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Aims/hypothesis Several studies have identified associations between type 2 diabetes and DNA methylation (DNAm). However, the causal role of these associations remains unclear. This study aimed to provide evidence for a causal relationship between DNAm and type 2 diabetes. Methods We used bidirectional two-sample Mendelian randomisation (2SMR) to evaluate causality at 58 CpG sites previously detected in a meta-analysis of epigenome-wide association studies (meta-EWAS) of prevalent type 2 diabetes in European populations. We retrieved genetic proxies for type 2 diabetes and DNAm from the largest genome-wide association study (GWAS) available. We also used data from the Avon Longitudinal Study of Parents and Children (ALSPAC, UK) when associations of interest were not available in the larger datasets. We identified 62 independent SNPs as proxies for type 2 diabetes, and 39 methylation quantitative trait loci as proxies for 30 of the 58 type 2 diabetes-related CpGs. We applied the Bonferroni correction for multiple testing and inferred causality based on pp Results We found strong evidence of a causal effect of DNAm at cg25536676 (DHCR24) on type 2 diabetes. An increase in transformed residuals of DNAm at this site was associated with a 43% (OR 1.43, 95% CI 1.15, 1.78, p=0.001) higher risk of type 2 diabetes. We inferred a likely causal direction for the remaining CpG sites assessed. In silico analyses showed that the CpGs analysed were enriched for expression quantitative trait methylation sites (eQTMs) and for specific traits, dependent on the direction of causality predicted by the 2SMR analysis. Conclusions/interpretation We identified one CpG mapping to a gene related to the metabolism of lipids (DHCR24) as a novel causal biomarker for risk of type 2 diabetes. CpGs within the same gene region have previously been associated with type 2 diabetes-related traits in observational studies (BMI, waist circumference, HDL-cholesterol, insulin) and in Mendelian randomisation analyses (LDL-cholesterol). Thus, we hypothesise that our candidate CpG in DHCR24 may be a causal mediator of the association between known modifiable risk factors and type 2 diabetes. Formal causal mediation analysis should be implemented to further validate this assumption. Graphical Abstract

Although there are some epigenome-wide association studies (EWAS) of insulin resistance, for most of them authors did not replicate their findings, and most are focused on populations of European ancestry, limiting the generalizability. In the Epigenetics in Pregnancy (EPIPREG; n = 294 Europeans and 162 South Asians) study, we conducted an EWAS of insulin resistance in maternal peripheral blood leukocytes, with replication in the Born in Bradford (n = 879; n = 430 Europeans and 449 South Asians), Methyl Epigenome Network Association (MENA) (n = 320), and Botnia (n = 56) cohorts. In EPIPREG, we identified six CpG sites inversely associated with insulin resistance across ancestry, of which five were replicated in independent cohorts (cg02988288, cg19693031, and cg26974062 in TXNIP; cg06690548 in SLC7A11; and cg04861640 in ZSCAN26). From methylation quantitative trait loci analysis in EPIPREG, we identified gene variants related to all five replicated cross-ancestry CpG sites, which were associated with several cardiometabolic phenotypes. Mediation analyses suggested that the gene variants regulate insulin resistance through DNA methylation. To conclude, our cross-ancestry EWAS identified five CpG sites related to lower insulin resistance.

