Microsatellite mutations in buccal cells are associated with aging and head and neck carcinoma

Abstract

Carcinogen exposure from tobacco smoking is the major cause of upper aerodigestive tract cancer, yet heavy smokers only have about a 10% life-time risk of developing one of these cancers. Current technologies allow only limited prediction of cancer risk and there are no approved screening methods applicable to the general population. We developed a method to assess somatic mutational load using small-pool PCR (SP-PCR) and analysed mutations in DNA isolated from cells obtained by mouth rinse. Mutation levels in the hypermutable tetranucleotide marker D7S1482 were analysed in specimens from 25 head and neck squamous carcinoma (HNSCC) cases and 31 controls and tested for associations with age, smoking history and cancer status. We found a significant association between mutation frequency and age (P ¼ 0.021, Generalized Linear Model (GLM), N ¼ 56), but no influence of smoking history. Cases had higher mutation frequencies than controls when corrected for the effects of age, a difference that was statistically significant in the subgroup of 10 HNSCC patients who were treated with surgery only (P ¼ 0.017, GLM, N ¼ 41). We also present evidence that cancer status is linked to levels of nonunique, and presumably clonally derived, mutations in D7S1482. Insertion mutations were observed in 833 (79%) of 1058 alleles, of which 457 (43%) could be explained by insertion of a single repeat unit; deletion mutations were found in 225 (21%) of tested alleles.In conclusion, we demonstrate that the sensitive detection of single molecule mutations in clinical specimens is feasible by SP-PCR. Our study confirms an earlier report that microsatellite mutations increase with age and is the first to provide evidence that these mutations may be associated with cancer status in individual subjects.

Buccal Epithelium, Cigarette Smoking, and Lung Cancer: Review of the Literature

“…They conducted a case-control study of 50 patients with lung cancer and 50 controls ( n = 100) matched by sex, age, and smoking history. Their results suggested a significant correlation between microsatellite frequency and age, a finding supported by previous studies [42] . However, they failed to identify a similar correlation between mutation frequency and lung cancer [43] .…”

Analysis of microsatellite mutations in buccal cells from a case-control study for lung cancer

“…The analysis strategy for the detection of microsatellite mutations was based on small-pool PCR whereby a known amount of DNA is diluted down to the single molecule level (approximately 9 pg/PCR, or 3 genome equivalents per well), from which specific amplicons are expanded using time-release PCR [18, 25]. In a single assay, hundreds of alleles are tested simultaneously and microsatellite mutations are visualized due to differences in retention time during capillary electrophoresis.…”

“…In this study, we used the tetranucleotide markers MycL1, D7S1482, and DXS891, which accumulate high mutation levels following environmental insult and can be measured reliably by DNA fragment analysis. Detailed experimental procedures and reproducibility for the markers used in this study (MycL1, D7S1482 and DXS981) have been described previously [18, 26]. In summary, 3 serial dilutions of 1:100, 1:1000 and 1:10,000 from the 5 ng/μl solution were each amplified in 48 wells using marker MycL1 and the number of blank wells was used to estimate the average number of molecules in each of the wells using a standard Poisson distribution.…”

“…Since DXS981 is an X-linked marker, this number was about half that in males. Scoring of microsatellite mutations was performed manually by examining of all of the capillary electrophoresis trace graphs using GeneMapper software, which uses peak height as a primary outcome measurement according to previously established criteria [18]. To distinguish PCR-induced slippage artifacts from true mutant molecules the signal from a given fragment length needed to be more than 50% that of the signal from the adjacent larger fragment (to the right) if present, and/or more than 10% of the signal from the adjacent smaller fragment (to the left) if present.…”

“…When studied in a model system, the corresponding repetitive sequences demonstrated increased sensitivity to DNA damage caused by carcinogen exposure [17]. Furthermore, we, and others, have linked advancing age with increased levels of microsatellite mutations, either in normal human blood lymphocytes [9] or normal buccal cells [18]. Taken together, results from these studies suggest that microsatellites are exquisitely susceptible to damage, and that tetranucleotide repeats may be particularly sensitive to the tobacco-related mutagens implicated in most lung malignancies.…”