Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator

Abstract Background The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene. Methods Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control. Results With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines. Conclusions We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val 158 Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications

Differential methylation relative to breast cancer subtype and matched normal tissue reveals distinct patterns

Due to the heterogeneous nature of breast cancer and the widespread use of single-gene studies, there is limited knowledge of multi-gene, locus-specific DNA methylation patterns in relation to molecular subtype and clinical features. We, therefore, quantified DNA methylation of 70 candidate gene loci in 140 breast tumors and matched normal tissues and determined associations with gene expression and tumor subtype. Using Sequenom’s EpiTYPER platform, approximately 1,200CpGs were interrogated and revealed six DNA methylation patterns in breast tumors relative to matched normal tissue. Differential methylation of several gene loci was observed within all molecular subtypes, while other patterns were subtype-dependent. Methylation of numerous gene loci was inversely correlated with gene expression, and in some cases, this correlation was only observed within specific breast tumor subtypes. Our findings were validated on a larger set of tumors and matched adjacent normal tissue from The Cancer Genome Atlas dataset, which utilized methylation data derived from both Illumina Infinium 27 and 450k arrays. These findings highlight the need to control for subtype when interpreting DNA methylation results, and the importance of interrogating multiple CpGs across varied gene regions.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2738-0) contains supplementary material, which is available to authorized users

Common Variants of Cytochrome P450 4F2 Exhibit Altered Vitamin E-ω-Hydroxylase Specific Activity12

Human cytochrome P450 4F2 (CYP4F2) catalyzes the ω-hydroxylation of the side chain of tocopherols (TOH) and tocotrienols (T3), the first step in their catabolism to polar metabolites excreted in urine. CYP4F2, in conjunction with α-TOH transfer protein, results in the conserved phenotype of selective retention of α-TOH. The purpose of this work was to determine the functional consequences of 2 common genetic variants in the human CYP4F2 gene on vitamin E-ω-hydroxylase specific activity using the 6 major dietary TOH and T3 as substrate. CYP4F2-mediated ω-hydroxylase specific activity was measured in microsomal preparations from insect cells that express wild-type or polymorphic variants of the human CYP4F2 protein. The W12G variant exhibited a greater enzyme specific activity (pmol product ⋅ min−1 ⋅ pmol CYP4F2−1) compared with wild-type enzyme for both TOH and T3, 230–275% of wild-type toward α, γ, and δ-TOH and 350% of wild-type toward α, γ, and δ-T3. In contrast, the V433M variant had lower enzyme specific activity toward TOH (42–66% of wild type) but was without a significant effect on the metabolism of T3. Because CYP4F2 is the only enzyme currently shown to metabolize vitamin E in humans, the observed substrate-dependent alterations in enzyme activity associated with these genetic variants may result in alterations in vitamin E status in individuals carrying these mutations and constitute a source of variability in vitamin E status

Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator

BACKGROUND: The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene. METHODS: Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control. RESULTS: With the exception of the CpG island in the 5’UTR and 1(st) exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val( 158 )Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val( 158 )Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines. CONCLUSIONS: We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val( 158 )Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications