Genomic, Proteomic, and Phenotypic Biomarkers of COVID-19 Severity: Protocol for a Retrospective Observational Study.

BACKGROUND: Health organizations and countries around the world have found it difficult to control the spread of COVID-19. To minimize the future impact on the UK National Health Service and improve patient care, there is a pressing need to identify individuals who are at a higher risk of being hospitalized because of severe COVID-19. Early targeted work was successful in identifying angiotensin-converting enzyme-2 receptors and type II transmembrane serine protease dependency as drivers of severe infection. Although a targeted approach highlights key pathways, a multiomics approach will provide a clearer and more comprehensive picture of severe COVID-19 etiology and progression. OBJECTIVE: The COVID-19 Response Study aims to carry out an integrated multiomics analysis to identify biomarkers in blood and saliva that could contribute to host susceptibility to SARS-CoV-2 and the development of severe COVID-19. METHODS: The COVID-19 Response Study aims to recruit 1000 people who recovered from SARS-CoV-2 infection in both community and hospital settings on the island of Ireland. This protocol describes the retrospective observational study component carried out in Northern Ireland (NI; Cohort A); the Republic of Ireland cohort will be described separately. For all NI participants (n=519), SARS-CoV-2 infection has been confirmed by reverse transcription-quantitative polymerase chain reaction. A prospective Cohort B of 40 patients is also being followed up at 1, 3, 6, and 12 months postinfection to assess longitudinal symptom frequency and immune response. Data will be sourced from whole blood, saliva samples, and clinical data from the electronic care records, the general health questionnaire, and a 12-item general health questionnaire mental health survey. Saliva and blood samples were processed to extract DNA and RNA before whole-genome sequencing, RNA sequencing, DNA methylation analysis, microbiome analysis, 16S ribosomal RNA gene sequencing, and proteomic analysis were performed on the plasma. Multiomics data will be combined with clinical data to produce sensitive and specific prognostic models for severity risk. RESULTS: An initial demographic and clinical profile of the NI Cohort A has been completed. A total of 249 hospitalized patients and 270 nonhospitalized patients were recruited, of whom 184 (64.3%) were female, and the mean age was 45.4 (SD 13) years. High levels of comorbidity were evident in the hospitalized cohort, with cardiovascular disease and metabolic and respiratory disorders being the most significant (P<.001), grouped according to the International Classification of Diseases 10 codes. CONCLUSIONS: This study will provide a comprehensive opportunity to study the mechanisms of COVID-19 severity in recontactable participants. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/50733.

Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation.

DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development.

Exercise is associated with younger methylome and transcriptome profiles in human skeletal muscle.

Exercise training prevents age-related decline in muscle function. Targeting epigenetic aging is a promising actionable mechanism and late-life exercise mitigates epigenetic aging in rodent muscle. Whether exercise training can decelerate, or reverse epigenetic aging in humans is unknown. Here, we performed a powerful meta-analysis of the methylome and transcriptome of an unprecedented number of human skeletal muscle samples (n = 3176). We show that: (1) individuals with higher baseline aerobic fitness have younger epigenetic and transcriptomic profiles, (2) exercise training leads to significant shifts of epigenetic and transcriptomic patterns toward a younger profile, and (3) muscle disuse "ages" the transcriptome. Higher fitness levels were associated with attenuated differential methylation and transcription during aging. Furthermore, both epigenetic and transcriptomic profiles shifted toward a younger state after exercise training interventions, while the transcriptome shifted toward an older state after forced muscle disuse. We demonstrate that exercise training targets many of the age-related transcripts and DNA methylation loci to maintain younger methylome and transcriptome profiles, specifically in genes related to muscle structure, metabolism, and mitochondrial function. Our comprehensive analysis will inform future studies aiming to identify the best combination of therapeutics and exercise regimes to optimize longevity.

STING activation in TET2-mutated hematopoietic stem/progenitor cells contributes to the increased self-renewal and neoplastic transformation.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.

Variations in psychological disorders, suicidality, and help-seeking behaviour among college students from different academic disciplines.

BACKGROUND: Elevated levels of suicidality, ADHD, mental ill-health and substance disorders are reported among college students globally, yet few receive treatment. Some faculties and courses appear to have more at-risk students than others. The current study aimed to determine if students commencing college in different academic disciplines were at a heightened risk for psychopathology, substance use disorders and suicidal behaviour, and examined variations in help-seeking behaviour. MATERIALS AND METHODS: The study utilised data collected from 1,829 first-year undergraduate students as part of the Student Psychological Intervention Trial (SPIT) which commenced in September 2019 across four Ulster University campuses in Northern Ireland and an Institute of Technology, in the North-West of Ireland. The SPIT study is part of the World Mental Health International College Student Initiative (WMH-ICS) which uses the WMH-CIDI to identify 12-month and lifetime disorders. RESULTS: Students from Life and Health Sciences reported the lowest rates of a range of psychological problems in the year prior to commencing college, while participants studying Arts and Humanities displayed the highest levels (e.g. depression 20.6%; social anxiety 38.8%). However, within faculty variations were found. For example, psychology students reported high rates, while nursing students reported low rates. Variations in help seeking behaviour were also revealed, with male students less likely to seek help. CONCLUSIONS: Detecting specific cohorts at risk of psychological disorders and suicidality is challenging. This study revealed that some academic disciplines have more vulnerable students than others, with many reluctant to seek help for their problems. It is important for educators to be aware of such issues and for colleges to provide information and support to students at risk. Tailored interventions and prevention strategies may be beneficial to address the needs of students from different disciplines.

