摘要
背景:聚合酶链式反应-直接测序分型技术被誉为HLA分型的金标准,在临床移植配型和骨髓库供者的大规模HLA分型中逐渐被广泛采用,但该方法的最大缺点是存在较高比例的模棱两可分型结果,故亟待寻找并建立适合中华骨髓库大规模供者HLA-测序模棱两可分型结果的解决方案。目的:调查中国南方汉族人群HLA-A,B,DRB1基因测序分型中模棱两可结果的分布状况,评估其可能的解决方案。设计、时间及地点:观察测量实验,2007-08/2008-08在深圳市血液中心输血医学研究所免疫遗传重点实验室完成。对象:选择深圳骨髓库的汉族骨髓供者416名,供者民族和籍贯按照自述原则确定。方法:采用聚合酶链反应-直接测序分型方法对416名汉族供者的HLA-A,B,DRB1基因进行测序分型,分析3个基因座模棱两可分型结果的分布情况,并分别采用高分辨聚合酶链反应-序列特异性引物法和杂合性模棱两可引物分离法进行解决;采用直接计数法统计基因频率,计算真实等位基因的相对概率。主要观察指标:416名供者HLA-A,B,DRB1基因直接测序分型结果的分布。HLA-A,B,DRB1基因测序模棱两可分型结果的分类及分布。序列特异性引物法及杂合性模棱两可引物分离法对模棱两可分型结果的解决能力。结果:80.29%的标本其HLA-A,B,DRB1基因出现模棱两可结果。80%左右的HLA-A,B基因和不足40%的HLA-DRB1基因模棱两可分型结果可以采用杂合性模棱两可引物分离法解决,其他则需要高分辨聚合酶链反应-序列特异性引物法解决。根据等位基因频率计算553个模棱两可等位基因组合中,75%的真实等位基因组合的相对概率大于98%。结论:杂合性模棱两可引物分离法和高分辨聚合酶链反应-序列特异性引物法分别对位于HLA-A,B和HLA-DRB1基因检测区内、外的模棱两可分型结果具有较高的解决能力,二者互为补充;在大规模供者HLA分型中应用
BACKGROUND： Polymerase chain reaction-sequence based typing （PCR-SBT） as a gold standard of human leucocyte antigen （HLA） typing, is widely used in clinical transplantation typing and HLA typing of donors from Chinese Marrow Donor Program. However, the high proportion of ambiguity typing is the most disadvantage of the method. Therefore, it is urgent to find an ideal solution for HLA typing OBJECTIVE： To investigate the ambiguity distribution of HLA-A, B and DRB1 loci by SBT method in Han population in southern China and evaluate its possible solutions. DESIGN, TIME AND SETTING： An observational measurement was performed at Key Laboratory of Immunogenetics, Institute of Transfusion Medicine, Shenzhen Blood Center, from August 2007 to August 2008. PARTICIPANTS： 416 Han marrow donors from Chinese Marrow Donor Program in Shenzhen were enrolled in the experiment. The nationality and native place of the donors were determined by self telling. METHODS： HLA-A, B and DRB1 loci of all donors were genotyped by PCR-SBT, and then the ambiguity distribution of the three loci was analyzed. The typing ambiguities were resolved by high-resolution polymerase chain reaction- sequence-specific primers （PCR-SSPs） and beterozygous ambiguity resolution primers （HARPs） methods, respectively. The frequency of genotypes was calculated with the direct count method, and then, the relative probability of true alleles was calculated. MAIN OUTCOME MEASURES： Distribution ofHLA-A, B and DRB1 loci in 416 donors by SBT method. Ambiguity distribution and classification of HLA-A, B and DRB1 loci by SBT method. Ability of high-resolution PCR-SSPs and HARPs methods to resolve ambiguous results. RESULTS： The ambiguity rate of the HLA-A, B and DRB1 loci among the 416 samples were 80.29%. About 80% HLA-A, 80% HLA-B and 〈 40% HLA-DRBI ambiguities could be solved by HARPs method, and the other by high-resolution PCR-SSPs method. According to calculation of the allele frequency data, the relative probability of 75% true alle
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第B12期10481-10486,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
深圳市科技计划资助项目(200802117)“中华骨髓库大规模供者HLA基因的PCR-SBT分型模式”
广东省医学科研基金资助项目(A2008622)“骨髓库供者HLA-SBT模棱两可分型结果的解决方案”~~
