Caroline L Relton

Pathogenic Mitochondrial DNA Mutations Are Common in the General Population

Mitochondrial DNA (mtDNA) mutations are a major cause of genetic disease, but their prevalence in the general population is not known. We determined the frequency of ten mitochondrial point mutations in 3168 neonatal-cord-blood samples from sequential live births, analyzing matched maternal-blood samples to estimate the de novo mutation rate. mtDNA mutations were detected in 15 offspring (0.54%, 95% CI = 0.30–0.89%). Of these live births, 0.00107% (95% CI = 0.00087–0.0127) harbored a mutation not detected in the mothers blood, providing an estimate of the de novo mutation rate. The most common mutation was m.3243A→G. m.14484T→C was only found on sub-branches of mtDNA haplogroup J. In conclusion, at least one in 200 healthy humans harbors a pathogenic mtDNA mutation that potentially causes disease in the offspring of female carriers. The exclusive detection of m.14484T→C on haplogroup J implicates the background mtDNA haplotype in mutagenesis. These findings emphasize the importance of developing new approaches to prevent transmission.

Induction of Epigenetic Alterations by Dietary and Other Environmental Factors

Dietary and other environmental factors induce epigenetic alterations which may have important consequences for cancer development. This chapter summarizes current knowledge of the impact of dietary, lifestyle, and environmental determinants of cancer risk and proposes that effects of these exposures might be mediated, at least in part, via epigenetic mechanisms. Evidence is presented to support the hypothesis that all recognized epigenetic marks (including DNA methylation, histone modification, and microRNA (miRNA) expression) are influenced by environmental exposures, including diet, tobacco, alcohol, physical activity, stress, environmental carcinogens, genetic factors, and infectious agents which play important roles in the etiology of cancer. Some of these epigenetic modifications change the expression of tumor suppressor genes and oncogenes and, therefore, may be causal for tumorigenesis. Further work is required to understand the mechanisms through which specific environmental factors produce epigenetic changes and to identify those changes which are likely to be causal in the pathogenesis of cancer and those which are secondary, or bystander, effects. Given the plasticity of epigenetic marks in response to cancer-related exposures, such epigenetic marks are attractive candidates for the development of surrogate endpoints which could be used in dietary or lifestyle intervention studies for cancer prevention. Future research should focus on identifying epigenetic marks which are (i) validated as biomarkers for the cancer under study; (ii) readily measured in easily accessible tissues, for example, blood, buccal cells, or stool; and (iii) altered in response to dietary or lifestyle interventions for which there is convincing evidence for a relationship with cancer risk.

Two-step epigenetic Mendelian randomization: a strategy for establishing the causal role of epigenetic processes in pathways to disease

The burgeoning interest in the field of epigenetics has precipitated the need to develop approaches to strengthen causal inference when considering the role of epigenetic mediators of environmental exposures on disease risk. Epigenetic markers, like any other molecular biomarker, are vulnerable to confounding and reverse causation. Here, we present a strategy, based on the well-established framework of Mendelian randomization, to interrogate the causal relationships between exposure, DNA methylation and outcome. The two-step approach first uses a genetic proxy for the exposure of interest to assess the causal relationship between exposure and methylation. A second step then utilizes a genetic proxy for DNA methylation to interrogate the causal relationship between DNA methylation and outcome. The rationale, origins, methodology, advantages and limitations of this novel strategy are presented.

Epigenetic epidemiology of common complex disease: prospects for prediction, prevention, and treatment.

As part of the PLoS Epigenetics Collection, Caroline Relton and George Davey Smith discuss the potential of epigenetics for the treatment and prevention of common complex diseases, including cancer.

