The individual SNP-serum retinol association P
-values from the initial GWAS are plotted by chromosome in Supplementary Material, Figure S1
. There were 10 SNPs on chromosomes 10 and 18 that reached genome-wide significance (P <
) for association with circulating retinol concentration. We examined the association between all SNPs with initial-scan P
-values below 10−5
= 121) in the replication set; no additional SNPs reached genome-wide significance after meta-analysis with these data. The two highly significant SNPs from chromosome 10 were in the gene neighborhood of the RBP4
gene, which encodes retinol-binding protein 4 (RBP4), one of the two major carriers of retinol in serum. The strongest signal in this region was for rs10882272 (P =
; Table and Fig. ). In the pooled analysis, the significance of an additional SNP that reached genome-wide significance in this region (rs11187545) was greatly attenuated when it was included in the conditional regression model with rs10882272, showing no evidence for independence and suggesting that the signals from the two SNPs originate from a common locus (even though the two SNPs are not highly correlated; r2
= 0.15) (Fig. ). Also shown in the figure are the recombination hotspots which support the signal most likely being from one allele. The underlying linkage disequilibrium, as demonstrated with both D
′ = 0.83 and the haplotype structure, indicate that the variants are well correlated and differ slightly in minor allele frequency.
SNPs associated with serum retinol at P< 5 × 10−7-GWAS and replication studies
LD structure of chromosome 10. P-values generated from ATBC and PLCO data. LR, the recombination rate on a logarithmic scale with 12 being ‘notable’ for a hotspot.
rs10882272 was independently significant in two of the replication sets (NHS-CGEMS, P =0.003; and InCH-males, P =0.025), as well as in the replication sets combined (P =9.49× 10−5), and it remained highly significant in the meta-analysis of the GWAS and replication data (P = 6.51× 10−15) (Table ). There was no heterogeneity across studies for this SNP (P =0.67, Table ). The estimated relative difference in mean retinol levels per copy of the rs10882272-C allele from the overall meta-analysis was a decrease of 3.0% (Table ), and we estimated that this SNP accounted for 0.5% of the variation in serum retinol levels.
We observed a cluster of eight SNPs on chromosome 18 that were significantly associated with serum retinol in the initial GWAS (P< 5× 10−8). These SNPs are near TTR, the gene encoding TTR, which dimerizes with RBP4 and is therefore also involved in retinol (as well as thyroid hormone) transport in circulation. The strongest signal in this region was for rs1667255 (Table and Fig. ). When the other seven SNPs that reached genome-wide significance in the pooled analysis (i.e. rs1667254, rs1616887, rs1667234, rs723744, rs4799585, rs9304102, rs1621308) were included in the regression model with rs1667255, their significance was greatly reduced, indicating signal from a common locus. rs1667255 did not reach statistical significance in the replication data set (P =0.08, Table ), but did exhibit highly significant heterogeneity in the strength of association across studies (P =0.0005, Table ), with similar magnitude of association in the ATBC and PLCO male cohorts, somewhat attenuated beta for two of the replication studies (NHS-CHD and InCH-males), and lack of association in the other replication studies of women. The combined meta-analysis yielded significance at the P =6.35× 10−14 level (Table ), and the random effects meta-analysis P-value was 0.085. Combining ATBC and PLCO cohorts, the estimated beta and standard deviation are 0.039 and 0.0046, while in the NHS and InCH-female set the estimated beta and standard deviation are 0.0065 and 0.0059. These yield a formal z-test P-value of 1.31× 10−5 for the difference in the strength of SNP association between the male and female samples. Comparing those with two copies of the minor allele to those with zero copy, the difference in mean retinol ranged from −2.3 to 8.7% across the GWAS and replication cohorts (Table ), and we estimated in the pooled PLCO and ATBC studies that this SNP accounted for 1.7% of the variation in circulating retinol levels. These results were unchanged when we restricted the analysis to participants who did not use supplemental vitamin A of any kind (data not shown).
LD structure of chromosome 18. P-values generated from ATBC and PLCO data. LR, the recombination rate on a logarithmic scale with 12 being ‘notable’ for a hotspot.
We conducted an analysis in the pooled discovery GWAS data combining the two identified SNPs (rs10882272 on chromosome 18, and rs1667255 on chromosome 10) and found that individuals with two copies of both variant alleles (i.e. rs1667255: C/C, and rs10882272: C/C) had 12.7–15.1% higher serum retinol than those who were homozygous for the common allele for both SNPs (ATBC: 15.1%, 95% CI: 14.3–18.4%; PLCO: 12.7%, 95% CI: 11.6–13.7%). We estimated that the two SNPs together accounted for 2.3% of the variance in serum retinol levels. A sensitivity analysis conducted among the 2184 controls from the ATBC and PLCO nested sets revealed identical regression parameter estimates to those obtained using all participants (per minor allele change in log retinol levels = −0.03 and 0.04 for rs10882272 and rs1667255, respectively).
The results above were unchanged when participants using supplemental vitamin A from either individual supplements or multivitamins (i.e. 10% in ATBC, 37% in PLCO and 41% in NHS) were excluded from the analysis (data not shown). We also tested the gene–serum retinol associations within strata of vitamin A supplement use: the betas, SEs and P-values for RBP4 rs10882272 among vitamin A supplement users and non-users in our GWAS sample were −0.030/0.012/P= 0.008 and −0.033/0.005/P= 1.41× 10−10, respectively. Similarly for TTR rs1667255, they were 0.040/0.012/P= 0.001 and 0.041/0.005/P= 3.38× 10−15, respectively. The gene–serum retinol associations were also identical for RBP4 for higher and lower baseline serum retinol strata (–0.010/0.005/P= 0.025 and −0.013/0.004/P= 0.002, respectively), but showed signal only in the above-the-median group for TTR (0.019/0.004/P= 1.31× 10−5) and not those with lower serum retinol levels (0.006/0.005/P= 0.24).