STUDY QUESTION Is cord blood DNA methylation associated with having been conceived by medically assisted reproduction? SUMMARY ANSWER This study does not provide strong evidence of an association of conception by medically assisted reproduction with variation in infant blood cell DNA methylation. WHAT IS KNOWN ALREADY Medically assisted reproduction consists of procedures used to help infertile/subfertile couples conceive, including ART. Due to its importance in gene regulation during early development programming, DNA methylation and its perturbations associated with medically assisted reproduction could reveal new insights into the biological effects of assisted reproductive technologies and potential adverse offspring outcomes. STUDY DESIGN, SIZE, DURATION We investigated the association of DNA methylation and medically assisted reproduction using a case–control study design (N = 205 medically assisted reproduction cases and N = 2439 naturally conceived controls in discovery cohorts; N = 149 ART cases and N = 58 non-ART controls in replication cohort). PARTICIPANTS/MATERIALS, SETTINGS, METHODS We assessed the association between medically assisted reproduction and DNA methylation at birth in cord blood (205 medically assisted conceptions and 2439 naturally conceived controls) at >450 000 CpG sites across the genome in two sub-samples of the UK Avon Longitudinal Study of Parents and Children (ALSPAC) and two sub-samples of the Norwegian Mother, Father and Child Cohort Study (MoBa) by meta-analysis. We explored replication of findings in the Australian Clinical review of the Health of adults conceived following Assisted Reproductive Technologies (CHART) study (N = 149 ART conceptions and N = 58 controls). MAIN RESULTS AND THE ROLE OF CHANCE The ALSPAC and MoBa meta-analysis revealed evidence of association between conception by medically assisted reproduction and DNA methylation (false-discovery-rate-corrected P-value < 0.05) at five CpG sites which are annotated to two genes (percentage difference in methylation per CpG, cg24051276: Beta = 0.23 (95% CI 0.15,0.31); cg00012522: Beta = 0.47 (95% CI 0.31, 0.63); cg17855264: Beta = 0.31 (95% CI 0.20, 0.43); cg17132421: Beta = 0.30 (95% CI 0.18, 0.42); cg18529845: Beta = 0.41 (95% CI 0.25, 0.57)). Methylation at three of these sites has been previously linked to cancer, aging, HIV infection and neurological diseases. None of these associations replicated in the CHART cohort. There was evidence of a functional role of medically assisted reproduction-induced hypermethylation at CpG sites located within regulatory regions as shown by putative transcription factor binding and chromatin remodelling. LIMITATIONS, REASONS FOR CAUTIONS While insufficient power is likely, heterogeneity in types of medically assisted reproduction procedures and between populations may also contribute. Larger studies might identify replicable variation in DNA methylation at birth due to medically assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS Newborns conceived with medically assisted procedures present with divergent DNA methylation in cord blood white cells. If these associations are true and causal, they might have long-term consequences for offspring health. STUDY FUNDING/COMPETING INTERESTS(S) This study has been supported by the US National Institute of Health (R01 DK10324), the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement no. 669545, European Union’s Horizon 2020 research and innovation programme under Grant agreement no. 733206 (LifeCycle) and the NIHR Biomedical Centre at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The UK Medical Research Council and Wellcome (Grant ref: 102215/2/13/2) and the University of Bristol provide core support for ALSPAC. Methylation data in the ALSPAC cohort were generated as part of the UK BBSRC funded (BB/I025751/1 and BB/I025263/1) Accessible Resource for Integrated Epigenomic Studies (ARIES, http://www.ariesepigenomics.org.uk). D.C., J.J., C.L.R. D.A.L and H.R.E. work in a Unit that is supported by the University of Bristol and the UK Medical Research Council (Grant nos. MC_UU_00011/1, MC_UU_00011/5 and MC_UU_00011/6). B.N. is supported by an NHMRC (Australia) Investigator Grant (1173314). ALSPAC GWAS data were generated by Sample Logistics and Genotyping Facilities at Wellcome Sanger Institute and LabCorp (Laboratory Corporation of America) using support from 23andMe. The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (Contract no. N01-ES-75558), NIH/NINDS (Grant nos. (i) UO1 NS 047537-01 and (ii) UO1 NS 047537-06A1). For this work, MoBa 1 and 2 were supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES-49019) and the Norwegian Research Council/BIOBANK (Grant no. 221097). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, Project no. 262700. D.A.L. has received support from national and international government and charity funders, as well as from Roche Diagnostics and Medtronic for research unrelated to this study. The other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Summary Background African populations are going through health transitions due to rapid urbanisation and international migration. However, the role of biological ageing in the emerging burden of cardiometabolic diseases among migrant and non-migrant Africans is unknown. We aimed to examine differences in epigenetic-age acceleration (EAA) as measured by four clocks (Horvath, Hannum, PhenoAge, and GrimAge) and their associations with cardiometabolic factors among migrant Ghanaians residing in Europe and non-migrant Ghanaians residing in Ghana using cross‑sectional data. Methods In this population-based cross-sectional RODAM substudy, recruitment of urban participants in Ghana was done in two cities (Kumasi and Obuasi), whereas recruitment in rural areas was done in 15 villages in the Ashanti region. In Europe, participants were recruited from the cities of Amsterdam (Netherlands), Berlin (Germany), and London (UK). The method and location of participant recruitment varied according to country and city. Participants were included in the RODAM study if they were older than 25 years, had completed the RODAM study questionnaire, were physically examined, and had blood samples taken. In the present subsample, data for DNA-methylation (DNAm) had to be available for the participants. We did not specify any exclusion criteria. We used genome-wide DNAm data from Ghanaians to quantify EAA. We assessed the correlation between DNAm-based age measures and chronological age, and then we did linear regressions to investigate the associations between EAA and body-mass index (BMI), fasting blood glucose (FBG), blood pressure, alcohol consumption, smoking status, physical activity, and one-carbon metabolism nutrients among migrant and non-migrant populations. We replicated our findings among rural–urban sibling pairs from the India Migration Study and among indigenous South Africans from the PURE-SA-NW study. Findings Between Feb 2, 2012, and Sept 30, 2014, 736 individuals participated in the RODAM epigenetics substudy, of which 12 (2%) were excluded during DNAm quality control, and a further 12 (2%) were excluded because of genotypic and phenotypic sex discordance. 712 (97%) of 736 participants were included in the analysis; 365 (51%) of these 712 participants were migrants and 347 (49%) were non-migrants. We found that migrant Ghanaians had lower EAA than non-migrant Ghanaians (intrinsic EAA Horvath –0·34 years vs 0·35 years; extrinsic EAA Hannum –0·86 years vs 0·90 years; PhenoAge acceleration –1·68 years vs 1·77 years; and GrimAge acceleration –0·18 years vs 0·19 years). Within migrant Ghanaians, higher FBG was positively associated with EAA measures, with the adjusted regression β for intrinsic EAA being 0·30 (95% CI 0·01 to 0·59) for migrants and 0·12 (−0·04 to 0·28) for non-migrants, for extrinsic EAA being 0·31 (0·05 to 0·56) for migrants and 0·08 (−0·06 to 0·22) for non-migrants, for PhenoAge acceleration being 0·39 (0·07 to 0·71) for migrants and 0·14 (−0·01 to 0·32) for non-migrants, and for GrimAge acceleration being 0·18 (0·01 to 0·34) for migrants and 0·12 (0·03 to 0·21) for non-migrants. Within non-migrant Ghanaians, higher BMI and vitamin-B9 (folate) intake were negatively associated with EAA measures. Our findings on FBG, BMI, and folate were replicated in the independent cohorts. Interpretation Our study shows that migration is negatively associated with EAA among Ghanaians. Moreover, cardiometabolic factors are differentially associated with EAA within migrant and non-migrant subgroups. Our results call for context-based interventions for cardiometabolic diseases among transitioning populations that account for the effects of biological ageing. Funding European Commission under the Framework Programme and European Research Council Consolidation.

