We found that NBS1 polymorphisms and haplotypes are associated with incidence of smoking-related cancers and that these associations may be modified by smoking status, supporting the notion that genetic variation within NBS1 plays a role in tobacco-related carcinogenesis. To our knowledge, many of these relationships have not been previously reported for cancers of the stomach, liver and UADT subsites (esophagus, oropharynx, larynx and nasopharynx).
We observed positive associations with the C
haplotype and cancers of the lung, oropharynx and larynx. In our study, we did not find consistent associations with rs709816 and rs1063054 in smoking-related cancer sites. However, haplotype analyses showed consistent positive associations across three of the four evaluated cancer sites, suggesting that either the variant C alleles in rs709816 and rs1063054 have a combined effect in tobacco-related tumorigenesis or that there is some other causal allele in LD with the C
haplotype. In addition, when stratified by smoking, we found that this positive association persisted among ever-smokers. Given that the associations were observed in cancer sites with direct contact to smoke inhalation (such as the oral cavity and lung), the C
haplotype may be involved in field cancerization (48
To our knowledge, these two SNPs have rarely been investigated in smoking-related cancers (14
). We are aware of one study investigating NBS1
haplotypes (rs1063045 and rs1805794, which are in LD with rs1061302) in lung cancer, in which the investigators found a positive association between lung cancer and the haplotype with recessive allele variants A and G (16
). While it is still possible these findings are in error given the rarity of the haplotype, the C
haplotype should be evaluated in studies with larger sample sizes and additional cancer sites, such as the esophagus, stomach and liver. The potential effect modification from smoking should be examined as well.
The potential functionality of rs709816 and rs1063054 remains unclear. However, the microRNA Target Site (PolymiRTS) database designates rs2735383, which is in LD (r2
> 0.99) with rs1063054, as one of the most probable targets for microRNA, miR-499 (49
), offering the possibility that alleles at this locus may influence NBS1
SNP rs1061302 was associated with cancers of the lung, larynx and liver in our study. Two NBS1
SNPs not included in our study, rs1805794 and rs1063045, were previously examined in relation to smoking-related cancers (14
). LD estimates from the Haploview program (51
) using HapMap data of the Centre d'Etude du Polymorphisme Humain (CEPH) population (52
) showed that both of these SNPs are in high LD (r2
> 0.99) with each other and with rs1061302. A recent meta-analysis of four bladder cancer studies reported that the variant allele of rs1805794 was positively associated with bladder cancer (OR
1.15; 95% CI: 1.01, 1.31) (53
). A meta-analysis of three lung cancer studies detected no association with this cancer site (OR
0.98; 95% CI: 0.51, 1.89) (53
). Since rs1805794 is in high LD with rs1061302, we expected to find similar results in our study. For bladder cancer, our point estimates for heterozygote and homozygote variant allele carriers of rs1061302 were in the positive direction but the CIs included the null. Similar positive associations were seen for smokers and non-smokers. The large CIs may be due to small sample size.
Interestingly, we found that the association between rs1061302 and lung cancer differed by smoking status, which may explain why we observed associations where the previous meta-analysis did not. For never-smokers, there was a clear dose–response relationship for the variant allele of rs1061302 and lung cancer. For smokers, rs1061302 heterozygotes showed an inverse relationship with lung cancer. Thus, it is possible that these smoking-dependent associations were masked in the previous meta-analysis by combining smokers and non-smokers. Additionally, our BFDP correction suggests that the observed rs1061302 association in smokers and non-smokers were probably not due to false discoveries (supplementary Table II
is available at Carcinogenesis
There are no published reports between rs1061302 and laryngeal or liver cancer. We observed that rs1061302 was inversely associated with laryngeal cancer and positively associated with liver cancer. The different direction of associations may reflect modification by major risk factors. Hepatitis infections are the primary risk factors for liver cancer (54
), whereas alcohol and smoking are considered risk factors for laryngeal and liver cancers (55
). To date, however, alcohol has not been established as a known risk factor for lung cancer (56
The associations between NBS1
polymorphisms and smoking-related cancers may be modified by smoking status, suggesting different tumorigenic pathways among smokers and never-smokers (57
). Our interaction analyses () support the idea that smoking status modifies the rate ratio for the associations for rs1061302 and rs709816 with lung cancer and for rs1063054 with stomach cancer. A study among a cohort of smokers found that rs6998169 was inversely associated with gene methylation (58
). Although our SNPs do not appear to be in LD with rs6998169, these findings suggest that genetic variation in NBS1
among smokers may alter the development of lung cancer by decreasing DNA methylation.
