Mutational spectra at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in T-lymphocytes of nonsmoking and smoking lung cancer patients

Molecular analysis of mutations at the hypoxanthine-guanine phosphoribosylt ransferase (HPRT) locus in peripheral blood T-lymphocytes can provide infor mation on mechanisms of somatic in vivo mutation in populations exposed to exogenous carcinogens and in individuals with inherent susceptibility to ca ncer and other diseases. To study possible mutational changes associated wi th smoking as a risk factor for lung cancer, we analyzed HPRT mutations in T-cells of newly diagnosed, nonsmoking and smoking lung cancer patients bef ore treatment. Reverse transcriptase polymerase chain reaction (RT-PCR) and DNA sequencing methods were used to identify 146 independent mutations, 73 each from 32 nonsmoking and 31 smoking cases. In 35 T-cell mutants, the HP RT cDNA showed loss of an entire exon, indicating a splicing mutation. Amon g the remaining 111 fully characterized mutations in the coding region, sin gle base pair (bp) substitutions DI predominated with 79% (48/61) in nonsmo kers and 90% (45/50) in smokers. Frameshift and small deletion (1-24 bp) mu tations were found in 18 mutants. The distribution of base pair substitutio ns was nonrandom, with significant clustering at previously identified hots pot positions 143, 197 and 617 in the HPRT coding sequence (P less than or equal to 0.008). One additional hotspot, GC --> TA at position 606, was obs erved only in smokers (P = 0.006), The frequency of GC > TA transversions w as higher in smokers (13%) than in nonsmokers (6%). Conversely, smokers had a lower frequency of GC > AT transitions (24%) than nonsmokers (35%). This smoking-associated shift of the: HPRT mutational spectrum, although not st atistically significant, is consistent with the in vitro mutagenicity of be nzo(a)pyrene (BaP), a prominent carcinogen of tobacco smoke, and with known differences in the TP53 mutational spectrum in lung turners of smokers and nonsmokers. Among nonsmokers, the HPRT mutational spectra in healthy popul ation controls and lung cancer patients were similar, but there was a margi nally significant difference (P = 0.07) in the distribution of base pair su bstitutions between smoking controls and patients. These results suggest th at (i) general mechanisms of somatic mutagenesis in individuals with possib le predisposition to cancer (e.g. nonsmoking lung cancer patients) are not different from those in normal healthy individuals, and (ii) the HPRT gene in T-cells is a useful reporter locus for smoking-associated somatic in viv o mutations occurring early in lung cancer development. (C) 2000 Elsevier S cience B.V. All rights reserved.