AimSeveral studies have identified associations between type 2 diabetes (T2D) and DNA methylation (DNAm). However, the causal role of these associations remains unclear. This study aims to provide evidence for a causal relationship between DNA methylation and T2D.MethodsWe implemented a bidirectional two-sample Mendelian randomization (2SMR) to evaluate causality at 58 CpG sites previously detected in a meta-analysis of epigenome-wide association studies (meta-EWAS) of prevalent T2D in Europeans. We retrieved genetic proxies for T2D and DNAm from the largest GWAS available. We also used data from the Avon Longitudinal Study of Parents and Children (ALSPAC, UK) when associations of interest were not available in the larger datasets. We identified 62 independent SNPs as proxies for T2D, and 39 methylation quantitative trait loci or mQTL as proxies for 30 of the 58 T2D-related CpGs. We applied correction for multiple testing using Bonferroni and inferred causality based on a P < 1.0×10−3 or P < 2.0×10−3 for the T2D⟶ DNAm direction, and the opposing DNAm ⟶ T2D direction of the 2SMR, respectively.ResultsWe found strong evidence of causality of DNAm at cg25536676 (DHCR24) on T2D, where an increase in transformed residuals of DNAm at this site were associated with 43% (OR=1.43, 95%CI=1.15-1.78, P=0.001) higher risk of T2D. We infer a likely causal direction for the remaining CpG sites assessed. In silico analyses showed that CpGs analyzed were enriched for eQTMs, and for specific traits dependent on the direction of causality predicted by 2SMR.ConclusionsWe identified one CpG mapping to a gene related with the metabolism of lipids (DHCR24), as a novel causal biomarker for the risk of T2D. CpGs within the same gene-region have previously been associated with T2D-related traits in observational studies (BMI, waist circumference, HDL-cholesterol, insulin) and in MR analyses (LDL-cholesterol). Thus, we hypothesize that our candidate CpG in DHCR24 may be a causal mediator of the association between known modifiable risk factors and T2D. Formal causal mediation analysis should be implemented to further validate this assumption.

Ethnic differences in non-communicable disease risk have been described between individuals of South Asian and European ethnicity that are only partially explained by genetics and other known risk factors. DNA methylation is one underexplored mechanism that may explain differences in disease risk. Currently there is little knowledge of how DNA methylation varies between South Asian and European ethnicities.This study characterised differences in blood DNA methylation between individuals of self-reported European and South Asian ethnicity from two UK-based cohorts; Southall and Brent Revisited (SABRE) and Born in Bradford (BiB). DNA methylation differences between ethnicities were widespread throughout the genome (n=16,433 CpG sites, 3.4% sites tested). Specifically, 76% of associations were attributable to ethnic differences in cell composition with fewer effects attributable to smoking and genetic variation. Ethnicity associated CpG sites were enriched for EWAS Catalog phenotypes including metabolites. This work highlights the need to consider ethnic diversity in epigenetic research.

Variation in DNA methylation (DNAm) is associated with multiple biological processes that track growth and development, ageing and age-related diseases. However, there is little understanding of what constitutes typical patterns of DNAm variation and how these patterns change across the life course. In this study, we synthesised a map of the human methylome across the life course, focussing on changes in variability and mean DNAm.Harmonizing DNAm datasets across eight longitudinal and cross-sectional UK-based studies, we meta-analysed n=13,215 blood samples from n=7,037 unique individuals from birth to 98 years of age. Changes in CpG-specific variability and means were described across the life course using a meta-regression framework. CpG-specific associations of variability or mean DNAm in relation to the likelihood of association with 100 traits linked to environmental exposures, health and disease were tested within and across ten developmental age bins across the life course.Age was linked to DNAm variability at 29,212 CpG sites. On average, we observed a 1.26 fold increase in DNAm variability per year across the life course. 33,730 CpGs displayed changes in mean DNAm, with 64% of these loci showing decreases in DNAm over time. CpG sites linked to traits were in general more variable across the life course.Our study provides, for the first time, a map of the human methylome across the life course, which is publicly accessible through a searchable online database. This resource allows researchers to query CpG-specific trajectories from birth to old age and link these to health and disease.

Background Biological aging estimators derived from DNA methylation data are heritable and correlate with morbidity and mortality. Consequently, identification of genetic and environmental contributors to the variation in these measures in populations has become a major goal in the field. Results Leveraging DNA methylation and SNP data from more than 40,000 individuals, we identify 137 genome-wide significant loci, of which 113 are novel, from genome-wide association study (GWAS) meta-analyses of four epigenetic clocks and epigenetic surrogate markers for granulocyte proportions and plasminogen activator inhibitor 1 levels, respectively. We find evidence for shared genetic loci associated with the Horvath clock and expression of transcripts encoding genes linked to lipid metabolism and immune function. Notably, these loci are independent of those reported to regulate DNA methylation levels at constituent clock CpGs. A polygenic score for GrimAge acceleration showed strong associations with adiposity-related traits, educational attainment, parental longevity, and C-reactive protein levels. Conclusion This study illuminates the genetic architecture underlying epigenetic aging and its shared genetic contributions with lifestyle factors and longevity.

