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 cells.

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 O 6 -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 O 6 -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 −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 .