Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.