Folic acid intervention during pregnancy alters DNA methylation, affecting neural target genes through two distinct mechanisms.

BACKGROUND: We previously showed that continued folic acid (FA) supplementation beyond the first trimester of pregnancy appears to have beneficial effects on neurocognitive performance in children followed for up to 11 years, but the biological mechanism for this effect has remained unclear. Using samples from our randomized controlled trial of folic acid supplementation in second and third trimester (FASSTT), where significant improvements in cognitive and psychosocial performance were demonstrated in children from mothers supplemented in pregnancy with 400 µg/day FA compared with placebo, we examined methylation patterns from cord blood (CB) using the EPIC array which covers approximately 850,000 cytosine-guanine (CG) sites across the genome. Genes showing significant differences were verified using pyrosequencing and mechanistic approaches used in vitro to determine effects on transcription. RESULTS: FA supplementation resulted in significant differences in methylation, particularly at brain-related genes. Further analysis showed these genes split into two groups. In one group, which included the CES1 gene, methylation changes at the promoters were important for regulating transcription. We also identified a second group which had a characteristic bimodal profile, with low promoter and high gene body (GB) methylation. In the latter, loss of methylation in the GB is linked to decreases in transcription: this group included the PRKAR1B/HEATR2 genes and the dopamine receptor regulator PDE4C. Overall, methylation in CB also showed good correlation with methylation profiles seen in a published data set of late gestation foetal brain samples. CONCLUSION: We show here clear alterations in DNA methylation at specific classes of neurodevelopmental genes in the same cohort of children, born to FA-supplemented mothers, who previously showed improved cognitive and psychosocial performance. Our results show measurable differences at neural genes which are important for transcriptional regulation and add to the supporting evidence for continued FA supplementation throughout later gestation. This trial was registered on 15 May 2013 at www.isrctn.com as ISRCTN19917787.

The Mediating Roles of Mental Health and Substance Use on Suicidal Behavior Among Undergraduate Students With ADHD.

OBJECTIVE: To evaluate the prevalence of suicidal ideation (SI), plans and attempts, and non-suicidal self-injury (NSSI) among students with attention deficit hyperactivity disorder (ADHD). Furthermore, we explored the mediating effects of depression, anxiety, alcohol and substance use on the association between ADHD and suicidal behaviors and NSSI. METHOD: Participants were first-year undergraduate students (n = 1,829) recruited as part of the World Mental Health International College Student Initiative. Participants completed validated clinical measures online. RESULTS: The prevalence of suicide behaviors and NSSI were significantly higher among students with ADHD than those without. Mediation analyses indicated that ADHD directly and indirectly increased suicidal behaviors and NSSI. While ADHD increased suicidal behaviors and NSSI through depression, ADHD and the co-variates age and gender also had indirect effects on suicidal behaviors via substance use. CONCLUSIONS: Specific predictors of risk were identified for students with ADHD which may inform the development of more targeted mental health and suicide prevention strategies across campuses.

Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development.

Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of ß-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.

Suicidal behaviours and mental health disorders among students commencing college.

The increase in psychological disorders and suicidal behaviour in students is a reason for growing concern. Some may start university with pre-existing problems, while others develop problems during this time. It is important to evaluate mental health and wellbeing early, identifying those at risk. The aim of this study was to compare mental health problems and help-seeking behaviour between students in Northern Ireland (NI) and the Republic of Ireland (ROI). Whilst geographically proximate, the institutions span a cross-border region with distinct education and healthcare systems. First-year undergraduate students (n = 1828) were recruited in September 2019 as part of the World Mental Health International College Student Initiative. Suicidal behaviour, mental health and substance disorders were investigated using the World Mental Health- Composite International Diagnostic Interview. Prevalence of disorders was high, with more ROI students experiencing problems than NI students. Students were significantly more likely to experience mental health problems if they were female (p<0.001), non-heterosexual (p<0.0001), and over the age of 21 (p<0.0001). These findings show that many students are starting university with high levels of psychopathology and suicidal behaviour, highlighting the importance of early intervention which may need to be tailored to different student populations.

Prognostic value of miR-21 for prostate cancer: a systematic review and meta-analysis.