Purpose of Review This review summarises recent advances in the field of epigenetics in order to understand the aetiology of type 2 diabetes (T2D). Recent Findings DNA methylation at a number of loci has been shown to be robustly associated with T2D, including TXNIP, ABCG1, CPT1A, and SREBF1. However, due to the cross-sectional nature of many epidemiological studies and predominant analysis in samples derived from blood rather than disease relevant tissues, inferring causality is difficult. We therefore outline the use of Mendelian randomisation (MR) as one method able to assess causality in epigenetic studies of T2D. Summary Epidemiological studies have been fruitful in identifying epigenetic markers of T2D. Triangulation of evidence including utilisation of MR is essential to delineate causal from non-causal biomarkers of disease. Understanding the causality of epigenetic markers in T2D more fully will aid prioritisation of CpG sites as early biomarkers to detect disease or in drug development to target epigenetic mechanisms in order to treat patients.

Aims: Epigenetic mechanisms regulate gene expression and may influence the pathogenesis of type 2 diabetes through the loss of insulin sensitivity. The aims of this study were to measure variation in DNA methylation at the type 2 diabetes locus KCNQ1 and assess its relationship with metabolic measures and with genotype. Methods: DNA methylation from whole blood DNA was quantified using pyrosequencing at 5 CpG sites at the KCNQ1 locus in 510 individuals without diabetes from the ‘Relationship between Insulin Sensitivity and Cardiovascular disease’ (RISC) cohort. Genotype data was analysed at the same locus in 1119 individuals in the same cohort. Insulin sensitivity was assessed by euglycaemic-hyperinsulinaemic clamp. Results: DNA methylation at the KCNQ1 locus was inversely associated with insulin sensitivity and serum adiponectin. This association was driven by a methylation-altering Single Nucleotide Polymorphism (SNP) (rs231840) which ablated a methylation site and reduced methylation levels. A second SNP (rs231357), in weak Linkage Disequilibrium (LD) with rs231840, was also associated with insulin sensitivity and DNA methylation. These SNPs have not been previously reported to be associated with type 2 diabetes risk or insulin sensitivity. Conclusion: Evidence indicates that genetic and epigenetic determinants at the KCNQ1 locus influence insulin sensitivity.

Background Type 2 diabetes (T2D) is a heterogeneous disease with well-known genetic and environmental risk factors contributing to its prevalence. Epigenetic mechanisms related to changes in DNA methylation (DNAm), may also contribute to T2D risk, but larger studies are required to discover novel markers, and to confirm existing ones. Results We performed a large meta-analysis of individual epigenome-wide association studies (EWAS) of prevalent T2D conducted in four European studies using peripheral blood DNAm. Analysis of differentially methylated regions (DMR) was also undertaken, based on the meta-analysis results. We found three novel CpGs associated with prevalent T2D in Europeans at cg00144180 (HDAC4), cg16765088 (near SYNM) and cg24704287 (near MIR23A) and confirmed three CpGs previously identified (mapping to TXNIP, ABCG1 and CPT1A). We also identified 77 T2D associated DMRs, most of them hypomethylated in T2D cases versus controls. In adjusted regressions among diabetic-free participants in ALSPAC, we found that all six CpGs identified in the meta-EWAS were associated with white cell-types. We estimated that these six CpGs captured 11% of the variation in T2D, which was similar to the variation explained by the model including only the common risk factors of BMI, sex, age and smoking (R2 = 10.6%). Conclusions This study identifies novel loci associated with T2D in Europeans. We also demonstrate associations of the same loci with other traits. Future studies should investigate if our findings are generalizable in non-European populations, and potential roles of these epigenetic markers in T2D etiology or in determining long term consequences of T2D.

Epigenome-wide association studies (EWAS) seek to quantify associations between traits/exposures and DNA methylation measured at thousands or millions of CpG sites across the genome. In recent years, the increase in availability of DNA methylation measures in population-based cohorts and case-control studies has resulted in a dramatic expansion of the number of EWAS being performed and published. To make this rich source of results more accessible, we have manually curated a database of CpG-trait associations (with p-4) from published EWAS, each assaying over 100,000 CpGs in at least 100 individuals. From January 7, 2022, The EWAS Catalog contained 1,737,746 associations from 2,686 EWAS. This includes 1,345,398 associations from 342 peer-reviewed publications. In addition, it also contains summary statistics for 392,348 associations from 427 EWAS, performed on data from the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Gene Expression Omnibus (GEO). The database is accompanied by a web-based tool and R package, giving researchers the opportunity to query EWAS associations quickly and easily, and gain insight into the molecular underpinnings of disease as well as the impact of traits and exposures on the DNA methylome. The EWAS Catalog data extraction team continue to update the database monthly and we encourage any EWAS authors to upload their summary statistics to our website. Details of how to upload data can be found here: http://www.ewascatalog.org/upload. The EWAS Catalog is available at http://www.ewascatalog.org.

