移植前白血病患者HLA区域杂合性缺失对HLA分型的影响

Effect of Heterozygosity Loss in HLA Region before Transplantation on HLA Typing in Patients with Leukemia

目的:研究移植前白血病患者初诊时期和缓解期HLA区域杂合性缺失(LOH)对HLA分型的影响。方法:用3种HLA基因分型常用的方法(SBT、SSO和SSP方法)分别对1例急性混合细胞白血病(MPAL)患者初诊静脉血样本1(骨髓原始细胞为81.7%)、样本2(骨髓原始细胞为93.07%)和缓解期静脉血样本3(骨髓原始细胞为<0.01%)进行HLA-A、B、C、DQB1、DRB1基因分型;通过2种DNA混合实验进一步确定SBT方法检测HLA杂合峰的检出阈值和检测HLA分型时对静脉血原始细胞比例的要求。结果:样本1和样本2的HLA-A、B、C、DQB1、DRB1均为纯合位点,其中样本1有些HLA位点的测序背景峰偏高,无法确定位点的具体结果;样本3的5个HLA座位除了C座位外均为杂合位点。DNA混合实验结果表明,用SBT方法检测HLA双等位基因杂合峰的最低检出限为20%;移植前白血病患者采用SBT方法检测HLA分型的要求是静脉血原始细胞<75%,若≥75%,则可能因为原始细胞HLA区域出现LOH,从而导致HLA位点漏检。结论:对于白血病初诊患者,特别是原始细胞≥75%,如果HLA某一位点分型结果为纯合位点,应该再次采集患者缓解期静脉血或者正常体细胞,如口腔黏膜细胞,重新对该位点进行复核,排除HLA区域发生LOH造成假性纯合的可能,以免HLA配型结果发生错误,影响移植供者的选择和最终的移植效果。

Objective： To investigate the effect of loss of heterozygosity（ LOH） in HLA region at initial diagnosis and remission of leukemia patient before transplantation on HLA typing. Methods： The HLA typing was performed in DNA extracted from peripheral blood obtained at diagnosis（ Sample 1 and Sample 2） and remission（ Sample 3） in one pretransplant male patient with mixedphenotype acute leukemia（ MPAL）. HLA typing for HLA-A,B,C,DQB1,DRB1 was performed by Sequence-based typing（ SBT）,Sequence-specific oligonucleotide probe hybridization（ SSO） and Sequence-specific primers（ SSP）. To define more precisely a cutoff limit for the detection of a heterozygous DNA present in a fraction of the cells by the SBT technology,DNA mixing experiments were performed. Results： SBT results showed that Sample 1 and Sample 2 were both homozygous HLA results at five loci（ lost one haplotype） although the sequencing background of Sample 1 was a little high. Except HLA-C locus was homozygous,Sample 3 was heterozygous HLA results at four loci. Based on DNA mixing experiments,a cutoff limit for the detection of heterozygous DNA was 20% by SBT technology,and a detection threshold for HLA-A,B,C,DQB1,DRB1 heterozygosity in blood samples was 〈75% blasts. Conclusion： Because LOH may be partial,any homozygous HLA result obtained during a blast crisis,especially ≥75% blasts,would have to be confirmed by a second typing on a buccal swab or on peripheral blood from the patient in complete remission.