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Background Previous studies have found that children born with a non-syndromic orofacial cleft have lower-than-average educational attainment. Differences could be due to a genetic predisposition to low intelligence and academic performance, factors arising due to the cleft phenotype (such as social stigmatization, impaired speech/language development) or confounding by the prenatal environment. A clearer understanding of this mechanism will inform interventions to improve educational attainment in individuals born with a cleft, which could substantially improve their quality of life. We assessed evidence for the hypothesis that common variant genetic liability to non-syndromic cleft lip with or without cleft palate (nsCL/P) influences educational attainment. Methods We performed a genome-wide association study (GWAS) meta-analysis of nsCL/P with 1692 nsCL/P cases and 4259 parental and unrelated controls. Using GWAS summary statistics, we performed Linkage Disequilibrium (LD)-score regression to estimate the genetic correlation between nsCL/P, educational attainment (GWAS n = 766 345) and intelligence (GWAS n = 257 828). We used two-sample Mendelian randomization to evaluate the causal effects of genetic liability to nsCL/P on educational attainment and intelligence. Results There was limited evidence for shared genetic aetiology or causal relationships between nsCL/P and educational attainment [genetic correlation (rg) −0.05, 95% confidence interval (CI) −0.12 to 0.01, P 0.13; MR estimate (βMR) −0.002, 95% CI −0.009 to 0.006, P 0.679) or intelligence (rg −0.04, 95% CI −0.13 to 0.04, P 0.34; βMR −0.009, 95% CI −0.02 to 0.002, P 0.11). Conclusions Common variants are unlikely to predispose individuals born with nsCL/P to low educational attainment or intelligence. This is an important first step towards understanding the aetiology of low educational attainment in this group.

A DNA bank has been created from the Millennium Cohort Study (MCS) saliva samples. A total of 23,336 samples are available, from 9,259 cohort members (4,630 males and 4,629 females), 8,898 mothers and 5,179 fathers. There are 4,533 mother, child, father ‘triads’. This paper describes the collection of the saliva samples from cohort members and their biological parents in the MCS. It analyses response rates and predictors of response, and details the DNA extraction, genotyping and imputation procedures performed on the data.Key messagesIt describes the collection of saliva samples in the Millennium Cohort Study, from cohort members and their resident biological parents.It analyses response rates and predictors of response.It details the DNA extraction, genotyping and imputation procedures performed on the data.

DNA methylation quantitative trait locus (mQTL) analyses on 32,851 participants identify genetic variants associated with DNA methylation at 420,509 sites in blood, resulting in a database of >270,000 independent mQTLs. Characterizing genetic influences on DNA methylation (DNAm) provides an opportunity to understand mechanisms underpinning gene regulation and disease. In the present study, we describe results of DNAm quantitative trait locus (mQTL) analyses on 32,851 participants, identifying genetic variants associated with DNAm at 420,509 DNAm sites in blood. We present a database of >270,000 independent mQTLs, of which 8.5% comprise long-range (trans) associations. Identified mQTL associations explain 15-17% of the additive genetic variance of DNAm. We show that the genetic architecture of DNAm levels is highly polygenic. Using shared genetic control between distal DNAm sites, we constructed networks, identifying 405 discrete genomic communities enriched for genomic annotations and complex traits. Shared genetic variants are associated with both DNAm levels and complex diseases, but only in a minority of cases do these associations reflect causal relationships from DNAm to trait or vice versa, indicating a more complex genotype-phenotype map than previously anticipated.

Characterizing genetic influences on DNA methylation (DNAm) provides an opportunity to understand mechanisms underpinning gene regulation and disease. Here we describe results of DNA methylation-quantitative trait loci (mQTL) analyses on 32,851 participants, identifying genetic variants associated with DNAm at 420,509 DNAm sites in blood. We present a database of >270,000 independent mQTL of which 8.5% comprise long-range (trans) associations. Identified mQTL associations explain 15-17% of the additive genetic variance of DNAm. We reveal that the genetic architecture of DNAm levels is highly polygenic and DNAm exhibits signatures of negative and positive natural selection. Using shared genetic control between distal DNAm sites we construct networks, identifying 405 discrete genomic communities enriched for genomic annotations and complex traits. Shared genetic factors are associated with both blood DNAm levels and complex diseases but in most cases these associations do not reflect causal relationships from DNAm to trait or vice versa indicating a more complex genotype-phenotype map than has previously been hypothesised.

