Zeng Jian-qiang, Xu Yun-ping, Wang Da-ming, Zou Hong-yan, Deng Zhi-hui, Yang Bao-cheng
Shenzhen Blood Center, Shenzhen Key Laboratory of Histocompatibility and Immunogenetics, Shenzhen, Guangdong, 518035, PR China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2009 Oct;26(5):562-6. doi: 10.3760/cma.j.issn.1003-9406.2009.05.019.
To analyze the possible reason for HLA-C allele dropout in routine sequence-based typing (SBT) and improve the accuracy of HLA-C SBT test.
A total of 620 randomly selected samples from healthy voluntary blood donors in Shenzhen were typed at HLA-C locus by sequence-based typing using the AlleleSEQR HLA-C plus sequence-based typing kit. Samples with no full match result were subjected to cloning and haplotype sequencing of the full-length HLA-C gene. If no novel mutations were found, samples were then retyped, using our self-designed PCR primer pair and PCR conditions replacing the AlleleSEQR HLA-C PCR reagents in the PCR set-up procedure so as to analyze the potential reasons for causing abnormal SBT result.
In the 620 samples typed at HLA-C locus using the AlleleSEQR HLA-C SBT commercial kit, 5 samples with no full match result were identified. The closest genotype showed one nucleotide mismatch with many different allele groups at different nucleotide position. Based on the PCR-SBT nucleotide sequence, heterozygous nucleotides were determined only in exon 4, whereas the nucleotides in exon 2 and 3 were all homozygotes. The results showed that HLA-Cw*0706 allele dropout existed in all the 5 samples with abnormal SBT results initially identified by AlleleSEQR HLA-C SBT kit, no novel mutation was found.
The results indicate that the PCR primer pair incompatible with DNA template may result in allele dropout in HLA-C SBT test. Based on the characterization of HLA-C full-length, it is essential to develop HLA-C SBT kit suitable for Chinese population in the future.
分析常规序列分型（SBT）中HLA - C等位基因缺失的可能原因，提高HLA - C SBT检测的准确性。
从深圳健康无偿献血者中随机选取620份样本，使用AlleleSEQR HLA - C plus序列分型试剂盒通过序列分型对HLA - C位点进行分型。对无完全匹配结果的样本进行全长HLA - C基因的克隆和单倍型测序。若未发现新的突变，则在PCR设置过程中使用自行设计的PCR引物对和PCR条件替代AlleleSEQR HLA - C PCR试剂对样本进行重新分型，以分析导致SBT结果异常的潜在原因。
在使用AlleleSEQR HLA - C SBT商业试剂盒对620份样本进行HLA - C位点分型时，鉴定出5份无完全匹配结果的样本。最接近的基因型在不同核苷酸位置与许多不同等位基因组显示一个核苷酸错配。基于PCR - SBT核苷酸序列，仅在外显子4中确定了杂合核苷酸，而外显子2和3中的核苷酸均为纯合子。结果表明，最初由AlleleSEQR HLA - C SBT试剂盒鉴定出的5份SBT结果异常的样本中均存在HLA - Cw*0706等位基因缺失，未发现新的突变。
结果表明，与DNA模板不兼容的PCR引物对可能导致HLA - C SBT检测中的等位基因缺失。基于HLA - C全长的特征，未来开发适合中国人群的HLA - C SBT试剂盒至关重要。
