We conducted a GWAS of serum alpha-tocopherol concentrations within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study cohort and replicated the findings in a combined meta-analysis with the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) Study and the Nurses’ Health Study (NHS) (Table 1 ). Similarly, a separate GWAS was performed for gamma-tocopherol concentrations using available data from the PLCO Study.
Briefly, the ATBC Study was a randomized, double-blind, placebo-controlled intervention trial conducted to determine whether supplementation with alpha-tocopherol, beta-carotene or both could prevent cancer (37 (link)). The participants were all male smokers at study entry, aged 50–69 years, and residents of southwestern Finland. Men were not eligible for study inclusion if they reported a history of cancer, had severe diseases limiting long-term participation or took supplements of vitamins E (>20 mg/day) or A (>20 000 IU/day) or beta-carotene (>6 mg/day). Fasting blood samples were collected at baseline and stored at −70°C until analyzed. Serum alpha-tocopherol levels were measured by high-performance liquid chromatography (38 (link)), with a coefficient of variation (CV) of 2.2%. Gamma-tocopherol was not measured at study baseline. Serum cholesterol levels were measured with an enzymatic assay by the CHOD-PAP method (Boehringer Mannhein) (39 (link)). GWAS data from the Illumina 550K platform were available for 4014 men that were also previously analyzed with respect to circulating vitamin D levels (12 (link)).
The PLCO Study was a multi-center trial conducted in the US to evaluate the effectiveness of cancer screening and examine early markers of cancer (40 (link)). PLCO male participants of Caucasian descent, aged 55–74 years, were included in the present GWAS (n= 992). Plasma concentrations of alpha- and gamma-tocopherol were measured by CLIA. The CVs for alpha- and gamma-tocopherol concentrations were 5.8 and 8.9%, respectively. Cholesterol was measured enzymatically by a standard procedure at 37°C on a Hitachi 912 autoanalyzer. GWAS genotyping used both the Illumina 317K and 240K platforms, and as a result, SNP coverage (relative to the Illumina 550K used for ATBC) for two of the loci associated with circulating alpha-tocopherol, was incomplete. Genotype imputation was therefore performed for rs964184 and rs11057830 using IMPUTE2 to identify SNPs with the 1000 genomes project June 2010 release and HapMap 3 release 2 as the reference set. The imputed SNPs had a high imputation quality score.
Data from the NHS, a cohort of US women, was also used to replicate the most significant findings (approximately 100 SNPs with P < 1 × 10−5 or higher) obtained in the original GWAS. For the NHS samples, plasma tocopherol levels were measured using reversed-phase, high-performance liquid chromatography. The CVs for each batch were ≤13% with the exception of one batch which had a CV of 22%. Total cholesterol was assayed from plasma using the enzymatic methods described by Allain et al. (41 (link)). The Affymetrix 6.0 platform was used for nested case–control studies of coronary heart disease (CHD; n= 425) and type 2 diabetes (T2D; n= 394), and Illumina 550K for breast cancer [Cancer Genetic Markers of Susceptibility (CGEMS); n= 1929]. Each study sample used the MACH to impute up to approximately 2.5 million autosomal SNPs with NCBI build 36 of Phase II HapMap CEU data (release 22) as the reference panel. All of the imputed SNPs had a high imputation quality score.
Prior to analysis, tocopherol levels were log-transformed to normalize the distributions. A linear model adjusted for age, BMI and cancer status was used to relate the log-transformed outcomes to a SNP by assuming an additive mode of inheritance. Furthermore, because it is well established that vitamin E levels are affected by circulating lipids, we further adjusted the analyses for total cholesterol. Because HDL cholesterol was available in the ATBC Study, we performed a sensitivity GWAS analysis that adjusted for ‘non-HDL’ cholesterol levels (total minus HDL, or essentially LDL + VLDL, which we would expect to more closely reflect triglycerides) and yielded SNP findings identical to those adjusted for total cholesterol. Additional models that included both total cholesterol and HDL also provided similar results. The likelihood ratio test was used to detect the association between the tocopherol levels and the SNPs, adjusting for the above covariates. To identify the independent effect of other SNPs in the region of the most significant SNP, the likelihood ratio test was again used in the initial GWAS sample with the most significant SNP and those covariates involved in the basic model. We used a fixed effects meta-analysis on the GWAS and replication studies. The meta-analysis was conducted by combining the study-specific beta-estimates weighted by the inverse of the corresponding variances. We performed a sensitivity analysis on the alpha-tocopherol GWAS, excluding subjects who reported any use of vitamin E supplements (including those from multivitamins); the identified SNPs remained significant and accentuated among non-users. Additionally adjusting the GWAS for HDL levels did not change study findings. Study protocols for ATBC, PLCO and NHS were approved by their respective institutional review boards and eligible participants provided written consent.
