造血干细胞移植志愿捐献者罕见基因人类白细胞抗原C*08:99的测序分析

Sequencing analysis of a rare human leukocyte antigen, C*08:99, from a volunteer donor of hematopoietic stem cell transplantation

背景:随着测序技术被广泛应用,器官移植配型的高分辨确认工作逐渐深入开展,人类白细胞抗原新等位基因不断涌现,但由于发现较晚,基因频率还不能准确计算,相关报道甚少,这些基因常被忽视,有时单纯根据基因频率判断分型结果,易造成分型结果的误判。目的:检测与分析1例造血干细胞移植志愿捐献者携带的罕见等位基因人类白细胞抗原C*08:99。方法:采用快速DNA提取试剂盒从全血样本中提取基因组DNA,经人类白细胞抗原C基因商品化测序分型试剂盒扩增,纯化后的扩增产物作为模板由试剂盒配套的第2,3和4外显子常规检测区正反向测序引物及自行研制的非常规检测区第5外显子正反向、第6外显子正向和第7外显子反向测序,经乙醇/醋酸钠/EDTA纯化的测序反应产物于ABI Prism^(TM) 3730测序仪电泳检测,相关的Assign 3.6+分析软件予以人类白细胞抗原高分辨水平基因分型。结果与结论:(1)将电泳后的数据导入Assign-SBT 3.6+分析软件,得出的分型结果为C*07:04,08:99。(2)结果证实,临床移植配型人类白细胞抗原C基因分型增加第5,6,7外显子区域外的多态性检测可提高分型结果的准确性,对临床组织配型工作具有重要意义。

BACKGROUND： As the sequencing technology has been widely used and high-resolution confirmation of organ transplant matching has been gradually developed, new human leukocyte antigen（HLA） alleles are emerging. However, the gene frequency of some genes cannot be calculated accurately, and there are rare reports. These genes are often ignored, and it is easy to misjudge their genotypes only according to gene frequency. OBJECTIVE： To test and analyze a rare allele, HLA-C＊08：99, from a volunteer donor of hematopoietic stem cell transplantation. METHODS： Genomic DNA was extracted automatically from the blood sample by using quick DNA purified kit and amplified by HLA-C locus commercial sequence-based typing kit. The purified PCR product was utilized as the DNA template in the sequencing reaction, and six direct sequencing reactions of PCR product covering exons 2, 3 and 4 in both directions were performed using commercial kit. Four direct sequencing reactions of PCR product covering exon 5 in both directions, exon 6 in forward direction and exon 7 in reverse direction were performed using in-house BigD ye terminator cycle sequencing reaction kit. Sequencing reaction products purified by ethanol/sodium acetate/ ethylenediaminetetraacetic acid method were sequenced by ABI PrismT M3730 DNA Sequencer. RESULTS AND CONCLUSION： The allele assignment was analyzed with Assign-SBT 3.6＋ software, and the sample HLA-C typing result was C＊07：04, 08：99. Increasing the sequencing analysis at exons 5, 6 and 7 of HLA-C locus will help to make clear the ambiguous SBT result and improve the accuracy of HLA-C typing when it is necessary, which shows important significance in clinical tissue matching.