Background Epigenetic data could help identify risk factors for orofacial clefts, either by revealing a causal role for epigenetic mechanisms in causing clefts or by capturing information about causal genetic or environmental factors. Given the evidence that different subtypes of orofacial cleft have distinct aetiologies, we explored whether children with different cleft subtypes showed distinct epigenetic profiles. Methods In whole-blood samples from 150 children from the Cleft Collective cohort study, we measured DNA methylation at over 450,000 sites on the genome. We then carried out epigenome-wide association studies (EWAS) to test the association between methylation at each site and cleft subtype (cleft lip only (CLO) n = 50; cleft palate only (CPO) n = 50; cleft lip and palate (CLP) n = 50). We also compared methylation in the blood to methylation in the lip or palate tissue using genome-wide data from the same 150 children and conducted an EWAS of CLO compared to CLP in lip tissue. Results We found four genomic regions in blood differentially methylated in CLO compared to CLP, 17 in CPO compared to CLP and 294 in CPO compared to CLO. Several regions mapped to genes that have previously been implicated in the development of orofacial clefts (for example, TBX1, COL11A2, HOXA2, PDGFRA), and over 250 associations were novel. Methylation in blood correlated with that in lip/palate at some regions. There were 14 regions differentially methylated in the lip tissue from children with CLO and CLP, with one region (near KIAA0415) showing up in both the blood and lip EWAS. Conclusions Our finding of distinct methylation profiles in different orofacial cleft (OFC) subtypes represents a promising first step in exploring the potential role of epigenetic modifications in the aetiology of OFCs and/or as clinically useful biomarkers of OFC subtypes. Electronic supplementary material The online version of this article (doi:10.1186/s13148-017-0362-2) contains supplementary material, which is available to authorized users.

Background Smoking status, alcohol consumption and HPV infection (acquired through sexual activity) are the predominant risk factors for oropharyngeal cancer and are thought to alter the prognosis of the disease. Here, we conducted single-site and differentially methylated region (DMR) epigenome-wide association studies (EWAS) of these factors, in addition to ∼ 3-year survival, using Illumina Methylation EPIC DNA methylation profiles from whole blood in 409 individuals as part of the Head and Neck 5000 (HN5000) study. Overlapping sites between each factor and survival were then assessed using two-step Mendelian randomization to assess whether methylation at these positions causally affected survival. Results Using the MethylationEPIC array in an OPC dataset, we found novel CpG associations with smoking, alcohol consumption and ~ 3-year survival. We found no CpG associations below our multiple testing threshold associated with HPV16 E6 serological response (used as a proxy for HPV infection). CpG site associations below our multiple-testing threshold (PBonferroni < 0.05) for both a prognostic factor and survival were observed at four gene regions: SPEG (smoking), GFI1 (smoking), PPT2 (smoking) and KHDC3L (alcohol consumption). Evidence for a causal effect of DNA methylation on survival was only observed in the SPEG gene region (HR per SD increase in methylation score 1.28, 95% CI 1.14 to 1.43, P 2.12 × 10−05). Conclusions Part of the effect of smoking on survival in those with oropharyngeal cancer may be mediated by methylation at the SPEG gene locus. Replication in data from independent datasets and data from HN5000 with longer follow-up times is needed to confirm these findings.

Importance Previous studies have found that children born with a non-syndromic form of cleft lip and/or palate have lower-than-average educational attainment. These differences could be due to a genetic predisposition to low intelligence and academic performance, factors arising due to the cleft phenotype (such as school absence, social stigmatization and impaired speech and language development), or confounding by the prenatal environment. If the association is not explained by a genetic predisposition, interventions and policies to improve educational attainment in individuals born with a cleft could have wide-ranging knock-on effects on their quality of life. Objective To assess evidence for the hypothesis that genetic liability to non-syndromic cleft lip with or without cleft palate (nsCL/P) influences educational attainment. Methods Using summary data from genome-wide association studies (GWAS), we performed Linkage Disequilibrium (LD)-score regression and two-sample Mendelian randomization to evaluate the relationship between genetic liability to nsCL/P (GWAS n=3,987) and educational attainment (GWAS n=766,345), and intelligence (GWAS n=257,828). Results There was little evidence for shared genetic aetiology between nsCL/P and educational attainment (rg −0.03, 95% CI −0.14 to 0.08, P 0.58; βMR 0.002, 95% CI −0.001 to 0.005, P 0.417) or intelligence (rg −0.01, 95% CI −0.12 to 0.10, P 0.85; βMR 0.002, 95% CI −0.010 to 0.014, P 0.669). Conclusions and relevance Individuals born with nsCL/P are unlikely to be genetically predisposed to low educational attainment or intelligence. Therefore, clinical-, school-, social- and family-level interventions to improve educational attainment could be effective. Research summary Evidence before this study Previous studies have found that children born with a cleft lip/palate tend to have lower-than-average educational attainment, even in the absence of other birth defects or known syndromes. These differences could be due to a genetic predisposition to low intelligence caused by undiagnosed congenital differences in brain structure or function. Alternatively, these differences might be explained by downstream factors related to having a cleft, such as higher school absences to attend healthcare appointments, social stigmatization, or impaired speech and language development. The differences might also be explained by confounding factors such as family socioeconomic position or parental health behaviours (for example smoking or drinking alcohol). None of these proposed explanations has been investigated in detail and it is currently unclear why individuals born with a cleft tend to do less well at school. Added value of this study Using a causal inference technique called Mendelian randomization, this study suggests that any association between non-syndromic cleft lip/palate and lower educational attainment is unlikely to be caused by a genetic predisposition to both cleft and low intelligence or academic achievement. Implications of all the available evidence This study provides reassurance that non-syndromic children born with cleft lip/palate are unlikely to be genetically predisposed to do poorly at school and have lower intelligence. Therefore, interventions and policies to improve educational attainment in children born with a cleft could be effective. The target of these interventions will depend on further research to understand the causes of lower educational attainment in this group. This highlights the need for a largescale longitudinal patient cohort study combining genetic data with detailed information on clinical, social, environmental and developmental factors. The Cleft Collective Cohort Study has been established to address this need.