Briefly, the ATBC Study was a randomized, double-blind, placebo-controlled intervention trial conducted to determine whether supplementation with alpha-tocopherol, beta-carotene or both could prevent cancer (37 (link)). The participants were all male smokers at study entry, aged 50–69 years, and residents of southwestern Finland. Men were not eligible for study inclusion if they reported a history of cancer, had severe diseases limiting long-term participation or took supplements of vitamins E (>20 mg/day) or A (>20 000 IU/day) or beta-carotene (>6 mg/day). Fasting blood samples were collected at baseline and stored at −70°C until analyzed. Serum alpha-tocopherol levels were measured by high-performance liquid chromatography (38 (link)), with a coefficient of variation (CV) of 2.2%. Gamma-tocopherol was not measured at study baseline. Serum cholesterol levels were measured with an enzymatic assay by the CHOD-PAP method (Boehringer Mannhein) (39 (link)). GWAS data from the Illumina 550K platform were available for 4014 men that were also previously analyzed with respect to circulating vitamin D levels (12 (link)).
The PLCO Study was a multi-center trial conducted in the US to evaluate the effectiveness of cancer screening and examine early markers of cancer (40 (link)). PLCO male participants of Caucasian descent, aged 55–74 years, were included in the present GWAS (n= 992). Plasma concentrations of alpha- and gamma-tocopherol were measured by CLIA. The CVs for alpha- and gamma-tocopherol concentrations were 5.8 and 8.9%, respectively. Cholesterol was measured enzymatically by a standard procedure at 37°C on a Hitachi 912 autoanalyzer. GWAS genotyping used both the Illumina 317K and 240K platforms, and as a result, SNP coverage (relative to the Illumina 550K used for ATBC) for two of the loci associated with circulating alpha-tocopherol, was incomplete. Genotype imputation was therefore performed for rs964184 and rs11057830 using IMPUTE2 to identify SNPs with the 1000 genomes project June 2010 release and HapMap 3 release 2 as the reference set. The imputed SNPs had a high imputation quality score.
Data from the NHS, a cohort of US women, was also used to replicate the most significant findings (approximately 100 SNPs with P < 1 × 10−5 or higher) obtained in the original GWAS. For the NHS samples, plasma tocopherol levels were measured using reversed-phase, high-performance liquid chromatography. The CVs for each batch were ≤13% with the exception of one batch which had a CV of 22%. Total cholesterol was assayed from plasma using the enzymatic methods described by Allain et al. (41 (link)). The Affymetrix 6.0 platform was used for nested case–control studies of coronary heart disease (CHD; n= 425) and type 2 diabetes (T2D; n= 394), and Illumina 550K for breast cancer [Cancer Genetic Markers of Susceptibility (CGEMS); n= 1929]. Each study sample used the MACH to impute up to approximately 2.5 million autosomal SNPs with NCBI build 36 of Phase II HapMap CEU data (release 22) as the reference panel. All of the imputed SNPs had a high imputation quality score.
Prior to analysis, tocopherol levels were log-transformed to normalize the distributions. A linear model adjusted for age, BMI and cancer status was used to relate the log-transformed outcomes to a SNP by assuming an additive mode of inheritance. Furthermore, because it is well established that vitamin E levels are affected by circulating lipids, we further adjusted the analyses for total cholesterol. Because HDL cholesterol was available in the ATBC Study, we performed a sensitivity GWAS analysis that adjusted for ‘non-HDL’ cholesterol levels (total minus HDL, or essentially LDL + VLDL, which we would expect to more closely reflect triglycerides) and yielded SNP findings identical to those adjusted for total cholesterol. Additional models that included both total cholesterol and HDL also provided similar results. The likelihood ratio test was used to detect the association between the tocopherol levels and the SNPs, adjusting for the above covariates. To identify the independent effect of other SNPs in the region of the most significant SNP, the likelihood ratio test was again used in the initial GWAS sample with the most significant SNP and those covariates involved in the basic model. We used a fixed effects meta-analysis on the GWAS and replication studies. The meta-analysis was conducted by combining the study-specific beta-estimates weighted by the inverse of the corresponding variances. We performed a sensitivity analysis on the alpha-tocopherol GWAS, excluding subjects who reported any use of vitamin E supplements (including those from multivitamins); the identified SNPs remained significant and accentuated among non-users. Additionally adjusting the GWAS for HDL levels did not change study findings. Study protocols for ATBC, PLCO and NHS were approved by their respective institutional review boards and eligible participants provided written consent.
