, six Y-STR loci were amplified by multiple PCR and the PCR products were detected by using ABI PrismTM 377 Sequencer. The haplotype frequencies at 6 Y-STR loci were determined in a total of 204 unrelated males from southern Han population of China. Ninety-three father/son pairs with demonstrated paternity and thirty-eight non-paternity father/son pairs were detected by using our Y-STR system. As a result, the number of alleles for DYS393、DYS19、DYS389Ⅱ、DYS390、DYS391and DYS385 were 5, 6, 8, 6, 4 and 44 , respectively. A total of 176 haplotypes at 6 Y-STR loci were found. Two father/son pairs with single Y-STR mutation were observed in the 93 father/son pairs with demonstrated paternity. Among the 38 non-paternity father/son pairs, one case with one Y-STR exclusion of paternity, one case with two Y-STR exclusions and 35 cases with 3 or more Y-STR exclusions were observed. Non-exclusion of paternity at 6 Y-STR loci was found only in one case. This result indicated that the six Y-STR loci were highly polymorphic and are suitable for personal identification and paternity testing.     

深圳无偿献血人群中李姓、王姓和张姓无关男性个体Y-STR单倍型遗传多态性

为研究姓氏群体Y染色体特异STR单倍型的遗传多态性, 采用PCR复合扩增和ABI PrismTM 3100基因测序仪荧光检测方法对DYS426等9个Y-STR基因座进行基因分型, 检测深圳地区李姓无关男性个体血样139份、王姓无关男性个体118份、张姓无关男性个体119份。结果在139份李姓群体中, 共检出126种单倍型, 其中118种单倍型仅出现1次, 检出频率最高的1种单倍型出现6次, 单倍型多样性为0.9974; 118份王姓无关男性样本中, 共检出105种单倍型, 其中94种单倍型仅出现1次, 检出频率最高的1种单倍型出现4次, 单倍型多样性为0.9953; 张姓无关男性样本中, 共检出101种单倍型, 其中88种单倍型仅出现1次, 检出频率最高的1种单倍型出现4次, 单倍型多样性为0.9964。结果表明: 深圳地区李、王、张姓氏无关男性个体Y-STR单倍型的遗传多态性丰富, 与以往的汉族无关男性群体遗传资料相比较, 差异不显著。     

中国汉族个体HLA-A、-B基因全长序列的测定及调控区多态性

文章利用20个中国汉族个体样本建立了稳定精确的HLA-A、-B基因全长序列的克隆测序方法, 获得HLA-A 10个等位基因4.2 kb序列, HLA-B 6个等位基因3.7 kb序列, 序列涵盖了两个基因的所有外显子、所有内含子、5′启动子区以及3′非翻译区(3′UTR)。A*1153是文章发现的一个新等位基因, B*151101的内含子序列、5个HLA-A以及2个HLA-B等位基因的5′启动子序列和3′UTR序列为国际上首次报道, 其他等位基因均延伸了IMGT/HLA数据库中释放的全长序列。文章首次在中国汉族个体中测定了IMGT/HLA数据库中没有覆盖的HLA-A、-B基因的上游5′启动子以及下游3′UTR区域的多态性模式。HLA-A基因5′启动子延伸区域共发现26个SNPs和一处3 bp(AAA/-)的插入/缺失, 3′UTR延伸区域共发现14个SNPs; HLA-B基因5′启动子延伸区域共发现5个SNPs和一处1 bp(T/-)的插入/缺失, 3′UTR延伸区域共发现8个SNPs。通过对两个基因的5′启动子、外显子以及3′UTR的系统发育树分析, 发现两个基因调控区与外显子的进化关系有所不同, HLA-A基因除A*24020101外, 其他等位基因两端调控区与外显子连锁比较紧密, HLA-B基因两端调控区与外显子之间则发生了较为频繁的重组事件。     

人类血小板抗原1～6系统同步基因分型的研究

为研究采用PCR—SSP技术,建立可靠的人类血小板抗原HPA-1,2,3,4,5,6系统的同步基因分型方法,并以所建立的方法研究血小板抗原。设计合成18条序列特异性引物,探索最佳退火温度,通过调整引物浓度、Mg2＋离子浓度,使HPA-1~6系统等位基因在同一条件下进行同步扩增和扩增产物在同一凝胶中进行同步电泳。引物的特异性和灵敏度采用基因型已知的质控DNA进行验证。应用此方法,对2000年度国际输血协会（ISBT）第十届血小板基因定型与血清学工作组送检的15份考核样本（其中血样2份,DNA样本13份）进行了基因分型。用此方法检测质控DNA,结果与已知的HPA基因型完全相符;15份第十届血小板基因定型与血清学工作组的考核样本的检测结果,与ISBT公布的结果完全相同,准确率达100%。Abstract: To set up the simultaneous genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR—SSP assay and use the genotyping method for the study of platelet antigens. In this study, 18 sequence-specific primers were designed and synthesized. The annealing temperature for all sequence-specific primer pair, the concentration of each primer pair and the concentration of Mg2+ were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The electrophoresis of PCR products was conducted simultaneously on the same agarose gel. Control DNA samples that genotypes known were used to confirm the sensitivity and specificity of each sequence-specific primer. 15 coded samples (including 2 blood samples and 13 DNA samples) distributed by 10TH Platelet Genotyping and Serology Workshop of the International Society of Blood Transfusion (ISBT) were typed for HPA-1 to 6 systems by this method. A concordance rate of 100 percent was observed between the results of control DNA samples typed by our PCR—SSP assay and the data of known specificity of control DNA. The results of 15 coded samples tested by our method agreed well with the results provided by ISBT report.     

汉族人群中22个HLA-Cw等位基因全长序列单核苷酸多态性分析

为探讨HLA-Cw (Human leukocyte antigen-Cw)基因全长序列分子遗传多态性, 文章对28个HLA-Cw基因型已知的汉族个体样本, 采用长距离PCR技术和高保真性的Pfu酶, 扩增HLA-Cw基因全长序列4.5 kb, 进行分子克隆和单倍体测序。采用群体遗传学研究方法分析了HLA-Cw等位基因全长序列中各亚区的单核苷酸多态性。结果表明: 在28个样本中共检测出22种等位基因, 序列均已提交GenBank和国际IMGT/HLA数据库并获得了认可; 其中Cw*0706、Cw*030301、Cw*140201的全长序列为首次报道, 尤其是Cw*0706内含子序列的获得, 能够重新设计对该等位基因测序分型的引物, 避免测序分型中可能对这一等位基因的漏检。将28个样本的56条单倍体序列用Clustal软件进行序列排比, 输入到DnaSP4.0进行多态性分析, 共发现244个SNPs, 10处插入/缺失多态性。对HLA-Cw等位基因各亚区多态性的分析, 发现第4内含子及以前并没有受到关注的第5外显子受到平衡选择的作用, 在进化中受到了选择压力, 预示着它们在免疫系统的进化过程中可能扮演着重要的角色。