Objectives Head and neck squamous cell carcinoma (HNSCC) is often associated with chronic systemic inflammation (SI). In the present study, we assessed if DNA methylation-derived SI (mdSI) indices: Neutrophil-to-Lymphocyte ratio (mdNLR) and Lymphocyte-to-Monocyte ratio (mdLMR) are associated with the presence of HNSCC and overall survival (OS). Materials and methods We used two peripheral blood DNA methylation datasets: an HNSCC case-control dataset (n = 183) and an HNSCC survival dataset (n = 407) to estimate mdSI indices. We then performed multivariate regressions to test the association between mdSI indices, HNSCC development and OS. Results Multivariate logistic regression revealed that elevated mdNLR was associated with increased odds of being an HNSCC case (OR = 3.25, 95% CI = 2.14–5.34, P = 4 × 10−7) while the converse was observed for mdLMR (OR = 0.88, 95% CI = 0.81–0.90, P = 2 × 10−3). In the HNSCC survival dataset, HPV16-E6 seropositive HNSCC cases had an elevated mdLMR (P = 9 × 10−5) and a lower mdNLR (P = 0.003) compared to seronegative patients. Multivariate Cox regression in the HNSCC survival dataset revealed that lower mdLMR (HR = 1.96, 95% CI = 1.30–2.95, P = 0.0013) but not lower mdNLR (HR = 0.68, 95% CI = 0.46–1.00, P = 0.0501) was associated with increased risk of death. Conclusion Our results indicate that mdSI estimated by DNA methylation data is associated with the presence of HNSCC and overall survival. The mdSI indices may be used as a valuable research tool to reliably estimate SI in the absence of cell-based estimates. Rigorous validation of our findings in large prospective studies is warranted in the future.

Data Resource Profile: Accessible Resource for Integrated Epigenomic Studies (ARIES) Caroline L Relton, Tom Gaunt, Wendy McArdle, Karen Ho, Aparna Duggirala, Hashem Shihab, Geoff Woodward, Oliver Lyttleton, David M Evans, Wolf Reik, Yu-Lee Paul, Gabriella Ficz, Susan E Ozanne, Anil Wipat, Keith Flanagan, Allyson Lister, Bastiaan T Heijmans, Susan M Ring and George Davey Smith MRC Integrative Epidemiology Unit, and School of Social and Community Medicine, University of Bristol, Bristol, UK, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK, University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, WA, Australia, Babraham Institute, Cambridge, UK, Wellcome Trust Sanger Institute, Cambridge, UK, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK, University of Cambridge Institute of Metabolic Sciences and MRC Metabolic Diseases Unit, Cambridge, UK, School of Computer Science, Newcastle University, Newcastle upon Tyne, UK and Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands

BackgroundThere is evidence that different subtypes of orofacial cleft have distinct aetiologies, although the precise molecular mechanisms underlying these are unknown. Given the key role of epigenetic processes such as DNA methylation in embryonic development, it is likely that aberrant DNA methylation may also play a part in the development of orofacial clefts.MethodsIn this study, we explored whether blood samples from children with different cleft subtypes showed distinct DNA methylation profiles.In whole blood samples from 150 children from the Cleft Collective cohort study, we measured DNA methylation at over 450,000 sites on the genome. We then carried out epigenome-wide association studies (EWAS) to test the association between methylation at each site and cleft subtype (cleft lip only CLO n=50; cleft palate only CPO n=50; cleft lip and palate CLP n=50).ResultsWe found four genomic regions differentially methylated in CLO compared to CLP, 17 in CPO compared to CLP and 294 in CPO compared to CLO. These regions included several mapping to genes that have previously been implicated in the development of orofacial clefts (for example,TBX1, COL11A2, HOXA2, PDGFRA) and over 250 novel associations.ConclusionOur finding of distinct methylation profiles in different cleft subtypes might reflect differences in their aetiologies, with DNA methylation either playing a causal role in development of OFC subtypes or reflecting causal genetic or environmental factors.