Aims: DNA methylation clocks are widely used to estimate biological age, although limited data are available on non-European ethnicities. This manuscript characterizes the behavior of five DNA methylation clocks in 120 older Black South African men. Methods: The age estimation accuracy of the Horvath, Hannum and skin and blood clocks and the relative age-related mortality risk and predicted time to death portrayed by the PhenoAge and GrimAge biomarkers are investigated, respectively. Results: The results confirm the tendency of DNA methylation clocks to underestimate the biological age of older individuals. GrimAge more accurately characterizes biological decline in this African cohort compared with PhenoAge owing to the unique inclusion of smoking-related damage in the GrimAge estimate. Conclusions: Each clock provides a different fraction of information regarding the aging body. It is essential to continue studying under-represented population groups to ensure methylation-derived indicators are robust and useful in all populations.

Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450 K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis.

BackgroundIncidence rates for melanoma and non-melanoma skin cancer (NMSC), which includes basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), have been steadily increasing in all populations. Populations of European ancestry exhibit the highest rates and therefore, have been widely studied. Pigmentation characteristics are well-known risk factors for skin cancer, particularly fair skin, red hair, blue eyes and the inability to tan. Polymorphisms in established pigmentation-related genes have been associated with these traits and with an increased risk of malignancy. However, the functional relationship between genetic variation and disease is still unclear, with the exception of red hair colour variants in the melanocortin 1 receptor (MC1R) gene.ObjectivesThe aim of this study was to explore the possibility that non-coding pigmentation SNPs are associated with pigmentary traits and skin cancer via DNA methylation (DNAm).Methods and ResultsUsing a meta-GWAS of whole blood DNAm from 36 European cohorts (N=27,750; the Genetics of DNA Methylation Consortium, GoDMC), we found that 19 out of 27 pigmentation-associated SNPs distributed within 10 genes (ASIP, BNC2, IRF4, HERC2, MC1R, OCA2, SLC24A4, SLC24A5, SLC45A2, TYR) were associated with 391 DNAm sites across 30 genomic regions. We selected 25 DNAm sites for further analysis.We examined the effect of the chosen DNAm sites on pigmentation traits, sun exposure phenotypes, and skin cancer, and on gene expression in whole blood. We found an association of decreased DNAm at cg07402062 with red hair in the Avon Longitudinal Study of Parents and Children (ALSPAC), and a strong positive association of DNAm at this and correlated sites with higher expression ofSPIRE2. Additionally, we investigated the association of gene expression in skin with pigmentation traits and skin cancer. The expression ofASIP,FAM83C,NCOA6,CDK10, andEXOC2was associated with hair colour, whilst that ofASIPandCDK10also had an effect on melanoma and BCC.ConclusionsOur results indicate that DNAm and expression of genes in the 16q24.3 and 20q11.22 regions, deserve to be further investigated as potential mediators of the relationship between genetic variants, pigmentation/sun exposure phenotypes, and some types of skin cancer.

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.

Biological ageing estimators derived from DNA methylation (DNAm) data are heritable and correlate with morbidity and mortality. Leveraging DNAm and SNP data from >41,000 individuals, we identify 137 genome-wide significant loci (113 novel) from meta-analyses of four epigenetic clocks and epigenetic surrogate markers for granulocyte proportions and plasminogen activator inhibitor 1 levels, respectively. We report strong genetic correlations with longevity and lifestyle factors such as smoking, education, and obesity. Significant associations are observed in polygenic risk score analysis and to a lesser extent in Mendelian randomization analyses. This study illuminates the genetic architecture underlying epigenetic ageing and its shared genetic contributions with lifestyle factors and longevity.

Urbanization coincides with a complex change in environmental exposure and a rapid increase in noncommunicable diseases (NCDs). Epigenetics, including DNA methylation (DNAm), is thought to mediate part of the association between genetic/environmental exposure and NCDs. The urban–rural divide provides a unique opportunity to investigate the effect of the combined presence of multiple forms of environmental exposure on DNAm and the related increase in disease risk. This review evaluates the ability of three epidemiological study designs (migration, income-comparative and urban–rural designs) to investigate the role of DNAm in the association between urbanization and the rise in NCD prevalence. We also discuss the ability of each study design to address the gaps in the current literature, including the complex methylation-mediated risk attributable to the cluster of forms of exposure characterizing urban and rural living, while providing a platform for developing countries to leverage their demographic discrepancies in future research ventures.

Background: High blood pressure is a major risk factor for cardiovascular disease and is influenced by both environmental and genetic factors. Epigenetic processes including DNA methylation potentially mediate the relationship between genetic factors, the environment and cardiovascular disease. Despite an increased risk of hypertension and cardiovascular disease in individuals of South Asians compared to Europeans, it is not clear whether associations between blood pressure and DNA methylation differ between these groups.Methods: We performed an epigenome-wide association study and differentially methylated region (DMR) analysis to identify DNA methylation sites and regions that were associated with systolic blood pressure, diastolic blood pressure and hypertension. We analyzed samples from 364 European and 348 South Asian men (first generation migrants to the UK) from the Southall And Brent REvisited cohort, measuring DNA methylation from blood using the Illumina Infinium® HumanMethylation450 BeadChip.Results: One CpG site was found to be associated with DBP in trans-ancestry analyses (i.e. both ethnic groups combined), while in Europeans alone seven CpG sites were associated with DBP. No associations were identified between DNA methylation and either SBP or hypertension. Comparison of effect sizes between South Asian and European EWAS for DBP, SBP and hypertension revealed little concordance between analyses. DMR analysis identified several regions with known relationships with CVD and its risk factors.Conclusion: This study identified differentially methylated sites and regions associated with blood pressure and revealed ethnic differences in these associations. These findings may point to molecular pathways which may explain the elevated cardiovascular disease risk experienced by those of South Asian ancestry when compared to Europeans.

