After the recent discovery that common genetic variation in 15q24–25.1 influences inherited risk of lung cancer (3
), we identified a second sequence variant at 15q24–25.1 associated with familial lung cancer (8
) and further validated this new association in large sporadic lung cancer populations. We showed that these two genetic variants on 15q24–25.1 have independent genetic effects on lung cancer risk. The second variant on 15q24–25.1, marked by rs481134, explains an additional 13.2% of the population attributable risk for lung cancer. These results further confirm the complexity of the chromosomal region 15q24–25.1 underlying lung cancer susceptibility.
Interestingly, the second variant did not show association with lung cancer in single-marker analysis. However, haplo-type analysis of SNPs rs1051730 and rs481134 provided stronger evidence for association with lung cancer. SNPs rs1051730 and rs481134 are in moderate LD (r2 = 0.30), which can mask or change the genetic effects of those loci in the association analysis. This may explain why the association of rs481134 with lung cancer was not detected in the single-marker analysis. The neutral haplotype G_T contains a wild-type allele at each variant, the protective haplotype G_C contains a wild-type allele at rs1051730 and a protective allele C at rs481134, whereas the haplotype A_C contains both a protective allele C at SNP rs481134 and a risk allele A at rs1051730. This suggests that the rs1051730 risk allele outweighs the rs481134 protective allele, and the overall effect of this haplotype increases risk of lung cancer. As a result, the protective effect of SNP rs481134 at haplotype A_C was masked by rs1051730, and thus its association with lung cancer was not detected in the single-marker analysis. Comparison of haplotypes G_T (neutral) and G_C (protective), which have the same allele G at rs1051730 but have different alleles (C/T) at rs481134, clearly showed that the allele C of rs481134 is associated with reduced risk of lung cancer. Similarly, comparison of haplotypes A_C (risk) with G_C (protective), which have the same allele C at rs481134 but have different alleles at rs1051730, showed that the allele A of rs1051730 is associated with increased risk of lung cancer. Actually, these comparisons are the rationale behind subsequent subgroup analyses for estimating marginal OR associated with each of these two variants.
To eliminate the effect masked by rs1051730, we performed an association analysis in a subgroup of subjects with genotype GG
at rs1051730 and identified a significant association of rs481134 with lung cancer. The allele C
of rs481134 is associated with reduced risk of lung cancer (), which is consistent with the haplotype analysis. These results showed that the two variants independently influence risk for lung cancer. Interestingly, the allelic OR associated with the risk allele of rs1051730 A
is much larger from subgroup analysis of subjects with genotype CC
at rs481134 than that estimated from the whole samples (OR, 1.60 versus 1.24; and ). This is because SNPs rs1051730 and rs481134 are in LD and they have opposite genetic effects on lung cancer risk. The effect of rs1051730 was offset by the effect of rs481134, and thus the effect size associated with rs1051730 was underestimated in previous studies (3
). Therefore, the marginal ORs associated with these two variants were used in the calculation of PAR for lung cancer. However, it should be noted that the estimated PAR for individual SNPs may not represent PAR in the general population because the marginal OR was used. The effect size associated with rs481134 is generally less significant than that of rs1051730 and may need to be further validated in future studies. In addition to their effects independently influencing risk of lung cancer, the two variants may exert a synergistic effect to modify the risk of lung cancer (Supplementary Table S4
). Our data emphasize the importance of the use of multilocus association models in association analyses.
Only three major haplotypes, G_T, A_C, and G_C, were observed at variants rs1051730 and rs481134 in all of the four populations. These three haplotypes accounted for more than 99% subjects in the samples. A very small number of individuals carry haplotype A_T. The risk for developing lung cancer is expected to be further increased among these individuals, although the number of individuals with this haplo-type was too small to formally estimate its effect size. In the subgroup analysis of subjects with genotype CC at rs481134, the allelic OR associated with the risk allele of rs1051730 A was estimated to be 1.60 (95% CI, 1.46–1.74). This allelic OR can be viewed as an indirect estimator of effect size of the haplotype A_T. Therefore, two risk haplotypes exist at 15q24–25.1: the common haplotype A_C confers moderate risk for increasing lung cancer and the rare haplotype A_T confers excessive risk for increasing lung cancer.
