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

原子弹幸存者 T 淋巴细胞体内 hprt 突变分析。

基本信息

批准号：
05454610
负责人：
KATO Takeshi
金额：
$ 3.97万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454610/
关键词：
Atomic Bomb Survivors Somatic Mutations Peripheral Blood T Cells HPRT Mutations Mutation Spectrum HPRT突然変異遺伝的影響 HPRT‐突然変異多重PCR RT-PCR 変異塩基配列の同定

项目摘要

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.

Akiyama等人报道了原子弹幸存者外周血T细胞中次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（hprt）基因的6-硫鸟嘌呤抗性（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。然而，对原子弹幸存者的TG^r突变T细胞的DNA进行Southern blot分析，未能显示对照组和幸存者组之间存在显著差异。为了进一步研究突变事件，我们开发了一种方法，该方法可以通过多重聚合酶链反应（PCR）和cDNA的RT-PCR和测序相结合来确定hprt位点的突变序列改变。检测的独立TG^r突变T细胞克隆数量为41个，来自接受低于0.005 Gy辐射的18名对照组个体，50个来自接受超过1.5 Gy辐射的24名暴露组个体（平均剂量2.45 <正负> 0.85 Gy）。通过多重PCR检测到的含有hprt外显子的基因组DNA片段的缺失或移位，在两组的10-15%的克隆中发现了明显的结构改变，这表明它们之间没有显著差异。从两组中恢复的hprtcdna中改变的序列类型不同。两组中碱基替换（-45%）、缺失或插入（-25%）和外显子跳变（-20%）的比例相似，表明两组基因组DNA的总体结构改变和hprt cDNA的序列改变均无显著差异。这些结果证实了我们之前的观察，即在暴露后的几十年里，原子弹诱导的hprt突变T细胞大多已经从外周血中消除。