Introduction: Human genetic research has succeeded in definitively identifying multiple genetic variants associated with risk for nicotine dependence and heavy smoking. To build on these advances, and to aid in reducing the prevalence of smoking and its consequent health harms, the next frontier is to identify genetic predictors of successful smoking cessation and also of the efficacy of smoking cessation treatments ("pharmacogenomics"). More broadly, additional biomarkers that can be quantified from biosamples also promise to aid "Precision Medicine" and the personalization of treatment, both pharmacological and behavioral. Aims and Methods: To motivate ongoing and future efforts, here we review several compelling genetic and biomarker findings related to smoking cessation and treatment. Results: These Key results involve genetic variants in the nicotinic receptor subunit gene CHRNA5, variants in the nicotine metabolism gene CYP2A6, and the nicotine metabolite ratio. We also summarize reports of epigenetic changes related to smoking behavior. Conclusions: The results to date demonstrate the value and utility of data generated from biosamples in clinical treatment trial settings. This article cross-references a companion paper in this issue that provides practical guidance on how to incorporate biosample collection into a planned clinical trial and discusses avenues for harmonizing data and fostering consortium-based, collaborative research on the pharmacogenomics of smoking cessation. Implications: Evidence is emerging that certain genotypes and biomarkers are associated with smoking cessation success and efficacy of smoking cessation treatments. We review key findings that open potential avenues for personalizing smoking cessation treatment according to an individual's genetic or metabolic profile. These results provide important incentive for smoking cessation researchers to collect biosamples and perform genotyping in research studies and clinical trials.

Background DNA methylation is associated with non-communicable diseases (NCDs) and related traits. Methylation data on continental African ancestries are currently scarce, even though there are known genetic and epigenetic differences between ancestral groups and a high burden of NCDs in Africans. Furthermore, the degree to which current literature can be extrapolated to the understudied African populations, who have limited resources to conduct independent large-scale analysis, is not yet known. To this end, this study examines the reproducibility of previously published epigenome-wide association studies of DNA methylation conducted in different ethinicities, on factors related to NCDs, by replicating findings in 120 South African Batswana men aged 45 to 88 years. In addition, novel associations between methylation and NCD-related factors are investigated using the Illumina EPIC BeadChip. Results Up to 86% of previously identified epigenome-wide associations with NCD-related traits (alcohol consumption, smoking, body mass index, waist circumference, C-reactive protein, blood lipids and age) overlapped with those observed here and a further 13% were directionally consistent. Only 1% of the replicated associations presented with effects opposite to findings in other ancestral groups. The majority of these inconcistencies were associated with population-specific genomic variance. In addition, we identified eight new 450K array CpG associations not previously reported in other ancestries, and 11 novel EPIC CpG associations with alcohol consumption. Conclusions The successful replication of existing EWAS findings in this African population demonstrates that blood-based 450K EWAS findings from commonly investigated ancestries can largely be extrapolated to ethnicities for which epigenetic data are not yet available. Possible population-specific differences in 14% of the tested associations do, however, motivate the need to include a diversity of ethnic groups in future epigenetic research. The novel associations found with the enhanced coverage of the Illumina EPIC array support its usefulness to expand epigenetic literature.

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.

SummaryRegulation of transcription occurs in a cell type specific manner by epigenetic mechanisms including DNA methylation and histone modifications. Methylation changes during stem cell differentiation may play a key role in lineage specification. We sought to characterise DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) in order to further our understanding of epigenetic regulation in chondrocytes. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis. We identified significant DNA hypomethylation during chondrogenesis including changes at many key cartilage gene loci. Importantly characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with adult cartilage and other tissue methylation profiles identified chondrocyte-specific regulatory regions. Taken together we have associated methylation at many CpGs with the chondrocyte phenotype.AbstractRegulation of transcription is determined in a cell type specific manner by epigenetic mechanisms including DNA methylation and histone modifications. Methylation changes during stem cell differentiation may play a role in lineage specification. Multipotent mesenchymal stem cell (MSC) differentiation into chondrocytes not only serves as a model for chondrocyte development but also provides an important source of cartilage for tissue engineering purposes. We sought to characterise DNA methylation changes during chondrogenesis to further understanding of epigenetic regulation but to also provide context for the changes identified during disease.DNA cytosine methylation changes during human MSC differentiation into chondrocytes were measured by Infinium 450K methylation array. Methylation changes at gene loci were contrasted with gene expression changes. Chromatin states of significant methylation loci were interpreted by intersection with chondrogenesis histone modification ChlP-seq data. Chondrogenic and cartilage specific hypomethylation was utilised in order to identify a chondrocyte methylome. Articular cartilage and tissue panel DNA methylation was compared and alterations during osteoarthritis cartilage disease classified.Significant DNA hypomethylation was identified following chondrogenic differentiation of MSCs including changes at many key cartilage gene loci. Highly upregulated genes during chondrogenesis were more likely to exhibit a reduction in DNA methylation. Characterisation of significant CpG loci indicated their predominant localisation in CpG poor regions which importantly are most likely to correspond to enhancer regions. Methylation level at certain CpGs following chondrogenesis corresponds to the level found in adult cartilage.Taken together, considerable demethylation changes to the epigenetic landscape occur during MSC chondrogenesis especially at sites marked by enhancer modifications. Comparison with other tissues, including healthy and OA cartilage, associates CpGs to the chondrocyte phenotype and provides context for changes in disease.

