【摘要】 目的改进以测序为基础(sequencing based typing,SBT)的HLA-DRB1基因分型方法,从而降低其分型成本和工作量。方法根据序列比对结果将DRB1基因分为6个簇(cluster),并设计6对簇特异引物(cluster specific primer,CSP),依据其对应的等位基因簇在人群中的频率高低,利用巢式PCR和CSPs渐进式地扩增,PCR产物纯化后进行测序分型。我们将此方法命名为以频率为基础的CSP-SBT法(Frequency based-CSP-SBT,FB-CSP-SBT)。结果用FB-CSP-SBT法对224个中国南方汉族冠心病患者临床样本进行HLA-DRB1等位基因分型,总共鉴定出31个等位基因型,覆盖了所有的HLA-DRB1等位基因谱系。其中,4个在人群中频率最高的簇依次为DR52(47.99%,包含DRB1*03、DRB1*08、DRB1*11、DRB1*12、DRB1*13和DRB1*14等6个DRB1等位基因谱系)、DR15(19.20%,包括DRB1*15和DRB1*16)、DR79(18.53%,包括DRB1*07和DRB1*09)和DR04(12.72%),占整个人群的98%以上。观察到的杂合性(heterozygosity)为0.9107,与前人报道的HLA-DRB1基因位点的杂合性相似,基因型分布符合Hardy-Weinberg平衡(P=0.3517)。对于每个样本,利用此方法对DRB1基因进行测序分型比传统的SBT至少减少6～8个PCR反应,同时还减少了测序反应的数目。结论用FB-CSP-SBT法可以准确地对HLA-DRB1基因进行分型,比传统的SBT法减少了工作量并降低了分型成本。更多还原

【Abstract】 Genotyping for HLA-DRB1 is required for many disease association studies and organ transplantation.However,conventional sequencing based typing(SBT) for HLA-DRB1 genotyping is laborious and tedious,and there are also many limitations in other methods.In this study,we improved the sequencing by progressively amplifying the samples with cluster specific primers(CSPs).Nested polymerase chain reaction(PCR) was used to prepare the products for SBT,which contains 1 outer primer pair(universal primer) and 6 inner primer pairs(CSPs).The 6 CSPs were ranked according to the reported frequency of their target alleles in the populations.The target alleles were more frequent,the corresponding CSPs were used first.Only the samples failed to be genotyped for both alleles were amplified with the CSPs for the less frequent alleles.The method was validated in a clinical sample set of 224 individuals.Total of 31 alleles were identified,which covered all DRB1 allele lineages.However,more than 98% alleles were identified using the CSPs for the most 4 popular clusters: DR52(DR03,DR08,DR11,DR12,DR13,and DR14),DR15(DR15 and DR16),DR79(DR07 and DR09) and DR04.The alleles in the cluster DR01 and DR10 were less than 2%.The observed heterozygosity was 0.9107,which was in agreement with expected values(0.9209) according to the Hardy-Weinberg equilibrium(P = 0.3517),suggesting this method could type DRB1 with less missing alleles.Genotyping for a large panel showed that this method greatly reduce the workload and decrease the cost of SBT for HLA-DRB1,which is cost-efficient and reliable.更多还原