The main finding of this report is that two-linked intronic SNPs of ATM exhibited a similar recessive pattern of association with increased NSCLC risk, which is strongly supported by the comet assay indicating that the variants might function through influencing the DNA damage/repair pathway of the host cell. No literature has investigated the associations of these 2 SNPs with any form of cancer or other diseases. The close proximity of the 2 SNPs (in introns 61 and 62) to the ATM PI3 kinase domain (exons 60 and 61) lends support to the hypothesis that these 2 SNPs might regulate ATM PI3 kinase activity by affecting either the kinase domain or FAT domain immediately before the kinase motif.
All SNPs conformed to Hardy–Weinberg Equilibrium (HWE) in both case patients and control subjects, except for ATM
08 and ATM
10, which showed significant deviation in controls (, 0.02 and 0.04, respectively). However, the divergence from HWE is not likely the result of genotyping errors since we observed complete concordance from genotyping quality controls. Moreover, neither of the 2 SNPs were still out of HWE when the Bonferroni-corrected significance threshold was applied to test the null hypothesis of HWE to detect genotyping errors.28
Similar results were obtained when we used the less conservative Benjamini–Hochberg step-up procedure to control for false discovery rate (FDR) of HWE at the 5% level (data not shown). More importantly, the results of our comet functional assays measuring the DNA damage/DRC indicated that the homozygous variant genotype of these 2 SNPs were associated with significantly higher levels of gamma-radiation-induced DNA damage, strongly suggesting that the elevated risk associated with them are biologically plausible rather than being attained by population selection bias.
Validation through functional assays lends supports to the conclusions drawn from genotyping data. However, such assays are scarce in most published cancer association studies. In the present report, we performed the comet assay, a single cell gel electrophoresis-based laboratory test frequently used to evaluate DNA damage/repair and genotoxicity,29
to assess the correlation of ATM
genotypes with the host DNA damage/repair capacity in the control group. We discovered that radiation-induced-, but not baseline-, DNA damage was significantly higher in the homozygous variant genotypes of both ATM
08 and ATM
10, when compared to genotypes with at least 1 wild-type allele. This was strongly consistent with the role played by ATM in monitoring radiation-induced DSB DNA damage and initiating the corresponding repair process.30
The delicate structure of the ATM protein has remained elusive, which has limited in-depth investigation of the interactions between ATM and relevant signal effectors implicated in DSB repair. It is clear that one of the converging effects of the ATM-mediated signal pathways is to induce cell cycle arrest and allow the cell to repair the damage. Therefore, the hypothesis that ATM
functional variants influence the function of the ATM protein on DNA repair coincide with the phenotypic assay results that the samples with homozygous variants of either ATM
08 or ATM
10 showed a significant increase in DNA damage as well as increased NSCLC risk. This finding was further supported by the data showing the associations of diplotype H2–H2, the only analyzed diplotype containing the homozygous variants of both ATM
08 and ATM
10, with elevated NSCLC risk. Previous reports have suggested that most mutations/variations in ATM
region exert aberrant functions by influencing ATM activity instead of its protein expression.31,32
Analysis of kinase activity of Ser1981
as well as the expression profile of the total ATM level using peripheral blood cells derived from subjects with different ATM
10 genotypes will cast more light on this hypothesis.
No significant gene-dose effect was identified for either ATM
08 or ATM
10. This may be because ATM
is a tumor suppressor gene and, thereby, 2 copies of the gene need to be inactivated before carcinogenesis ensues. This recessive functional fashion is a common theme of many tumor suppressor genes and the “two-hit” hypothesis of carcinogenesis is the most convincing explanation for its working mechanism.33
We also noticed that the elevated risks for ATM
08 and ATM
10 were higher in former smokers than in current smokers. An unambiguous hypothesis regarding interaction between smoking and ATM
genetic variants has yet to be developed. The genetic effects of ATM
variants observed in this study may have been overwhelmed by the strong smoking exposure in current smokers. Moreover, as the results of the stratified analyses were based on relatively small sample size, this result needs to be interpreted with caution and requires validation in larger studies. Among the 6 intronic SNPs analyzed, ATM
07 and ATM
09 have been associated with risk of breast cancer15,16
; however, our data did not suggest that any of these was related to NSCLC risk. ATM
05, the only nsSNP currently identified as having an MAF ≥10%, has been implicated in various types of cancer18–20
but showed no effect in this study. Furthermore, haplotypes constructed using only potential functional SNPs (nsSNPs and regulatory SNPs, including ATM
05 and ATM
11) did not reveal any association with NSCLC risk (data not shown).
In a recent case-control study, Kim et al
. described the association of rs664143 (ATM
09) with increased lung cancer risk in a Korean population.22
This SNP did not show an evident link with NSCLC risk in our study, possibly due to the remarkable difference in the allelic frequency between these 2 populations.22
In addition, we restricted our case patients to NSCLC, whereas Kim et al
. investigated all lung cancer subtypes.
In summary, our study presents the first epidemiological data describing the associations of 2 common ATM polymorphisms (ATM08 and ATM10) and elevated NSCLC risk. The hypothesis that these 2 SNPs function through influencing ATM kinase activity was underscored by the independent phenotypic assay analyzing DNA repair profiles. Nevertheless, we cannot rule out the possibility that other rare ATM polymorphisms in LD with these 2 SNPs are the real causative agents. Further investigations exploring the molecular mechanism of the proposed ATM functional variations as well as the downstream signal transducers mediating the observed effects are warranted to provide a new dimension to our current understanding of the significance of ATM sequence variants in a more inclusive picture of lung carcinogenesis.