Prenatal exposure to maternal smoking and offspring DNA methylation across the lifecourse: findings from the Avon Longitudinal Study of Parents and Children (ALSPAC)

Maternal smoking during pregnancy has been found to influence newborn DNA methylation in genes involved in fundamental developmental processes. It is pertinent to understand the degree to which the offspring methylome is sensitive to the intensity and duration of prenatal smoking. An investigation of the persistence of offspring methylation associated with maternal smoking and the relative roles of the intrauterine and postnatal environment is also warranted. In the Avon Longitudinal Study of Parents and Children, we investigated associations between prenatal exposure to maternal smoking and offspring DNA methylation at multiple time points in approximately 800 mother–offspring pairs. In cord blood, methylation at 15 CpG sites in seven gene regions (AHRR, MYO1G, GFI1, CYP1A1, CNTNAP2, KLF13 and ATP9A) was associated with maternal smoking, and a dose-dependent response was observed in relation to smoking duration and intensity. Longitudinal analysis of blood DNA methylation in serial samples at birth, age 7 and 17 years demonstrated that some CpG sites showed reversibility of methylation (GFI1, KLF13 and ATP9A), whereas others showed persistently perturbed patterns (AHRR, MYO1G, CYP1A1 and CNTNAP2). Of those showing persistence, we explored the effect of postnatal smoke exposure and found that the major contribution to altered methylation was attributed to a critical window of in utero exposure. A comparison of paternal and maternal smoking and offspring methylation showed consistently stronger maternal associations, providing further evidence for causal intrauterine mechanisms. These findings emphasize the sensitivity of the methylome to maternal smoking during early development and the long-term impact of such exposure.

Gene–gene interaction in folate-related genes and risk of neural tube defects in a UK population

Objective: To investigate the contribution of polymorphic variation in genes involved in the folate-dependent homocysteine pathway in the aetiology of neural tube defects (NTD). Design: Case-control association study. Subjects: A total of 530 individuals from families affected by NTD, 645 maternal controls, and 602 healthy newborn controls from the northern UK. Main outcome measures: Seven polymorphisms in six genes coding for proteins in the folate-dependent homocysteine pathway (MTHFR 677C→T, MTHFR 1298A→C, MTRR 66A→G, SHMT 1420C→T, CβS 844ins68, GCPII 1561C→T, RFC-1 80G→A). The impact of each polymorphism and the effect of gene–gene interactions (epistasis) upon risk of NTD were assessed using logistic regression analysis. Results: The MTHFR 677C→T polymorphism was shown to represent a risk factor in NTD cases (CC v CT+TT odds ratio (OR) 2.03 [95% confidence interval (CI) 1.09, 3.79] p = 0.025) and the MTRR 66A→G polymorphism was shown to exert a protective effect in NTD cases (AA v AG+GG OR 0.31 [95% CI 0.10, 0.94] p = 0.04). When statistical tests for interaction were conducted, three genotype combinations in cases (MTRR/GCPII; MTHFR 677/CβS; MTHFR 677/MTRR) and one combination in case mothers (CβS/RFC-1) were shown to elevate NTD risk. Maternal–fetal interaction was also detected when offspring carried the MTHFR 677C→T variant and mothers carried the MTRR 66A→G variant, resulting in a significantly elevated risk of NTD. Conclusion: Both independent genetic effects and gene–gene interaction were observed in relation to NTD risk. Multi-locus rather than single locus analysis might be preferable to gain an accurate assessment of genetic susceptibility to NTD.

Association Between Genetic Variants on Chromosome 15q25 Locus and Objective Measures of Tobacco Exposure

