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The Mutational Spectrum of the HPRT Gene from Human T Cells in Vivo Shares a Significant Concordant Set of Hot Spots with MNNG-treated Human Cells¹

Division of Cardiology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [A. T-M.]; Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [A. T-M., J. G-G., W. G. T.]; Genome Therapeutics, Corp., Waltham, Massachusetts 02453 [L. L. L.]; and Division of Gastroenterology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [C. L. G.]

The preponderance of G:C to A:T transitions in inherited and somatic human mutations has led to the hypothesis that some of these mutations arise as a result of formation of O⁶-methylguanine in DNA. To test this hypothesis, the fine structure map of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)-induced mutations was determined in human lymphoblastoid cells in the human hypoxanthine-guanine-phosphoribosyltransferase (HPRT) gene and compared with HPRT mutations observed in somatic T lymphocytes from normal individuals. Human TK6 cells, which are methylguanine methyltransferase deficient (MGMT^-), were treated with the methylating agent MNNG to create a level of O⁶-methylguanine in cellular DNA equal to that found in normal human tissues. A total of 676 bp of the HPRT gene was scanned using constant denaturing capillary electrophoresis and high-fidelity PCR. MNNG induced 14 predominant hot spots, all which were G:C to A:T transitions. Thirteen of these 14 MNNG-induced hot spots were found among the in vivo set, and 10 of the MNNG-induced hot spots were among 75 putative in vivo hot spots (mutations observed two or more times in vivo). Using a hypergeometric test for concordance, the MNNG-induced hot spots were found to be a significant subset of the putative in vivo hot spots (P < 4 x 10^-7). The set of shared hot spots comprise some 18% of the HPRT in vivo hot spot spectrum and strongly suggest that MNNG-induced hot spots in vitro share a common mutational pathway with a significant subset of somatic mutations in vivo.

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