Analysis of in vivo hprt mutations in T lymphocytes from atomic bomb survivors.

Akiyama等人，报道了原子弹爆炸幸存者外周血T细胞次黄嘌呤-鸟嘌呤磷酸核糖转移酶（hprt）基因6-硫代鸟嘌呤抗性（TG^r）突变频率升高，突变频率与辐射剂量之间存在轻微但显著的正相关。对原子弹爆炸幸存者的TG^r突变T细胞DNA进行Southern印迹分析，结果显示对照组和幸存者组之间没有显著差异。为了进一步研究突变事件，我们开发了一种方法，通过该方法，可以通过i）多重聚合酶链反应（PCR）和ii）cDNA的RT-PCR和测序的组合来确定hprt基因座处的突变序列改变。在对照组（18人，接受剂量低于0.005戈伊）和暴露组（24人，接受剂量超过1.5戈伊，平均剂量为2.45 ± 0.85戈伊）中，检测到41个独立的TG^r突变T细胞克隆<plus-minus>。通过多重PCR检测到的总体结构改变是基因组DNA中含有hprt外显子的片段的丢失或移位，在来自两组的10-15%的克隆中发现，因此表明它们之间没有显著差异。从两组中回收的HPRTcDNA中的改变的序列是各种类型的。在两组中发现了相似比例的碱基置换（~ 45%）、缺失或插入（~ 25%）和外显子跳跃（~ 20%），表明两组的基因组DNA中的总体结构改变和hprt cDNA中的序列改变均无显著差异。这些结果证实了我们先前的观察，即原子弹诱导的HPRT突变型T细胞在暴露后的几十年里大多已从外周血中消除。

Akiyama et al., repoted an elevated frequency of 6-thioguanine-resistant (TG^r) mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in T cells of peripheral blood from atomic bomb survivors and a slight, but significant, positive correlation between the frequency of mutation and radiation dose. Southern blot analysis of DNA from TG^r mutant T cells of atomic bomb survivors, gowever, failed to show a significant difference between the control and survivor groups. To investigate further the mutational events, we have developed a method by which mutational sequence alterations at the hprt locus can be determined by the combination of, i) the multiplex polymerase chain reaction (PCR) and ii) the RT-PCR of cDNA and sequencing. The numbers of independent TG^r mutant T cell clones examined were 41 from a control group of 18 individuals who had received less than 0.005 Gy, and 50 from an exposed group of 24 individuals who had received more than 1.5 Gy (mean dose, 2.45 <plus-minus> 0.85 Gy) . Gross structural alterations, which were detected by multiplex PCR as a loss of or shift in hprt exon-containing fragments of genomic DNA,were found in 10-15% of the clones from both groups, thus indicating there was no significant difference between them. The altered sequences in the HPRTcDNAs recovered from both groups were of various types. Similar proportions of base substitutions (-45%) , deletions or insertions (-25%) , and exon skipping (-20%) were found in both groups, indicating that neither the gross structural alterations in the genomic DNA nor sequence alterations in the hprt cDNA of both groups differ significantly. These results confirm our previous observation that A-bomb-induced HPRT-mutant T cells mostly have been eliminated from peripheral blood over the decades that have elapsed since exposure.