Background Genome-wide association studies have identified associations of genetic variants at 17q21 near ORMDL3 with childhood asthma. Objectives We sought to determine whether associations in this region are specific to particular asthma phenotypes and specific to ORMDL3 . Methods We examined associations between 244 independent single nucleotide polymorphisms (SNPs) plus 13 previously identified asthma-related SNPs in the region between 34 and 36 Mb on chromosome 17 and early wheezing phenotypes, doctor-diagnosed asthma and atopy at 7½ years, and bronchial hyperresponsiveness and lung function at 8½ years in 7045 children from the Avon Longitudinal Study of Parents and Children birth cohort study. With this, cis expression quantitative trait loci signals for the same SNPs were assessed in 875 samples across genes in the same region. Results The strongest evidence for phenotypic association was seen for persistent wheezing (rs8076131 near ORMDL3 : relative risk ratio [RRR], 1.60 [95% CI, 1.40-1.84], P = 1.4 × 10 −11 ; rs2305480 near GSDML : RRR, 1.60 [95% CI, 1.39-1.83], P = 1.5 × 10 −11 ; and rs9303277 near IKZF3 : RRR, 1.57 [95% CI, 1.37-1.79], P = 4.4 × 10 −11 ). Similar but less precisely estimated effects were seen for intermediate-onset wheeze, but there was little evidence of associations with other wheezing phenotypes. There was some evidence of associations with bronchial hyperresponsiveness. SNPs across the whole region show strong evidence of association with differential levels of expression at GSDML , IKZF3 , and MED24 , as well as ORMDL3. Conclusions Associations of SNPs in the 17q21 locus are specific to asthma and specific wheezing phenotypes and are not explained by associations with intermediate phenotypes, such as atopy or lung function.

Background The influence of genetic variation on complex diseases is potentially mediated through a range of highly dynamic epigenetic processes exhibiting temporal variation during development and later life. Here we present a catalogue of the genetic influences on DNA methylation (methylation quantitative trait loci (mQTL)) at five different life stages in human blood: children at birth, childhood, adolescence and their mothers during pregnancy and middle age. Results We show that genetic effects on methylation are highly stable across the life course and that developmental change in the genetic contribution to variation in methylation occurs primarily through increases in environmental or stochastic effects. Though we map a large proportion of the cis-acting genetic variation, a much larger component of genetic effects influencing methylation are acting in trans. However, only 7 % of discovered mQTL are trans-effects, suggesting that the trans component is highly polygenic. Finally, we estimate the contribution of mQTL to variation in complex traits and infer that methylation may have a causal role consistent with an infinitesimal model in which many methylation sites each have a small influence, amounting to a large overall contribution. Conclusions DNA methylation contains a significant heritable component that remains consistent across the lifespan. Our results suggest that the genetic component of methylation may have a causal role in complex traits. The database of mQTL presented here provide a rich resource for those interested in investigating the role of methylation in disease. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0926-z) contains supplementary material, which is available to authorized users.

