Fuscoe J C, Vira L K, Collard D D, Moore M M
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Environ Mol Mutagen. 1997;29(1):28-35. doi: 10.1002/(sici)1098-2280(1997)29:1<28::aid-em4>3.0.co;2-9.
V(D)J recombinase is normally involved in the highly regulated rearrangement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-receptor genes. Occasionally, this tightly controlled process acts on inappropriate places in the genome and results in deletions and translocations. Some of these illegitimate V(D)J recombinase-mediated events have been implicated in the genetic changes associated with several forms of leukemia and lymphoid malignancy. We have developed a sensitive, specific polymerase chain reaction (PCR)-based assay to quantify such events in the peripheral blood cells of humans. This assay detects a V(D)J recombinase-mediated deletion in the hprt gene, which codes for a housekeeping enzyme and is not implicated in cancer development. Alterations in this gene serve as a surrogate indicator for these illegitimate events, which may be occurring throughout the genome. The assay involves a hemi-nested PCR with two sets of primers. Multiple replicates of genomic DNA (each representing 4 x 10(5) cells) are amplified with specific primers under conditions in which a single copy will give a detectable PCR product. Poisson statistics are then used to estimate the deletion mutant frequency. The frequency of cells with the hprt deletion among 20 healthy young adults ranged from <1.3 x 10(-7) to 4.1 x 10(-7) and was compared with the frequency of t(14;18) previously determined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA sequences at the deletion junctions were determined and provided evidence for multiple independent mutations in some individuals. This assay may serve as a biomarker for the level of illegitimate V(D)J recombination occurring in peripheral blood cells of humans.
V(D)J重组酶通常参与免疫球蛋白和T细胞受体基因片段（分别在B细胞和T细胞中）的高度调控重排，以形成功能性抗体基因和T细胞受体基因。偶尔，这个严格控制的过程会作用于基因组中的不适当位置，导致缺失和易位。这些非法的V(D)J重组酶介导的事件中的一些与几种形式的白血病和淋巴恶性肿瘤相关的基因变化有关。我们开发了一种基于聚合酶链反应（PCR）的灵敏、特异的检测方法，用于定量人类外周血细胞中的此类事件。该检测方法检测hprt基因中V(D)J重组酶介导的缺失，hprt基因编码一种管家酶，与癌症发展无关。该基因的改变可作为这些可能在整个基因组中发生的非法事件的替代指标。该检测方法涉及使用两组引物的半巢式PCR。在单拷贝能产生可检测PCR产物的条件下，用特异性引物扩增基因组DNA的多个复制品（每个代表4×10⁵个细胞）。然后使用泊松统计来估计缺失突变频率。20名健康年轻成年人中hprt缺失细胞的频率范围为<1.3×10⁻⁷至4.1×10⁻⁷，并与之前在这些个体中确定的t(14;18)频率进行比较。这两种基因组重排测量频率之间未发现相关性。确定了缺失连接处的DNA序列，并为一些个体中的多个独立突变提供了证据。该检测方法可作为人类外周血细胞中非法V(D)J重组水平的生物标志物。
