Fetal–juvenile origins of point mutations in the adult human tracheal–bronchial epithelium: Absence of detectable effects of age, gender or smoking status

Abstract

Allele-specific mismatch amplification mutation assays (MAMA) of anatomically distinct sectors of the upper bronchial tracts of nine nonsmokers revealed many numerically dispersed clusters of the point mutations C742T, G746T, G747T of the TP53 gene, G35T of the KRAS gene and G508A of the HPRT1 gene. Assays of these five mutations in six smokers have yielded quantitatively similar results. One hundred and eighty four micro-anatomical sectors of 0.5–6 × 10⁶ tracheal–bronchial epithelial cells represented en toto the equivalent of approximately 1.7 human smokers’ bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers above the 95% upper confidence limits of historical background controls were found in 198 of 425 sector assays. No significant differences (P = 0.1) for negative sector fractions, mutant fractions, distributions of mutant cluster size or anatomical positions were observed for smoking status, gender or age (38–76 year). Based on the modal cluster size of mitochondrial point mutants, the size of the adult bronchial epithelial maintenance turnover unit was estimated to be about 32 cells. When data from all 15 lungs were combined the log 2 of nuclear mutant cluster size plotted against log 2 of the number of clusters of a given cluster size displayed a slope of ∼1.1 over a range of cluster sizes from ∼2⁶ to 2¹⁵ mutant copies. A parsimonious interpretation of these nuclear and previously reported data for lung epithelial mitochondrial point mutant clusters is that they arose from mutations in stem cells at a high but constant rate per stem cell doubling during at least ten stem cell doublings of the later fetal–juvenile period. The upper and lower decile range of summed point mutant fractions among lungs was about 7.5-fold, suggesting an important source of stratification in the population with regard to risk of tumor initiation.

Introduction

A causal relationship has been established between point and other mutations of a particular tumor suppressor gene at a fraction significantly greater than expected by chance for a few specific cancer types e.g. APC and colonic adenocarcinoma, VHL and renal carcinoma, PTCH and basal cell carcinoma [1]. Indications of causal relationships between point mutations in tissue of apparently normal histology and environmental mutagens are less clear save for the example of skin cell mutations and sunlight wherein forms of point mutation associated with solar irradiation have been found in TP53 and PTCH [2]. Absent direct measurement of mutations before and after exposure to environmental carcinogens it is not possible to determine if the carcinogen acted by induction of oncomutations by selection of pre-existing oncomutants or both [3].

To test the relationship in humans between a second clear environmental carcinogen and point mutagenesis the total number and micronatomical clustering of five specific point mutations have now been measured in pathologically normal lungs of male and female cigarette smokers and nonsmokers ranging in age from 38 to 76 years. To measure point mutations in lung tissue we used “mismatch amplification mutation assay” (MAMA), a form of allele-specific PCR assay that permits detection of specific point mutations from tissue DNA at levels of ∼10⁻⁵ mutant copies/total copies [4]. This form of assay had been previously used to discover that a specific G to A transition of H-RAS found in most methylnitrosourea-induced rat breast cancers existed in clusters of mutant cells prior to carcinogen treatment and that said clusters were not increased in number by carcinogen treatment [5], [6]. Because no lung tumor suppressor genes have been identified, MAMAs were devised for five specific point mutations in three separate genes, based on the assumption that mutagens would act indiscriminately among genes regardless of their role, if any, in lung carcinogenesis: C742T (codon 248, CGG, arginine → TGG, tryptophan) G746T(codon 249, AGG, arginine → ATG, methionine) and G747T(codon 249, AGG, arginine → AGT, serine) in the TP53 gene, G35T (codon 12, GGT, glycine → GTT, valine) in the KRAS gene and G508A (codon 170, CGA, arginine → TGA, stop) in the HPRT1 gene. In a series of 463 mutation assays of micro-anatomical samples of the tracheal–bronchial region of nine nonsmokers about half of all microanatomical sectors were found to contain detectable multiple copy clusters of any of the five mutations assayed [7]. Herein we report the results from a similar number of sector assays from smokers’ lungs. Formal statistical tests of the null hypothesis that there are no statistically significant differences dependent upon patient gender, age or smoking status have been developed and applied. The distribution of mutant cluster number as function of cluster size has been analyzed to discover if it contains information relevant to the size of hypothetical lung epithelial maintenance turnover units and age at which mutations arise.