Background Two single-nucleotide polymorphisms, rs1051730 and rs16969968, located within the nicotinic acetylcholine receptor gene cluster on chromosome 15q25 locus, are associated with heaviness of smoking, risk for lung cancer, and other smoking-related health outcomes. Previous studies have typically relied on self-reported smoking behavior, which may not fully capture interindividual variation in tobacco exposure. Methods We investigated the association of rs1051730 and rs16969968 genotype (referred to as rs1051730–rs16969968, because these are in perfect linkage disequilibrium and interchangeable) with both self-reported daily cigarette consumption and biochemically measured plasma or serum cotinine levels among cigarette smokers. Summary estimates and descriptive statistical data for 12 364 subjects were obtained from six independent studies, and 2932 smokers were included in the analyses. Linear regression was used to calculate the per-allele association of rs1051730–rs16969968 genotype with cigarette consumption and cotinine levels in current smokers for each study. Meta-analysis of per-allele associations was conducted using a random effects method. The likely resulting association between genotype and lung cancer risk was assessed using published data on the association between cotinine levels and lung cancer risk. All statistical tests were two-sided. Results Pooled per-allele associations showed that current smokers with one or two copies of the rs1051730–rs16969968 risk allele had increased self-reported cigarette consumption (mean increase in unadjusted number of cigarettes per day per allele = 1.0 cigarette, 95% confidence interval [CI] = 0.57 to 1.43 cigarettes, P = 5.22 × 10−6) and cotinine levels (mean increase in unadjusted cotinine levels per allele = 138.72 nmol/L, 95% CI = 97.91 to 179.53 nmol/L, P = 2.71 × 10−11). The increase in cotinine levels indicated an increased risk of lung cancer with each additional copy of the rs1051730–rs16969968 risk allele (per-allele odds ratio = 1.31, 95% CI = 1.21 to 1.42). Conclusions Our data show a stronger association of rs1051730–rs16969968 genotype with objective measures of tobacco exposure compared with self-reported cigarette consumption. The association of these variants with lung cancer risk is likely to be mediated largely, if not wholly, via tobacco exposure.

Epigenome-wide association of DNA methylation markers in peripheral blood from Indian Asians and Europeans with incident type 2 diabetes: a nested case-control study.

BACKGROUND Indian Asians, who make up a quarter of the worlds population, are at high risk of developing type 2 diabetes. We investigated whether DNA methylation is associated with future type 2 diabetes incidence in Indian Asians and whether differences in methylation patterns between Indian Asians and Europeans are associated with, and could be used to predict, differences in the magnitude of risk of developing type 2 diabetes. METHODS We did a nested case-control study of DNA methylation in Indian Asians and Europeans with incident type 2 diabetes who were identified from the 8-year follow-up of 25 372 participants in the London Life Sciences Prospective Population (LOLIPOP) study. Patients were recruited between May 1, 2002, and Sept 12, 2008. We did epigenome-wide association analysis using samples from Indian Asians with incident type 2 diabetes and age-matched and sex-matched Indian Asian controls, followed by replication testing of top-ranking signals in Europeans. For both discovery and replication, DNA methylation was measured in the baseline blood sample, which was collected before the onset of type 2 diabetes. Epigenome-wide significance was set at p<1 × 10(-7). We compared methylation levels between Indian Asian and European controls without type 2 diabetes at baseline to estimate the potential contribution of DNA methylation to increased risk of future type 2 diabetes incidence among Indian Asians. FINDINGS 1608 (11·9%) of 13 535 Indian Asians and 306 (4·3%) of 7066 Europeans developed type 2 diabetes over a mean of 8·5 years (SD 1·8) of follow-up. The age-adjusted and sex-adjusted incidence of type 2 diabetes was 3·1 times (95% CI 2·8-3·6; p<0·0001) higher among Indian Asians than among Europeans, and remained 2·5 times (2·1-2·9; p<0·0001) higher after adjustment for adiposity, physical activity, family history of type 2 diabetes, and baseline glycaemic measures. The mean absolute difference in methylation level between type 2 diabetes cases and controls ranged from 0·5% (SD 0·1) to 1·1% (0·2). Methylation markers at five loci were associated with future type 2 diabetes incidence; the relative risk per 1% increase in methylation was 1·09 (95% CI 1·07-1·11; p=1·3 × 10(-17)) for ABCG1, 0·94 (0·92-0·95; p=4·2 × 10(-11)) for PHOSPHO1, 0·94 (0·92-0·96; p=1·4 × 10(-9)) for SOCS3, 1·07 (1·04-1·09; p=2·1 × 10(-10)) for SREBF1, and 0·92 (0·90-0·94; p=1·2 × 10(-17)) for TXNIP. A methylation score combining results for the five loci was associated with future type 2 diabetes incidence (relative risk quartile 4 vs quartile 1 3·51, 95% CI 2·79-4·42; p=1·3 × 10(-26)), and was independent of established risk factors. Methylation score was higher among Indian Asians than Europeans (p=1 × 10(-34)). INTERPRETATION DNA methylation might provide new insights into the pathways underlying type 2 diabetes and offer new opportunities for risk stratification and prevention of type 2 diabetes among Indian Asians. FUNDING The European Union, the UK National Institute for Health Research, the Wellcome Trust, the UK Medical Research Council, Action on Hearing Loss, the UK Biotechnology and Biological Sciences Research Council, the Oak Foundation, the Economic and Social Research Council, Helmholtz Zentrum Munchen, the German Research Center for Environmental Health, the German Federal Ministry of Education and Research, the German Center for Diabetes Research, the Munich Center for Health Sciences, the Ministry of Science and Research of the State of North Rhine-Westphalia, and the German Federal Ministry of Health.