The 15q25.1 lung cancer susceptibility locus contains IREB2, LOC123688, PSMA4, CHRNA5, CHRNA3
, and CHRNB4
(). The nAChR genes encode for subunits of the nicotinic acetylcholine receptors, seem biologically relevant, and are attractive candidates. Recent candidate-gene studies of nicotine dependence also identified several variants in the CHRNA5-CHRNA3-CHRNB4
gene cluster on 15q24–25.1 that alter risk for nicotine dependence, including a missense SNP, rs16969968 (a change from aspartic acid to asparagine at codon 398 in CHRNA5; refs. 14
). SNP rs16969968 shows high LD with rs1051730 (r2
= 0.99) from the lung cancer association analyses. Nicotinic receptors containing the missense variant α4β2α5N398 of CHRNA5 exhibit reduced response to the nicotinic agonist epibatidine compared with receptors containing the more common variant α4β2α5D398 (16
). Interestingly, SNP rs588765, in high LD with rs481134 (r2
= 0.98), was initially identified to be associated with mRNA levels of CHRNA5
in brain. The major allele of rs588765 is associated with higher expression of CHRNA5
, conferring reduced risk of nicotine dependence (8
). The same two genetic variants are also associated with lung cancer risk. These data point to the candidacy of CHRNA5
for the 15q24–25.1 locus underlying lung cancer susceptibility, although the other five candidate genes in this high LD region cannot be completely excluded ().
Genetic variation in CHRNA5
can affect its functionality in two ways: (a
) rendering the receptors differentially responsive to their ligands and thus affecting their downstream signal transduction (17
), and (b
) modifying expression and thus affecting the composition of nAChR subunits that govern the acute response to agonists, such as endogenous acetylcholine and exogenous nicotine (18
). The mechanisms of regulation of CHRNA5 function or amount may underlie the genesis of lung cancer in smokers in two ways (Supplementary Fig. S1
). First, polymorphisms in CHRNA5
may affect nicotine dependence and propensity to smoke and to develop lung cancer. Smokers who are addicted to nicotine usually use more tobacco than those who are not, and thus they have a higher likelihood of developing lung cancer resulting from increased exposure to carcinogens found in tobacco smoke. This mechanism suggests a possible indirect genetic factor (e.g., through smoking behavior) contributing to lung cancer. Second, nAChRs that are functionally present in human lung airway epithelial cells and in lung carcinomas may play a direct, functional role in lung carcinogenesis (19
). In addition to nicotine, nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N
-nitrosonornicotine (NNN) are two major carcinogens found in tobacco smoke. NNK and NNN are agonists for α-bungarotoxin α7 nAChR and the heteromeric, epibatidine-sensitive α-β nAChRs, respectively (20
). The affinity of NNK for the α7 nAChR was found to be 1,300 times higher than that of nicotine, whereas the affinity of NNN for heteromeric α-β nAChRs was 5,000 times higher than that of nicotine (20
). Recent studies have shown that all these individual constituents of tobacco smoke can stimulate nAChR signaling in nonneuronal cells, including regulation of cell proliferation, angiogenesis, migration, invasion, and secretion (19
). This mechanism suggests a possible direct gene-environment interaction factor (e.g., cell proliferation and apoptosis) contributing to lung cancer. That is, given the same amount of tobacco exposure, tobacco-induced nonneuronal nAChR signaling responds differentially in smokers with different variants in CHRNA5. This may account for individual differences in lung cancer susceptibility to the same environmental risk factors such as tobacco smoking. It is also possible that both of these two mechanisms exist during lung carcinogenesis. Defining the contribution of both mechanisms warrants further studies.