Epigenetics encapsulates a group of molecular mechanisms including DNA methylation, histone modification and microRNAs (miRNAs). Gestational diabetes (GDM) increases the risk of adverse perinatal outcomes and is associated with future offspring risk of obesity and type 2 diabetes. It has been hypothesised that epigenetic mechanisms mediate an effect of GDM on offspring adiposity and type 2 diabetes and this could provide a modifiable mechanism to reduce type 2 diabetes in the next generation. Evidence for this hypothesis is lacking. Epigenetic epidemiology could also contribute to reducing type 2 diabetes by identifying biomarkers that accurately predict risk of GDM and its associated future adverse outcomes. We reviewed published human studies that explored associations between any of maternal GDM, type 2 diabetes, gestational fasting or post-load glucose and any epigenetic marker (DNA methylation, histone modification or miRNA). Of the 81 relevant studies we identified, most focused on the potential role of epigenetic mechanisms in mediating intrauterine effects of GDM on offspring outcomes. Studies were small (median total number of participants 58; median number of GDM cases 27) and most did not attempt replication. The most common epigenetic measure analysed was DNA methylation. Most studies that aimed to explore epigenetic mediation examined associations of in utero exposure to GDM with offspring cord or infant blood/placenta DNA methylation. Exploration of any causal effect, or effect on downstream offspring outcomes, was lacking. There is a need for more robust methods to explore the role of epigenetic mechanisms as possible mediators of effects of exposure to GDM on future risk of obesity and type 2 diabetes. Research to identify epigenetic biomarkers to improve identification of women at risk of GDM and its associated adverse (maternal and offspring) outcomes is currently rare but could contribute to future tools for accurate risk stratification. Electronic supplementary material The online version of this article (10.1007/s00125-019-05011-8) contains peer-reviewed but unedited supplementary material including a slideset of the figures for download, which is available to authorised users.

Background Indian Asians, who make up a quarter of the world's 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

Several studies have investigated the relationship between genetic variation and DNA methylation with respect to type 2 diabetes, but it is unknown if DNA methylation is a mediator in the disease pathway or if it is altered in response to disease state. This study uses genotypic information as a causal anchor to help decipher the likely role of DNA methylation measured in peripheral blood in the etiology of type 2 diabetes. Illumina HumanMethylation450 BeadChip data were generated on 1,018 young individuals from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. In stage 1, 118 unique associations between published type 2 diabetes single nucleotide polymorphisms (SNPs) and genome-wide methylation (methylation quantitative trait loci [mQTLs]) were identified. In stage 2, a further 226 mQTLs were identified between 202 additional independent non–type 2 diabetes SNPs and CpGs identified in stage 1. Where possible, associations were replicated in independent cohorts of similar age. We discovered that around half of known type 2 diabetes SNPs are associated with variation in DNA methylation and postulated that methylation could either be on a causal pathway to future disease or could be a noncausal biomarker. For one locus (KCNQ1), we were able to provide further evidence that methylation is likely to be on the causal pathway to disease in later life.

Objective Titin gene (TTN) mutations have been described in eight families with hereditary myopathy with early respiratory failure (HMERF). Some of the original patients had features resembling myofibrillar myopathy (MFM), arguing that TTN mutations could be a much more common cause of inherited muscle disease, especially in presence of early respiratory involvement. Methods We studied 127 undiagnosed patients with clinical presentation compatible with MFM. Sanger sequencing for the two previously described TTN mutations in HMERF (p.C30071R in the 119th fibronectin-3 (FN3) domain, and p.R32450W in the kinase domain) was performed in all patients. Patients with mutations had detailed review of their clinical records, muscle MRI findings and muscle pathology. Results We identified five new families with the p.C30071R mutation who were clinically similar to previously reported cases, and muscle pathology demonstrated diagnostic features of MFM. Two further families had novel variants in the 119th FN3 domain (p.P30091L and p.N30145K). No patients were identified with mutations at position p.32450. Conclusions Mutations in TTN are a cause of MFM, and titinopathy is more common than previously thought. The finding of the p.C30071R mutation in 3.9% of our study population is likely due to a British founder effect. The occurrence of novel FN3 domain variants, although still of uncertain pathogenicity, suggests that other mutations in this domain may cause MFM, and that the disease is likely to be globally distributed. We suggest that HMERF due to mutations in the TTN gene be nosologically classified as MFM-titinopathy.

Objective To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. Methods The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. Results The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling. Conclusion MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.