The NBS1 protein has three known functional regions: N-terminus (a.a. position 1–196), central region (a.a. position 278–343) and C-terminus (a.a. position 665–693) (59
). The C-terminus is the binding site for the MRN complex (59
) and DSBs cannot be detected in the non-homologous end joining pathway or repaired by the homologous recombination pathway without this complex (6
). Given that rs1061302 codes for a.a. 672 in the C-terminus and that we found associations between rs1061302 and lung, larynx and liver cancers, SNPs in LD with rs1061302 or within this region may inadvertently affect proper binding of the MRN complex and alter its ability to accurately repair or detect DNA DSBs. Although rs1061302 is a synonymous polymorphism, the altered nucleotide may affect messenger RNA stability (60
), splicing (62
) or the translation rate (63
). Confirmation studies are needed to determine the true ‘functional’ SNP.
Our candidate SNP selection approach, which relied heavily on previously reported SNPs, is a limitation of this study. We may have missed important genetic variations in NBS1
. However, an analysis of SNP genotype data from the International HapMap project (52
) suggests that the SNPs included in our study may be adequate proxies for the majority of genetic variation in NBS1. We used LDSelect (64
) to bin SNPs in the NBS1 gene region (all exons, introns and 2 kb flanking sequence) that were in high LD (r2
≥ 0.80) in the CEPH population. The rs1061302 SNP captured information on 31 other SNPs, rs1063054 captured information on 13 additional SNPs and rs709816 captured information on another 9 SNPs. Thus, of the 63 NBS1
SNPs with a minor allele frequency >5% genotyped in the HapMap project, our three selected SNPs captured information on ~89% of the SNPs.
The small sample sizes or low participation rates observed for some of the cancer sites may be a consequence of low survival for many of these cancers (liver, stomach and esophagus) (66
). If NBS1
polymorphisms are associated with survival, our study findings may be affected by survival bias. To our knowledge, only a breast cancer study investigated such an hypothesis and found no association among rs709816, rs1805794, rs1063045 and rs1061302 in breast cancer survival (P
0.65, 0.24, 0.53 and 0.40, respectively) (67
). Additionally, the small sample sizes of the Taixing City and MSKCC studies limited the precision of our estimates and assessment of effect modification by smoking status. Subsequent studies evaluating SNP function or potential SNP–SNP interactions between NBS1
and other genes in the non-homologous end joining or homologous recombination pathway may elucidate potential biological mechanisms.
Ours is the first study that we are aware of to investigate the association between NBS1 SNPs and haplotypes with nine smoking-related cancers. We had a range of racial/ethnic groups, enabling us to examine NBS1 SNPs in different populations, and our sample size was fairly large for lung and UADT cancer sites. We took several precautions to minimize genotype misclassification and our quality control assessment indicated a high degree of accuracy. We also used the BFDP correction to account for multiple comparisons. Our findings suggest that NBS1 is probably influential in the development of lung, UADT, stomach, liver and bladder cancers and that smoking status may alter the direction of the association. The association of the CAC haplotype with cancers of the lung, UADT, oropharynx and larynx indicates that this haplotype or SNPs in LD with this haplotype may alter tobacco smoking-related DSB repair ability. The role of SNPs that influence microRNA function or those that code for the C-terminus portion of NBS1 should be considered and evaluated in future studies.