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背景:人类白细胞抗原基因测序分型中,当某些等位基因间的差异碱基位于测序范围外时,无法得到清晰的等位基因结果。目的:分析中国人群人类白细胞抗原A、B、DRB1基因测序分型中模棱两可等位基因的分布规律,探讨其在中华骨髓库大样本人类白细胞抗原A、B、DRB1基因的测序分型中的解决方案。设计、时间及地点:采用随机抽样方法对研究样本进行人类白细胞抗原A、B、DRB1基因的测序分型,并对其中的模棱两可等位基因进行验证性实验,于2006-01/2008-06在深圳市血液中心完成。材料:658名深圳骨髓库汉族供者乙二胺四乙酸盐抗凝血5mL。方法:采用聚合酶链式反应--测序分型方法对658名深圳骨髓库供者的人类白细胞抗原A、B和DRB1基因进行高分辨分型,并采用针对相应位点的高分辨聚合酶链式反应-序列特异性引物方法对其中由于碱基差异位于检测区外而产生的模棱两可等位基因进行鉴别。主要观察指标:模棱两可等位基因的分布及确认。结果:658份标本中,人类白细胞抗原A、B和DRB1三个基因座的模棱两可等位基因分别为9个(2种)、140个(5种)、406个(8种),占等位基因总数的14.06%(555/3948)。高分辨聚合酶链式反应-序列特异性引物鉴定结果显示,中国报道的DRB1*1401被全部确认为DRB1*1454;12例B*0705/B*0706中,有1例被确认为B*0706,其他13种等位基因的鉴定结果分别为A*6801、A*7402、B*2705、B*3501、B*4402、B*5801、DRB1*0101、DRB1*0406、DRB1*0803、DRB1*1101、DRB1*1201、DRB1*1302和DRB1*1502。结论:在中华骨髓库大样本人类白细胞抗原基因的测序分型中可以应用直接鉴别的方法区分模棱两可等位基因,但在临床移植前的高分辨确认试验中则需要采用实验方法进行精确分型。
Background: In human leukocyte antigen sequencing, no clear allele results are obtained when the difference bases between certain alleles are outside the sequencing range. OBJECTIVE: To analyze the distribution of ambiguous alleles in Chinese human leukocyte antigen A, B and DRB1 gene sequencing and to find their solutions in the sequencing typing of human leukocyte antigen A, B and DRB1 Program. DESIGN, TIME AND SETTING: The genotyping and sequencing of human leukocyte antigen A, B and DRB1 genes were carried out on a random sample of samples. The confirmatory experiment was carried out on the ambiguous alleles among them. Shenzhen Blood Center completed. Materials: 658 Shenzhen bone marrow donor Han ethylenediamine tetraacetate anticoagulant 5mL. Methods: The human leukocyte antigen A, B and DRB1 genes of 658 human bone marrow donors from Shenzhen were genotyped by polymerase chain reaction - sequencing and genotyping by high resolution polymerase chain reaction The reaction-sequence-specific primer method identifies ambiguous alleles where the base differences are outside the detection zone. MAIN OUTCOME MEASURES: Distribution and confirmation of ambiguous alleles. Results: Among the 658 specimens, there were 9 (2), 140 (5) and 406 (8) equivocal alleles at the three loci of human leukocyte antigen A, B and DRB1 14.06% (555/3948) of the total number of genes. The results of high-resolution polymerase chain reaction-sequence-specific primers showed that DRB1 * 1401 reported in China was all confirmed as DRB1 * 1454; 1 of 12 cases of B * 0705 / B * 0706 was identified as B * 0706 , And the other 13 alleles were A * 6801, A * 7402, B * 2705, B * 3501, B * 4402, B * 5801, DRB1 * 0101, DRB1 * 0406, DRB1 * 0803, DRB1 * 1101, DRB1 * 1201, DRB1 * 1302 and DRB1 * 1502. Conclusion: The method of direct discrimination can be used to distinguish ambiguous alleles in the sequencing of human leukocyte antigen gene of large sample of bone marrow in China. However, in the high-resolution confirmatory test before clinical transplantation, the accurate typing is required by experimental methods.