Full text and MPG-specific services(opens in a new window)| Export | Download | Add to List | More... Cleft Palate-Craniofacial Journal Volume 53, Issue 3, May 2016, Pages 317-325 Opportunities and challenges in establishing a cohort study: An example from cleft lip/palate research in the United Kingdom (Article) Stock, N.M.a , Humphries, K.b, St. Pourcain, B.b, Bailey, M.b, Persson, M.a, Ho, K.M.b, Ring, S.b, Marsh, C.c, Albery, L.c, Rumsey, N.a, Sandy, J.b a Centre for Appearance Research, University of the West of England, Coldharbour Lane, Bristol, United Kingdom b Faculty of Medicine and Dentistry, University of Bristol, United Kingdom c South West Cleft Service, University Hospitals Bristol NHS Foundation Trust, United Kingdom Hide additional affiliations View references (32) Abstract Background: Cleft lip and/or palate (CL/P) is one of the most common birth conditions in the world, but little is known about its causes. Professional opinion remains divided as to which treatments may be the most beneficial for patients with CL/P, and the factors that contribute to psychological adjustment are poorly understood. The use of different methodological approaches and tools plays a key role in hampering efforts to address discrepancies within the evidence base. A new UK-wide program of research, The Cleft Collective, was established to combat many of these methodological challenges and to address some of the key research questions important to all CL/P stakeholders. Objective: To describe the establishment of CL/P cohort studies in the United Kingdom and to consider the many opportunities this resource will generate. Results: To date, protocols have been developed and implemented within most UK cleft teams. Biological samples, environmental information, and data pertaining to parental psychological well-being and child development are being collected successfully. Recruitment is currently on track to meet the ambitious target of approximately 9800 individuals from just more than 3000 families. Conclusions: The Cleft Collective cohort studies represent a significant step forward for research in the field of CL/P. The data collected will form a comprehensive resource of information about individuals with CL/P and their families. This resource will provide the basis for many future projects and collaborations, both in the United Kingdom and around the world.

Gene expression is a heritable cellular phenotype that defines the function of a cell and can lead to diseases in case of misregulation. In order to detect genetic variations affecting gene expression, we performed association analysis of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) with gene expression measured in 869 lymphoblastoid cell lines of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort in cis and in trans. We discovered that 3,534 genes (false discovery rate (FDR) = 5%) are affected by an expression quantitative trait locus (eQTL) in cis and 48 genes are affected in trans. We observed that CNVs are more likely to be eQTLs than SNPs. In addition, we found that variants associated to complex traits and diseases are enriched for trans-eQTLs and that trans-eQTLs are enriched for cis-eQTLs. As a variant affecting both a gene in cis and in trans suggests that the cis gene is functionally linked to the trans gene expression, we looked specifically for trans effects of cis-eQTLs. We discovered that 26 cis-eQTLs are associated to 92 genes in trans with the cis-eQTLs of the transcriptions factors BATF3 and HMX2 affecting the most genes. We then explored if the variation of the level of expression of the cis genes were causally affecting the level of expression of the trans genes and discovered several causal relationships between variation in the level of expression of the cis gene and variation of the level of expression of the trans gene. This analysis shows that a large sample size allows the discovery of secondary effects of human variations on gene expression that can be used to construct short directed gene regulatory networks., Author Summary Humans differ in their genetic sequences at millions of positions but only a subset of these differences have a functional effect. In order to detect functional genetic differences, we assessed the impact of common genetic variants on gene expression in 869 individuals and discovered that the expression of many genes is affected by common variants in cis or in trans. We show that the effect of some variants on gene expression cannot be detected in other tissues, highlighting the tissue specificity of gene regulation. In addition, we show that variants associated to common diseases are more likely to affect gene expression in cis and in trans. Finally, we show that variants affecting gene expression in cis often affect gene expression in trans, which suggests that the trans effects are due to the cis genes expression. We tested this hypothesis and discovered several cases of genes regulated in trans by a cis regulated gene in a causal manner. This shows that a population-based strategy with a large number of individuals has the potential to detect secondary effects of common variants that can be used to construct short directed regulatory networks.

Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many expression quantitative trait locus (eQTL) studies, typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono-and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis effect on expression cannot be accounted for by common cis variants, a finding that reveals the contribution of low-frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene, and we identify several replicating trans variants that act predominantly in a tissue-restricted manner and may regulate the transcription of many genes. © 2012 Nature America, Inc. All rights reserved.

Background DNA methylation is strongly associated with smoking status at multiple sites across the genome. Studies have largely been restricted to European origin individuals yet the greatest increase in smoking is occurring in low income countries, such as the Indian subcontinent. We determined whether there are differences between South Asians and Europeans in smoking related loci, and if a smoking score, combining all smoking related DNA methylation scores, could differentiate smokers from non-smokers. Results Illumina HM450k BeadChip arrays were performed on 192 samples from the Southall And Brent REvisited (SABRE) cohort. Differential methylation in smokers was identified in 29 individual CpG sites at 18 unique loci. Interaction between smoking status and ethnic group was identified at the AHRR locus. Ethnic differences in DNA methylation were identified in non-smokers at two further loci, 6p21.33 and GNG12. With the exception of GFI1 and MYO1G these differences were largely unaffected by adjustment for cell composition. A smoking score based on methylation profile was constructed. Current smokers were identified with 100% sensitivity and 97% specificity in Europeans and with 80% sensitivity and 95% specificity in South Asians. Conclusions Differences in ethnic groups were identified in both single CpG sites and combined smoking score. The smoking score is a valuable tool for identification of true current smoking behaviour. Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.