DNA Methylation Patterns in Cord Blood DNA and Body Size in Childhood

Background Epigenetic markings acquired in early life may have phenotypic consequences later in development through their role in transcriptional regulation with relevance to the developmental origins of diseases including obesity. The goal of this study was to investigate whether DNA methylation levels at birth are associated with body size later in childhood. Principal Findings A study design involving two birth cohorts was used to conduct transcription profiling followed by DNA methylation analysis in peripheral blood. Gene expression analysis was undertaken in 24 individuals whose biological samples and clinical data were collected at a mean ± standard deviation (SD) age of 12.35 (0.95) years, the upper and lower tertiles of body mass index (BMI) were compared with a mean (SD) BMI difference of 9.86 (2.37) kg/m2. This generated a panel of differentially expressed genes for DNA methylation analysis which was then undertaken in cord blood DNA in 178 individuals with body composition data prospectively collected at a mean (SD) age of 9.83 (0.23) years. Twenty-nine differentially expressed genes (>1.2-fold and p<10−4) were analysed to determine DNA methylation levels at 1–3 sites per gene. Five genes were unmethylated and DNA methylation in the remaining 24 genes was analysed using linear regression with bootstrapping. Methylation in 9 of the 24 (37.5%) genes studied was associated with at least one index of body composition (BMI, fat mass, lean mass, height) at age 9 years, although only one of these associations remained after correction for multiple testing (ALPL with height, p Corrected = 0.017). Conclusions DNA methylation patterns in cord blood show some association with altered gene expression, body size and composition in childhood. The observed relationship is correlative and despite suggestion of a mechanistic epigenetic link between in utero life and later phenotype, further investigation is required to establish causality.

Type 2 Diabetes TCF7L2 Risk Genotypes Alter Birth Weight: A Study of 24,053 Individuals

The role of genes in normal birth-weight variation is poorly understood, and it has been suggested that the genetic component of fetal growth is small. Type 2 diabetes genes may influence birth weight through maternal genotype, by increasing maternal glycemia in pregnancy, or through fetal genotype, by altering fetal insulin secretion. We aimed to assess the role of the recently described type 2 diabetes gene TCF7L2 in birth weight. We genotyped the polymorphism rs7903146 in 15,709 individuals whose birth weight was available from six studies and in 8,344 mothers from three studies. Each fetal copy of the predisposing allele was associated with an 18-g (95% confidence interval [CI] 7-29 g) increase in birth weight (P=.001) and each maternal copy with a 30-g (95% CI 15-45 g) increase in offspring birth weight (P=2.8x10-5). Stratification by fetal genotype suggested that the association was driven by maternal genotype (31-g [95% CI 9-48 g] increase per allele; corrected P=.003). Analysis of diabetes-related traits in 10,314 nondiabetic individuals suggested the most likely mechanism is that the risk allele reduces maternal insulin secretion (disposition index reduced by ~0.15 standard deviation; P=1x10-4), which results in increased maternal glycemia in pregnancy and hence increased offspring birth weight. We combined information with the other common variant known to alter fetal growth, the -30G-->A polymorphism of glucokinase (rs1799884). The 4% of offspring born to mothers carrying three or four risk alleles were 119 g (95% CI 62-172 g) heavier than were the 32% born to mothers with none (for overall trend, P=2x10-7), comparable to the impact of maternal smoking during pregnancy. In conclusion, we have identified the first type 2 diabetes-susceptibility allele to be reproducibly associated with birth weight. Common gene variants can substantially influence normal birth-weight variation.