测序分型中2例样本HLA-DQB1等位基因第2外显子PCR扩增丢失及其原因分析

Analysis for 2 samples with HLA-DQB1 allele dropout at exon 2 in sequence-based typing

目的探讨HLA-DQB1基因测序分型时等位基因漏检和丢失的原因，以进一步提高HLA测序分型的准确性。方法对2000份HLA高分辨组织配型血样，采用AlleleSEQRHLA-DQ脚测序分型试剂盒进行常规检测。对序列峰图“异常”及无完全匹配结果的样本，进一步采用PCR-rSSO流式磁珠法复检，同时采用自行设计的特异性引物进行PCR产物分子克隆和测序，进行单倍型序列分析。结果在2000份经AlleleSEQRHLA-DQ剧测序分型的样本中，共发现2例样本序列峰图“异常”。经PCR-rSSO流式磁珠法和自行设计的引物测序复检，确认其为杂合型样本。PCR产物分子克隆和单倍型序列分析证实，第1份样本为第2外显子PCR扩增丢失HLA-DQB1 ＊02：02等位基因序列。第2份样本为第2外显子PCR扩增丢失HLA-DQB1 ＊02：01：01等位基因序列，未发现新的碱基突变。结论HLA-DQB1基因测序分型时，因DQB1基因存在优势扩增现象，可导致HLA-DQB1第2外显子等位基因的漏检和丢失。上述发现将为HLA精确分型提供借鉴。

Objective To explore the reason for HLA-DQB1 allele dropout during routine sequence- based typing（SBT） in order to improve the accuracy of typing. Methods Two thousand samples derived from HLA high-resolution typing laboratory were typed for HLA-DQB1 locus using an AlleleSEQR HLA- DQB1 SBT kit. Non-conclusive results and “abnormal” sequencing samples were retyped using a LABType rSSO HD HLA-DQB1 kit and further analyzed with both sequence-specific primers and group-specific primers and sequenced for haplotype analysis. Results Among the 2000 samples, 2 samples with no conclusive result were identified. The heterozygosity was confirmed with both the LAB Type SSO HD HLA-DQB1 kit and PCR-SBT in house method. Subsequent HLA-DQB1 cloning and haplotype sequencing have elucidated that HLA-DQB1 ＊ 02：02 dropped out at exon 2 for the first sample and HLA-DQB1 ＊ 02： 01：01 dropped out at exon 2 for the second sample during PCR amplification. No novel nucleotide mutation was found. Conclusion Our results indicated that preferential amplification at exon 2 of DQB1 may result in allele dropout in exon 2 sequences during HLA-DQB1 SBT test. This may provide useful information for HLA genotyping.