Over the last two decades, the mutation of mitochondrial DNA (mtDNA) has emerged as a major cause of inherited human disease. The disorders present clinically in at least 1 in 10,000 adults, but pathogenic mutations are found in approximately 1 in 200 of the background population. Mitochondrial DNA is maternally inherited and there can be marked phenotypic variability within the same family. Heteroplasmy is a significant factor and environmental toxins also appear to modulate the phenotype. Although genetic and biochemical studies have provided part of the explanation, a comprehensive understanding of the incomplete penetrance of these diseases is lacking--both at the population and family levels. Here, we review the potential role of epigenetic factors in the pathogenesis of mtDNA diseases and the contribution that epidemiological approaches can make to improve our understanding in this area. Despite being previously dismissed, there is an emerging evidence that mitochondria contain the machinery required to epigenetically modify mtDNA expression. In addition, the increased production of reactive oxygen species seen in several mtDNA diseases could lead to the epigenetic modification of the nuclear genome, including chromatin remodelling and alterations to DNA methylation and microRNA expression, thus contributing to the diverse pathophysiology observed in this group of diseases. These observations open the door to future studies investigating the role of mtDNA methylation in human disease.

Epigenetic mechanisms are believed to play an important role in disease, development and ageing with early life representing a window of particular epigenomic plasticity. The knowledge upon which these claims are based is beginning to expand. This review summarises evidence pointing to the determinants of epigenetic patterns, their juxtaposition at the interface of the environment, their influence on gene function and the relevance of this information to child health.

Summary Background Genetic factors have a role in the pathogenesis of ischaemic stroke, but the main genes involved have yet to be defined. Mitochondrial mechanisms have been implicated in the pathophysiology of acute stroke, but the role of mitochondrial DNA (mtDNA) has not been comprehensively studied. We investigated whether there is an association between mtDNA haplotypes and incidence of stroke. Methods The major European mtDNA haplogroups were identified in two independent subpopulations (n=950) from a study of occurrence of transient ischaemic attack (TIA) and ischaemic stroke and were compared with those of patients with acute coronary syndromes from the same populations (n=340) and with those of independent population controls (n=2939). Findings The presence of mtDNA sub-haplogroup K was significantly less frequent in patients with TIA or stroke than in controls in both subpopulations separately and in a pooled analysis (odds ratio 0·54, 95% CI 0·39–0·75, p

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 mother's 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.

In light of previous research showing that different types of relationships affect levels of testosterone in men, this study examined whether categorizing relationship types according to relationship length can shed further light on variations in levels of testosterone. Salivary testosterone samples were obtained from a sample of men and details about their relationship status, sociosexual orientation, extra-pair sexual interest, and their perceptions of their relationships were recorded. Using a median split analysis, participants who indicated that they had been in their relationship for less than 12 months were categorized as being in “new relationships” and those in longer relationships being categorized as in long-term relationships. Results showed that levels of testosterone of single men and men in new relationships did not differ, but both had significantly greater levels of testosterone than men in long-term relationships. Differences in levels of testosterone were unrelated to sociosexual orientation and extra-pair sexual interest. These findings support the evolutionary explanation of levels of testosterone in men varying in accordance with their internal motivation to seek new potential mates.

Comparisons of migrant, native and host populations are particularly useful in elucidating the balance between genetic and environmental influences on disease risk. In this issue, Campanella and colleagues 1 have conducted a novel study of epigenetics in Italian migrants and non-migrants from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Italy. Differences in DNA methylation were identified at several CpG sites when comparing migrant and host populations, including after adjustment for available dietary and lifestyle factors. They hypothesized that patterns they observed fit with an adaptive mechanism to counter the mismatch between early life exposures and later life environment experienced in the migrant population. The role of epigenetics and its interaction with the genome to mediate the changing health outcomes observed in many migrant groups is clearly important. However, migrant studies are complex. The effects of changing environments on health outcomes vary not only according to ethnicity and regions of origin and destination, but also according to who is migrating, why and when they migrate and what health outcome is being measured and over what period. We discuss a small selection of historic migrant studies in order to illustrate their utility and interpretation, alongside aspects of the new epigenetic study by

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.

Family and twin studies provide strong evidence of a major genetic influence in autism, but the underlying gene defects have yet to be characterized. The mothers of boys with autism share autistic traits, raising the possibility of a maternally inherited factor. Mitochondrial DNA (mtDNA) is almost exclusively inherited down the maternal line. We therefore explored the possibility that a particular mtDNA lineage contributes to the risk of developing autism. The mtDNA haplogroup was determined in 162 autism probands, and compared to two sets of population controls. Results show no compelling evidence of an association of any mitochondrial haplogroup in autism.

The m.8993T-->C MTATP6 mutation of mitochondrial DNA (mtDNA) usually causes mitochondrial disease in childhood, but was recently described in a family with adult onset ataxia and polyneuropathy. Cytochrome c oxidase muscle histochemistry, which is the standard clinical investigation for mitochondrial disease in adults, is usually normal in patients with MTATP6 mutations. This raises the possibility that these cases have been missed in the past. We therefore studied 308 patients with unexplained ataxia and 96 patients with suspected Charcot-Marie-Tooth disease to determine whether the m.8993T-->C MTATP6 mutation is common in unexplained inherited ataxia and/or polyneuropathy. We identified a three-generation family with the m.8993T-->C mutation of mtDNA. One subject had episodic ataxia (EA) and transient hemipareses, broadening the phenotype. However, no further cases were identified in an additional cohort of 191 patients with suspected EA. In conclusion, m.8993T-->C MTATP6 should be considered in patients with unexplained ataxia, CMT or EA, but cases are uncommon.