Øivind Midttun

450K epigenome-wide scan identifies differential DNA methylation in newborns related to maternal smoking during pregnancy.

Background: Epigenetic modifications, such as DNA methylation, due to in utero exposures may play a critical role in early programming for childhood and adult illness. Maternal smoking is a major risk factor for multiple adverse health outcomes in children, but the underlying mechanisms are unclear. Objective: We investigated epigenome-wide methylation in cord blood of newborns in relation to maternal smoking during pregnancy. Methods: We examined maternal plasma cotinine (an objective biomarker of smoking) measured during pregnancy in relation to DNA methylation at 473,844 CpG sites (CpGs) in 1,062 newborn cord blood samples from the Norwegian Mother and Child Cohort Study (MoBa) using the Infinium HumanMethylation450 BeadChip (450K). Results: We found differential DNA methylation at epigenome-wide statistical significance (p-value < 1.06 × 10–7) for 26 CpGs mapped to 10 genes. We replicated findings for CpGs in AHRR, CYP1A1, and GFI1 at strict Bonferroni-corrected statistical significance in a U.S. birth cohort. AHRR and CYP1A1 play a key role in the aryl hydrocarbon receptor signaling pathway, which mediates the detoxification of the components of tobacco smoke. GFI1 is involved in diverse developmental processes but has not previously been implicated in responses to tobacco smoke. Conclusions: We identified a set of genes with methylation changes present at birth in children whose mothers smoked during pregnancy. This is the first study of differential methylation across the genome in relation to maternal smoking during pregnancy using the 450K platform. Our findings implicate epigenetic mechanisms in the pathogenesis of the adverse health outcomes associated with this important in utero exposure.

The Methylenetetrahydrofolate Reductase 677C→T Polymorphism as a Modulator of a B Vitamin Network with Major Effects on Homocysteine Metabolism

Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C-->T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 micromol/liter, 10.9 micromol/liter, and 13.3 micromol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C-->T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P<or=.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 micromol/liter for folate, 4.1 micromol/liter for riboflavin, 3.2 micromol/liter for cobalamin, and 2.1 micromol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR-B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations.

Serum B vitamin levels and risk of lung cancer.

CONTEXT B vitamins and factors related to 1-carbon metabolism help to maintain DNA integrity and regulate gene expression and may affect cancer risk. OBJECTIVE To investigate if 1-carbon metabolism factors are associated with onset of lung cancer. DESIGN, SETTING, AND PARTICIPANTS The European Prospective Investigation into Cancer and Nutrition (EPIC) recruited 519,978 participants from 10 countries between 1992 and 2000, of whom 385,747 donated blood. By 2006, 899 lung cancer cases were identified and 1770 control participants were individually matched by country, sex, date of birth, and date of blood collection. Serum levels were measured for 6 factors of 1-carbon metabolism and cotinine. MAIN OUTCOME MEASURE Odds ratios (ORs) of lung cancer by serum levels of 4 B vitamins (B(2), B(6), folate [B(9)], and B(12)), methionine, and homocysteine. RESULTS Within the entire EPIC cohort, the age-standardized incidence rates of lung cancer (standardized to the world population, aged 35-79 years) were 6.6, 44.9, and 156.1 per 100,000 person-years among never, former, and current smokers for men, respectively. The corresponding incidence rates for women were 7.1, 23.9, and 100.9 per 100,000 person-years, respectively. After accounting for smoking, a lower risk for lung cancer was seen for elevated serum levels of B(6) (fourth vs first quartile OR, 0.44; 95% confidence interval [CI], 0.33-0.60; P for trend <.000001), as well as for serum methionine (fourth vs first quartile OR, 0.52; 95% CI, 0.39-0.69; P for trend <.000001). Similar and consistent decreases in risk were observed in never, former, and current smokers, indicating that results were not due to confounding by smoking. The magnitude of risk was also constant with increasing length of follow-up, indicating that the associations were not explained by preclinical disease. A lower risk was also seen for serum folate (fourth vs first quartile OR, 0.68; 95% CI, 0.51-0.90; P for trend = .001), although this was apparent only for former and current smokers. When participants were classified by median levels of serum methionine and B(6), having above-median levels of both was associated with a lower lung cancer risk overall (OR, 0.41; 95% CI, 0.31-0.54), as well as separately among never (OR, 0.36; 95% CI, 0.18-0.72), former (OR, 0.51; 95% CI, 0.34-0.76), and current smokers (OR, 0.42; 95% CI, 0.27-0.65). CONCLUSION Serum levels of vitamin B(6) and methionine were inversely associated with risk of lung cancer.

