Leukocyte Oncogenic Base Substitution Mutation Frequencies
Leukocyte Oncogenic Base Substitution Mutation Frequencies
By Ronald A. Tapp, MS and Vincent L. Wilson, PhD
The possibility that an individual’s relative predisposition to cancer or other disease may be identifiable by the presence and pattern of rare but detectable mutations in easily accessible tissues such as peripheral blood leukocytes (PBL) has an aesthetic appeal. However, there are numerous issues that are poorly understood, including the background frequency of mutation in leukocytes and other tissues. In the absence of a genome destabilizing disease, such as a constitutional DNA repair deficiency, cancer, or a significant exposure to one or more genotoxic agents, these mutations are rare. For single genes, the null mutation frequency is generally accepted to be about one in a million cells.1,2 Abnormal chromosomal translocations or gene rearrangements have been reported to be present at similar frequencies.3 These mutations do not necessarily represent important oncogenic mutations.
Single Base Substitution Mutations
Oncogenic mutations include a broad spectrum of fixed changes in the genomic complement, including chromosomal aberrations, gene rearrangements, gene amplification, insertions, deletions, and single base substitutions. Single base substitution mutations represent up to 85% of the oncogenic mutations occurring in many tumor-suppressing genes (i.e., p53 and RB) and proto-oncogenes in cancers. Using Needle-in-a-Haystack PCR/RE/LCR mutation detection and identification procedures, single base substitution mutations in multiple oncogenic sites have been studied in PBL specimens obtained from a limited number of apparently normal individuals.4,5 Mutations were detected in eight of these normal individuals at the level of one cell (one allele) in 106 cells, although the specific frequency of each mutation was not determined. A total of 17 independent, rare single base substitution mutations were identified. These rare mutations were scattered over five different bases in three different loci, including codon 12 of Harvey-ras, codon 248 of p53, and codons 12 and 13 of N-ras. Aside from two individuals, as noted below, the data from this small population suggest a background base substitution mutation frequency of less than or equal to one cell in a million in circulating leukocytes.5
More than one-half of the 17 single base substitution mutations were found in two individuals, as represented by mutations detected in four out of five and four out of four base sites analyzed, respectively. These two individuals were considered to either have been recently exposed to a significant genotoxic dose or exhibit a hypermutable phenotype in one or more cellular clones.
Hypermutable Phenotype
One of the hallmarks of cancer is the expression of a hypermutable (or mutator) phenotype resulting in the accumulation of multiple DNA alterations at the nucleotide and/or chromosomal levels.6,7 The genetic basis for the generation of the hypermutable phenotype is under study, and putative candidates include a group of genes known as gatekeepers.8,9 Mutations in these genes are known to cause significant cellular and biological changes. Theoretically, one or more stem cells within the bone marrow could have inherited such a mutation that leads to the expression of a hypermutable phenotype in the two individuals observed by Wilson et al.5 The production of such hypermutable cells could readily result from exogenous or endogenous genotoxic exposure or from a constitutional predisposing genetic error. Regardless of the cause, the resulting effect may lead to one or more clones of genomically unstable cells.
Genomic Instabilities
Genomic instabilities appear to be a common characteristic of cancer that may be represented in different forms, such as microsatellite, chromosomal, or epigenetic instability.9-12 These instabilities do not include single base substitution mutations which account for the majority of the activating and "deactivating" mutations in proto-oncogenes and tumor-suppressing genes.13-14 Single base instabilities are likely to be at least as important as the instabilities noted above. Single base instabilities may be present in a large proportion of cancers; however, they have not been previously identified due to the lack of methods to evaluate this marker.
Mutation Frequencies
Based on recent literature, a significant proportion of the human population may be represented by susceptible individuals. In addition to the one in 10 (2 out of 19) identified as harboring a hypermutable phenotype by Wilson et al, Shen et al reported that approximately 10% of the healthy individuals studied had inherited variant alleles of DNA repair genes.5,15 A few of these variant alleles have been associated with breast cancer.16 A lower proportion of "normal" individuals harboring higher than expected mutation frequencies was observed with the glycophorin A (GPA) mutation assay.17 Two of the individuals continued to display abnormally high GPA mutation frequencies upon further sampling. However, individuals with high GPA mutation frequencies did not display significantly high illegitimate V(D)J rearrangement frequencies.18
Conclusion
Further studies are needed to determine the extent of hypermutability and/or genomic instabilities within individuals expressing an abnormally high mutation frequency with one of these sensitive assays. It is quite possible that individuals may harbor a clone that carries only one form of genomic instability with limiting mutational consequences. The importance or relative health risk to individuals harboring high frequencies of mutations still needs to be determined. Albertini et al followed an individual harboring a hypermutable T-cell clone for several years without observation of any obvious adverse health effects.19 However, such a high frequency of hypermutable phenotypes within the human population is surprising unless these individuals are susceptible to mutagenic processes. Systematic population studies need to be conducted to elucidate the significance of these observations for cancer development. (Mr. Tapp is a PhD candidate in the Department of Veterinary Physiology, Pharmacology, and Toxicology, in the School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA. Dr. Wilson is Associate Professor, Claiborne Chair of Environmental Toxicology, and Chair, Interdepartmental Studies, Concentration in Toxicology, Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA.)
References
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