Ballinger Scott W, Judice Stephen A, Nicklas Janice A, Albertini Richard J, O'Neill J Patrick
University of Vermont, Genetics Laboratory, Burlington 05401, USA.
Environ Mol Mutagen. 2002;40(2):85-92. doi: 10.1002/em.10099.
V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase.
7号染色体p15处的T细胞受体(TCR)γ可变区(GV)基因与7号染色体q35处的TCRβ连接区(BJ)基因之间,由V(D)J重组酶介导的重组会导致形成一个杂交TCR基因。这些TCRγ/β基因座间重排发生在经典的V(D)J重组信号序列(RSS)处,并且由于这些基因座呈反向排列,会导致染色体结构中可检测到的倒位事件,即inv(7)(p15;q35)。在淋巴系统恶性肿瘤中也发现了由V(D)J重组酶介导的涉及癌基因以及TCR或免疫球蛋白基因的类似重排。已有文献描述了能够跨inv(7)基因组重组连接序列进行聚合酶链反应(PCR)扩增的寡核苷酸引物。Southern印迹分析主要用于确认产物的GV/BJ杂交性质,而关于这些重组的DNA序列的信息有限。我们对外周血样本中单核细胞的全基因组DNA进行了这种PCR方法的改良,以提高特异性,并对11名个体(3名成人、1名儿童、6名新生儿和1名共济失调毛细血管扩张症(AT)患者)的25个样本中TCR基因的GV1和BJ1家族之间重组产生的易位连接点进行直接测序。基于先前的研究,我们将重点放在新生儿样本上,先前的研究表明新生儿中主要的次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HPRT)突变是由V(D)J重组酶介导的缺失事件,并且这些突变的频率会随着年龄的增长而降低。尽管所使用的基于稀释系列的PCR检测方法并未产生明确定义的定量终点,但本研究结果强烈表明,新生儿中inv(7)重组的发生频率与成人相当或更低。与PCR引物对一致,所有测序产物均包含一个GV1和一个BJ1片段,并且大多数还包含一个BD1片段。GV1s2和1s4是最常发现的GV1基因(分别为8个和9个样本),BJ1s5和1s6是最常发现的BJ1基因(分别为9个和10个样本)。这些结果证明了该方法在评估由V(D)J重组酶介导的GV/BJ基因座间重排方面的有效性。