Systemic Markers of Interferon-γ–Mediated Immune Activation and Long-Term Prognosis in Patients With Stable Coronary Artery Disease

Objective—Interferon &ggr; (IFN-&ggr;) is centrally involved in atherosclerosis-related inflammation, but its activity cannot be reliably assessed by systemic measurements. In activated macrophages, IFN-&ggr; stimulates production of neopterin and conversion of tryptophan to kynurenine. We evaluated the relationships of plasma neopterin and plasma kyunernine:tryptophan ratio (KTR) to long-term prognosis in patients with stable angina pectoris and angiographically verified significant coronary artery disease. Methods and Results—Samples were obtained from 2380 patients with a mean age of 63.7 years; 77.3% were men. During a median follow-up of 56 months, 10.8% of patients experienced a major coronary event (MCE), and 9.5% died. For MCE, each SD increment of neopterin and KTR (logarithmically transformed) was associated with multivariable adjusted hazard ratios and 95% CIs of 1.28 (1.10 to 1.48) and 1.28 (1.12 to 1.48), respectively. The corresponding hazard ratios (95% CIs) for all-cause mortality were 1.40 (1.21 to 1.62) (neopterin) and 1.23 (1.06 to 1.43) (KTR). Conclusion—In patients with stable angina pectoris, systemic markers of IFN-&ggr; activity, plasma neopterin, and plasma KTR provide similar risk estimates for MCE and mortality. Our results support experimental data linking IFN-&ggr; to acute atherosclerotic complications.

Self-reported smoking status and plasma cotinine concentrations among pregnant women in the Norwegian Mother and Child Cohort Study

Introduction:Underreporting of smoking in epidemiologic studies is common and may constitute a validity problem, leading to biased association measures. In this prospective study, we validated self-reported tobacco use against nicotine exposure assessed by plasma cotinine in the Norwegian Mother and Child Cohort Study (MoBa).Methods:The study was based on a subsample of 2,997 women in the MoBa study who delivered infants during the period 2002–2003. Self-reported tobacco use (test variable) and plasma cotinine concentrations (gold standard) were assessed at approximately gestational week 18.Results:Daily smoking was reported by 9% of the women, occasional smoking by 4%, and nonsmoking by 86% of the women. Sensitivity and specificity for self-reported smoking status were calculated using a cotinine cut-off estimated from the study population (30 nmol/l). Plasma cotinine concentrations ≥30 nmol/l were found in 94% of self-reported daily smokers, 66% of occasional smokers, and 2% of nonsmokers. After the numbers of self-reported nonsmokers with cotinine concentrations above the cut-off limit were added, the daily smoking prevalence increased from 9 to 11%. The sensitivity and specificity for self-reported daily smoking, using 30 nmol/l as the cut-off concentration, were 82 and 99%, respectively.Discussion:These findings suggest that self-reported tobacco use is a valid marker for tobacco exposure in the MoBa cohort.

Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns

Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illuminas HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring.

Quantitative profiling of folate and one-carbon metabolism in large-scale epidemiological studies by mass spectrometry

Abstract Background: Derangements of one-carbon metabolism have been related to the development of chronic diseases. Metabolic profiling as part of epidemiological studies in this area should include intermediates involved in the transfer of one-carbon units, cofactors for the relevant enzymes and markers of inflammation, kidney function and smoking. Methods: We established five platforms that measured 6–16 analytes each. Platforms A (gas chromatography-mass spectrometry; GC-MS) and B (gas chromatography-tandem mass spectrometry; GC-MS/MS) involved methylchloroformate derivatization of primary amines, thiols and carboxylic acids. Platform C determined basic compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using an ether-linked phenyl reversed-phase column. Platforms D and E (LC-MS/MS) exploited the efficient ionization and high sensitivity obtained for a wide range of analytes, using a mobile phase containing a high concentration of acetic acid. The chromatographic run times ranged from 3 to 8 min. Results: The analyte concentrations ranged from 0.2 nmol/L to 400 μmol/L. Platforms A and B both measured methylmalonic acid, total homocysteine and related amino acids. Platform B also included sarcosine, cystathionine, tryptophan and kynurenine. Platform C was optimized for the measurement of choline and betaine, but also included arginine, asymmetric and symmetric dimethylarginine and creatinine. A diversity of low abundance compounds mainly occurring in the nanomolar range were measured on platform D. These were vitamin B2 and B6 species, neopterin, cotinine and tryptophan metabolites. Platform E measured folates and folate catabolites. Conclusions: Approximately 40 analytes related to one-carbon metabolism were determined in less than 1 mL of plasma/serum using five complementary analytical platforms. As a method control, several metabolites were measured on two or more platforms. Logistics and data handling were carried out by specially designed software. This strategy allows profiling of one-carbon metabolism in large-scale epidemiological studies. Clin Chem Lab Med 2007;45:1737–45.

