Table shows the selected characteristics of the study participants in each phase.
Characteristics of the study populations
In the discovery phase, we tested the association of 539 437 SNPs that passed strict quality control measures in 673 healthy participants. These individuals were from the control population in a recently published GWAS of bladder cancer risk for whom the BLM sensitivity measurement was available (16
). The quantile–quantile plot for the multiplicity adjusted P-
values showed no evidence for a systematic inflation of P
-values (genomic inflation factor λ = 0.969) (Supplementary Material, Fig. S1
). We further determined the potential effect of population substructure by using two approaches: one including further adjustment for strata identified by PLINK and the other including further adjustment for residual population structure using the top four principal components by EIGENSTRAT. These two approaches showed similar strength of associations with no evidence for the inflation of the multiplicity adjusted P-
values (λ = 0.969 for PLINK and λ = 0.968 for EIGENSTRAT, Supplementary Material, Fig. S1
), indicating that the effect of population substructure was negligible.
In the first replication phase, we selected SNPs with multiplicity adjusted P
level in the discovery phase to be validated in an independent set of 575 individuals who had BLM sensitivity data from the control population of a recent GWAS (using Illumina's Humanhap300 chip) of lung cancer risk (17
). A total of 554 SNPs from discovery set were significantly associated with BLM sensitivity satisfying these criteria (Fig. ). Among these 554 SNPs, 287 were covered by the HumanHap300 chip, 245 were imputed from HapMap release 22, build 36, and 22 SNPs were not HapMap SNPs and hence not included in the analysis. All the imputed SNPs showed high quality with an average predicted r2
of 0.93 between imputed genotypes and the true underlying genotypes.
Manhattan plot presenting genome-wide association results for 539 437 autosomal SNPs that passed the strict quality control measures and had >20 subjects carrying at least one variant allele.
We then performed a second validation using TaqMan assay in 259 healthy individuals from additional controls of the ongoing bladder and lung case control studies for the 13 SNPs that were significantly associated with BLM sensitivity at P
< 0.05 in the first replication phase (Supplement Material, Table S1
). There was no significant heterogeneity between the discovery and first replication phase (P
for heterogeneity > 0.1) for these 13 SNPs. Since SNP rs10093667 failed the TaqMan design, only 12 SNPs were genotyped in this second replication phase. Out of the 12 SNPs, 4 SNPs were imputed in the first replication phase. Therefore, we also performed direct genotyping using TaqMan assays for these four SNPs in the first replication phase (Supplement Material, Table S2
Overall, SNP rs8093763 showed the strongest evidence of association with BLM sensitivity in a recessive model (combined P = 2.64 × 10−8, Table ). We observed significantly lower BLM-induced chromatid breaks (BICB) for genotypes containing wild-type allele compared with the homozygous variant genotype in the discovery set (0.71 versus 0.90, P = 3.77 × 10−5) and in the first replication phase (0.61 versus 0.84, P = 7.00 × 10−5). The result of replication phase 2 was not statistically significant (0.65 versus 0.68, P = 0.44). There was no significant evidence for heterogeneity among the three phases (P for heterogeneity = 0.23). The second strongest association was observed for rs708547 on chromosome 4q12 using the recessive model (combined P = 8.79 × 10−7, P for heterogeneity = 0.93, Table ). The genotypes with at least one wild-type allele exhibited consistently higher BICB than the homozygous variant genotype in the discovery set (0.74 versus 0.56, P = 2.94 × 10−4), in replication phase 1 (0.63 versus 0.47, P = 1.12 × 10−2) and in replication phase 2 (0.66 versus 0.51, P = 3.29 × 10−2). We also observed a consistent association for SNP rs4662834 on chromosome 2q21 using the dominant model (combined P = 4.89 × 10−6, P for heterogeneity = 0.72, Table ). The common homozygous genotype had higher BICB than variant allele carrying genotypes in the discovery set (0.79 versus 0.70, P = 1.54 × 10−4), in replication phase 1 (0.67 versus 0.59, P = 2.37 × 10−2) and in replication phase 2 (0.69 versus 0.63, P = 1.68 × 10−1).
SNPs with the strongest associations with BLM sensitivity in combined analysis of three phases
Rs8093763 is located on chromosome 18q21. Linkage disequilibrium (LD) analysis of the HapMap SNPs in its vicinity showed that it maps to a region of low LD structure, and no SNPs in the HapMap database are in strong LD (r2> 0.6) with rs8093763. We performed imputations for SNPs in the HapMap database within 1Mb of rs8093763 and computed the combined P-value for the discovery set and replication phase 1 (Fig. ). Rs8093763 remained the most significant SNP in the region.
Figure 2. The locus of 18q21 encompassing rs8093763. (A) Results of SNP association from combined discovery set and replication phase 1: observed results from genotyped SNPs are in red and imputed results are in blue. Screen shot of all known genes in this region (more ...)
There are a few genes in this region, the nearest being PMAIP1/Noxa
, about 64 kb from rs8093763. To support the biological plausibility of our observation, we queried the Gene Expression Database (GEO; http://www.ncbi.nlm.nih.gov/geo/
) for data sets obtained from BLM-treated samples. We identified one data set (GDS1714) that compared gene expression differences of 14 lymphoblastoid cell lines (7 BLM-sensitive and 7 BLM-insensitive based on mutagen sensitivity assays) at baseline and after a 4 h exposure to BLM (13
). The mean expression of PMAIP1/Noxa
was higher in BLM sensitive (mean ± SD: 0.99 ± 0.58) than in insensitive cell lines (0.64 ± 0.60) at baseline, although the difference was not statistically significant (P
= 0.28) possibly due to small sample size. In BLM-insensitive cells, the expression of PMAIP1/Noxa
was little changed after BLM treatment (before versus after: 0.64 ± 0.60 versus 0.70 ± 0.41) (P
= 0.80); however, in BLM-sensitive cells, the expression of PMAIP1/Noxa
was apparently increased (before versus after: 0.99 ± 0.58 versus 1.37 ± 0.59) (P
= 0.24), although again the data did not reach statistical significance possibly due to small sample size. When we compared the expression of PMAIP1/Noxa
after BLM treatment, BLM-sensitive cells exhibited significantly higher expression (1.37 ± 0.59) than in BLM-insensitive cells (0.70 ± 0.41) (P
= 0.03). We also performed same analyses of other genes (Fig. ) in this region, but none of them showed similar trend as PMAIP1/Noxa
(data not shown).