The Inklings Remembered: A Conversation with Colin Havard

In late 2011, the authors met with Colin Havard, son of Inkling Dr. Robert E. “Humphrey” Havard and recorded his reminiscences about his father, his Catholic faith, his friendships with J.R.R. Tolkien and the Lewis brothers in particular, and the Inklings and practicing medicine in Oxford in general. As the lone Inkling from a scientific background, he brought a unique perspective to the group’s discussions

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

Genome-wide association study identifies common variants associated with circulating vitamin E levels

In genome-wide association studies (GWAS) of common genetic variants associated with circulating alpha- and gamma-tocopherol concentrations in two adult cohorts comprising 5006 men of European descent, we observed three loci associated with alpha-tocopherol levels, two novel single-nucleotide polymorphisms (SNPs), rs2108622 on 19pter-p13.11 (P= 1.7 × 10−8) and rs11057830 on 12q24.31 (P= 2.0 × 10−8) and confirmed a previously reported locus marked by rs964184 on 11q23.3 (P= 2.7 × 10−10). The three SNPs have been reported to be associated with lipid metabolism and/or regulation. We replicated these findings in a combined meta-analysis with two independent samples, P= 7.8 × 10−12 (rs964184 on 11q23.3 near BUD13, ZNF259 and APOA1/C3/A4/A5), P= 1.4 × 10−10 (rs2108622 on 19pter-p13.11 near CYP4F2) and P= 8.2 × 10−9 (rs11057830 on 12q24.31 near SCARB1). Combined, these SNPs explain 1.7% of the residual variance in log alpha-tocopherol levels. In one of the two male GWAS cohorts (n= 992), no SNPs were significantly associated with gamma-tocopherol concentrations after including data from the replication sample for 71 independent SNPs with P< 1 × 10−4 identified

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

The Role Of Vitamin E Hydroxylases In Vitamin E Metabolism And Status

Vitamin E is a group of compounds that are considered to be the most important lipophilic antioxidants, however there is still much unknown about the biological actions of the various forms of vitamin E as well as the mechanisms that influence the concentration of vitamin E forms in tissues. Despite the common predominance of mainly [gamma]-tocopherol ([gamma]-TOH) in the diet, [alpha]-TOH is present in serum and tissues at levels 5-6 times that of [gamma]-TOH. The biological rational for this selectivity remains an enigma. The focus of this work was on the selective postabsorptive catabolism of non-[alpha]-TOH forms via the vitamin E-[omega]-oxidation pathway. Cytochrome P450 4F2 (CYP4F2) is the only known human enzyme shown to display TOH-[omega]-hydroxylase activity. In an effort to investigate the role of TOH-[omega]-hydroxylase activity in vitamin E metabolism and status, the functional murine ortholog of CYP4F2 was identified and the consequences of its deletion on vitamin E metabolism and status were determined. In vivo and in vitro studies revealed Cyp4f14 to be the major, but not the only, vitamin E-[omega]-hydroxylase in mice, and to have critical function in regulating body-wide vitamin E status. Disruption of Cyp4f14 expression resulted in hyper-accumulation of [gamma]-TOH in mice fed a soybean oil diet in which the major tocopherol was [gamma]-TOH. Supplementation of Cyp4f14-/- mice with high levels of [delta]- and [gamma]-TOH exacerbated the tissue enrichment of these forms of vitamin E. Through the use of metabolic cage studies, previously unappreciated mechanisms of vitamin E elimination were discovered, which served to counterbalance the metabolic deficit observed in Cyp4f14-/- mice. Fecal elimination of unmetabolized TOHs was determined to be a high capacity mechanism to be minimize diet induced accumulation of TOHs, especially at high dietary levels. Additionally, novel [omega]-1 and [omega]-2 vitamin E hydroxylase activities were discovered and were found to quantitatively important vitamin E elimination mechanisms. Cyp4f14-/- mice also revealed the existence of other hepatic TOH-[omega]-hydroxylase enzyme(s). Therefore genetically modified mice, in which no CYP activity was present in the liver, were utilized in order to eliminate all hepatic vitamin E metabolism. Metabolic cage studies revealed the presence of vitamin E hydroxylase activity in non-hepatic tissues. Mouse and human small intestine mucosa were found to have TOH-[omega]-hydoxylase activity, representing at least one site of extra-hepatic vitamin E metabolism. Lastly, the use of cell culture studies demonstrated that two polymorphisms in CYP4F2 functionally alter TOH-[omega]-hydroxylase activity, which may play a role in vitamin E status in humans. Overall, the current works lends new insights into the physiological role of the TOH-[omega]oxidation pathway as well as reveals novel mechanisms of vitamin E metabolism in both mice and humans, which play an important role in the regulation of vitamin E status