Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14:18) translocations as a measure of ill egitimate V(D)J recombination. We determined the baseline frequencies of th ese two mutations in mononuclear leukocyte DNA from the umbilical cord bloo d of newborns and from the peripheral blood of adults, In an initial group of 21 newborns, no t(14;18) translocations were detected (< 0.049 X 10(-7)) . The frequency of HPRT exons 2 + 3 deletions was 0.10 X 10(-7) per mononuc lear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%-70%) and previous results using the T-cell cloning assay (similar to 2-3 X 10(-7) per clonable T-cell), Phytohemagglutinin (PHA), a s used in the T-cell cloning assay, was examined for its effect on the freq uencies of these mutation events in mononuclear leukocytes from an addition al 11 newborns and from 12 adults. There was no significant effect of PI-W on t(14;18) translocations which were rare among the newborns (1 detected a mong 2.7 X 10(8) leukocytes analyzed), and which occurred at frequencies fr om < 1 X 10(-7) (undetected) to 1.6 X 10(-4) among the adults. The extremel y high frequencies of t(14;18)-bearing cells in three adults were due mainl y to in vivo expansion of two to six clones. However. PHA appeared to stimu late a modest (although not significant) increase in the frequency of HPRT exons 2 + 3 deletions in the leukocytes of the newborns, from 0.07 X 10(-7) to 0.23 X 10(-7). We show that both the direct PCR assay and the T-cell cl oning assay detect similar frequencies of HPRT exons 2 + 3 deletions when c alculations are normalized to blood volume, indicating that the apparent di screpancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effect s of environmental genotoxic agents on this clinically important recombinat ion mechanism. (C) 1999 Elsevier Science B.V. All rights reserved.