The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene 
have been repeatedly found in sectors of human tumors by direct DNA sequencing. 
The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral 
T lymphocytes by clonal expansion under selective conditions. To discover if 
these mutations also occur frequently in normal tissues from which tumors arise, 
we have developed and validated allele-specific mismatch amplification mutation 
assays (MAMA) for each mutation. Reconstruction experiments demonstrated 
linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type 
alleles. The cumulative distributions of all negative controls established 
robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed 
depending on the mutation. One hundred and seventy-seven micro-anatomical 
samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from 
nine non-smokers were assayed representing en toto the equivalent of 
approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically 
significant mutant copy numbers were found in 257 of 463 assays. Clusters of 
mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five 
point mutations were detected at mutant fractions of >10(-5) in two or more 
lungs, we infer that they are mutational hotspots generated in lung epithelial 
stem cells. As the cancer-associated mutations did not differ in cluster size 
distribution from the HPRT1 mutation, we infer that none of the mutations 
conferred a growth advantage to somatic heterozygous clusters or maintenance 
turnover units. Specific mutants appeared in very large copy numbers, 
1000-35,000, in 18/257 positive assays. Various hypotheses to account for the 
observed cluster size distributions are offered.