摘要目的 探讨HLA-C基因测序分型时等位基因漏检和丢失的原因,以提高HLA测序分型的成功率和准确性.方法 620份随机选择的深圳健康捐血者血样,采用AlleleSEQR HLA-C plus测序分型试剂盒进行检测,对无完全匹配、分型结果"异常"的标本,采用分子克隆和测序方法进行全长单倍体序列分析;对未检出新的碱基点突变的样本,进一步采用自行设计的PCR引物和AlleleSEQR试剂盒中的测序引物进行再测序,分析测序分型结果"异常"的原因.结果 620份经AlleleSEQR HLA-C测序分型的样本中,发现5例样本无完全匹配的基因型,与之最接近的多种等位基因型均存在单个碱基的不匹配;并且在第4外显子出现碱基杂合,但第2和第3外显子区域内无杂合碱基.经分子克隆和单倍体测序,以及采用自行设计的PCR引物和AlleleSEQR试剂盒中的测序引物再测序,证实了5例标本均存在Cw * 0706等位基因漏检和丢失现象,未发现新的碱基点突变.结论 HLA-C基因测序分型时,因PCR引物与模板DNA不匹配会导致等位基因的漏检和丢失.根据中国人群HLA-C分子全长序列特点,开发适合于中国人群的测序分型试剂十分必要.

abstractsObjective 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. Methods 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 t'or causing "abnormal" SBT result. Results 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. Conclusion 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.