Prognostic Impact of Vitamin B6 Metabolism in Lung Cancer

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

Maternal smoking and DNA methylation in newborns: In utero effect or epigenetic inheritance?

Background: Maternal smoking in pregnancy is associated with adverse health outcomes in children, including cancers; underlying mechanisms may include epigenetic modifications. Using Illuminas 450K array, we previously identified differential DNA methylation related to maternal smoking during pregnancy at 26 CpG sites (CpGs) in 10 genes in newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). Whether these methylation signals in newborns reflect in utero exposure only or possibly epigenetic inheritance of smoking-related modifications is unclear. Methods: We therefore evaluated the impact of the timing of mothers smoking (before or during pregnancy using cotinine measured at 18 weeks gestation), the fathers smoking before conception, and the grandmothers smoking during her pregnancy with the mother on methylation at these 26 CpGs in 1,042 MoBa newborns. We used robust linear regression, adjusting for covariates, applying Bonferroni correction. Results: The strongest and only statistically significant associations were observed for sustained smoking by the mother during pregnancy through at least gestational week 18 (P < 1.6 × 10−5 for all 26 CpGs). We observed no statistically significant differential methylation due to smoking by the mother before pregnancy or that ceased by week 18, fathers smoking before conception, or grandmothers smoking while pregnant with the mother. Conclusions: Differential methylation at these CpGs in newborns seems to reflect sustained in utero exposure rather than epigenetic inheritance. Impact: Smoking cessation in early pregnancy may negate effects on methylation. Analyses of maternal smoking during pregnancy and offspring health outcomes, including cancer, limited to ever smoking might miss true associations. Cancer Epidemiol Biomarkers Prev; 23(6); 1007–17. ©2014 AACR.

Kinetic Modeling of Storage Effects on Biomarkers Related to B Vitamin Status and One-Carbon Metabolism

BACKGROUND Biomarkers and metabolites related to B vitamin function and one-carbon metabolism have been studied as predictors of chronic diseases in studies based on samples stored in biobanks. For most biomarkers, stability data are lacking or fragmentary. METHODS Degradation and accumulation kinetics of 32 biomarkers were determined at 23 °C in serum and plasma (EDTA, heparin, and citrate) collected from 16 individuals and stored for up to 8 days. In frozen serum (-25 °C), stability was studied cross-sectionally in 650 archival samples stored for up to 29 years. Concentration vs time curves were fitted to monoexponential, biexponential, linear, and nonlinear models. RESULTS For many biomarkers, stability was highest in EDTA plasma. Storage effects were similar at room temperature and at -25 °C; notable exceptions were methionine, which could be recovered as methionine sulfoxide, and cystathionine, which decreased in frozen samples. Cobalamin, betaine, dimethylglycine, sarcosine, total homocysteine, total cysteine, tryptophan, asymetric and symmetric dimethyl argenine, creatinine, and methylmalonic acid were essentially stable under all conditions. Most B vitamins (folate and vitamins B2 and B6) were unstable; choline increased markedly, and some amino acids also increased, particularly in serum. The kynurenines showed variable stability. For many biomarkers, degradation (folate and flavin mononucleotide) or accumulation (pyridoxal, riboflavin, choline, amino acids) kinetics at room temperature were non-first order. CONCLUSIONS Data on stability and deterioration kinetics for individual biomarkers are required to optimize procedures for handling serum and plasma, and for addressing preanalytical bias in epidemiological and clinical studies.