QUANTIFICATION OF HPRT GENE DELETIONS MEDIATED BY ILLEGITIMATE V(D)J RECOMBINATION IN PERIPHERAL-BLOOD CELLS OF HUMANS

V(D)J recombinase is normally involved in the highly regulated rearran gement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-rec eptor genes. Occasionally, this tightly controlled process acts on ina ppropriate places in the genome and results in deletions and transloca tions. Some of these illegitimate V(D)J recombinase-mediated events ha ve been implicated in the genetic changes associated with several form s 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. Alte rations in this gene serve as a surrogate indicator for these illegiti mate events, which may be occurring throughout the genome. The essay i nvolves a hemi-nested PCR with two sets of primers. Multiple replicate s of genomic DNA (each representing 4 x 10(5) cells) are amplified wit h 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 de letion among 20 healthy young adults ranged from <1.3 x 10(-7) to 4.1 x 10 and was compared with the frequency of t(14; 18) previously deter mined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA se quences at the deletion junctions were determined and provided evidenc e for multiple independent mutations in some individuals. This assay m ay serve as a biomorker For the level of illegitimate V(D)J recombinat ion occurring in peripheral blood cells of humans. (C) 1997 Wiley-Liss , Inc.