Elevated levels of miR-21 expression are associated with many cancers, suggesting it may be a promising clinical biomarker. In prostate cancer (PCa), however, there is still no consensus about the usefulness of miR-21 as an indicator of disease progression. This systematic review and meta-analysis was conducted to investigate the value of miR-21 expression as a prognostic measurement in PCa patients. Medline (Ovid), EMBASE, Web of Science, Scopus and Cochrane Library databases were systematically searched for relevant publications between 2010 to 2021. Studies exploring the relationship between miR-21 expression, PCa prognosis and clinicopathological factors were selected for review. Those reporting hazard ratio (HR) and 95% confidence intervals (CIs) were subject to meta-analyses. Fixed-effect models were employed to calculated pooled HRs and 95% CIs. Risk of bias in each study was assessed using QUIPS tool. Certainty of evidence in each meta-analysis was assessed using GRADE guidelines. A total of 64 studies were included in the systematic review. Of these, 11 were eligible for inclusion in meta-analysis. Meta-analyses revealed that high miR-21 expression was associated with poor prognosis: HR = 1.58 (95% CI = 1.19-2.09) for biochemical recurrence, MODERATE certainty; HR = 1.46 (95% CI = 1.06-2.01) for death, VERY LOW certainty; and HR = 1.26 (95% CI = 0.70-2.27) for disease progression, VERY LOW certainty. Qualitative summary revealed elevated miR-21 expression was significantly positively associated with PCa stage, Gleason score and risk groups. This systematic review and meta-analysis suggests that elevated levels of miR-21 are associated with poor prognosis in PCa patients. miR-21 expression may therefore be a useful prognostic biomarker in this disease.

Effects of maternal folic acid supplementation during the second and third trimesters of pregnancy on neurocognitive development in the child: an 11-year follow-up from a randomised controlled trial.

BACKGROUND: Maternal folic acid (FA) supplementation before and in early pregnancy prevents neural tube defects (NTD), but it is uncertain whether continuing FA after the first trimester has benefits on offspring health. We aimed to evaluate the effect of FA supplementation throughout pregnancy on cognitive performance and brain function in the child. METHODS: Follow-up investigation of 11-year-old children, residing in Northern Ireland, whose mothers had participated in a randomised trial of Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) in pregnancy and received 400 µg/day FA or placebo from the 14th gestational week. Cognitive performance (Full Scale Intelligence Quotient, Verbal Comprehension, Working Memory, Perceptual Reasoning, and Processing Speed) was assessed using the Wechsler Intelligence Scale for Children. Neuronal function was assessed using magnetoencephalographic (MEG) brain imaging. RESULTS: Of 119 mother-child pairs in the FASSTT trial, 68 children were assessed for neurocognitive performance at 11-year follow-up (Dec 2017 to Nov 2018). Children of mothers randomised to FA compared with placebo scored significantly higher in two Processing Speed tests, i.e. symbol search (mean difference 2.9 points, 95% CI 0.3 to 5.5, p = 0.03) and cancellation (11.3 points, 2.5 to 20.1, p = 0.04), whereas the positive effect on Verbal Comprehension was significant in girls only (6.5 points, 1.2 to 11.8, p = 0.03). MEG assessment of neuronal responses to a language task showed increased power at the Beta (13-30 Hz, p = 0.01) and High Gamma (49-70 Hz, p = 0.04) bands in children from FA-supplemented mothers, suggesting more efficient semantic processing of language. CONCLUSIONS: Continued FA supplementation in pregnancy beyond the early period currently recommended to prevent NTD can benefit neurocognitive development of the child. MEG provides a non-invasive tool in paediatric research to objectively assess functional brain activity in response to nutrition and other interventions. TRIAL REGISTRATION: ISRCTN ISRCTN19917787 . Registered on 15 May 2013.

Low birth weight female piglets show altered intestinal development, gene expression, and epigenetic changes at key developmental loci.

Intestinal development is compromised in low birth weight (LBW) pigs, negatively impacting their growth, health, and resilience. We investigated the molecular mechanisms of the altered intestinal maturation observed in neonatal and juvenile LBW female piglets by comparing the changes in intestinal morphology, gene expression, and methylation in LBW versus normal birth weight (NBW) female piglets. A total of 16 LBW/NBW sibling pairs were sacrificed at 0 hours, 8 hours, 10 days, and 8 weeks of age. The gastrointestinal tract was weighed, measured, and the small intestine was sampled for histomorphology, gene expression, and methylation analyses. Impaired intestinal development, with shorter villi and shallower crypts, was observed in LBW female piglets. The expression of intestinal development markers (ALPI and OLFM) rapidly peaked after birth in NBW but not in LBW female piglets. The lower expression of genes involved in nutrient digestion (ANPEP and SI) and barrier function (OCLN and CLDN4) in LBW, together with their delayed development of intestinal villi and crypts could help to explain the compromised health and growth potential of LBW female piglets. The changes in methylation observed in LBW in key regulators of intestinal development (OLFM4 and FZD5) suggest long-term effects of BW on intestinal gene expression, development, and function. Accordingly, experimental demethylation induced in IPEC-J2 cells led to increased expression of intestinal genes (MGA, DPP4, and GLUT2). Overall, we have identified the alterations in transcription or epigenetic marking at a number of genes critical to intestinal development, which may contribute to both the short- and long-term failure of LBW female piglets to thrive.

The metabolic-epigenetic nexus in type 2 diabetes mellitus.

The prevalence of type 2 diabetes mellitus (T2DM) continues to rise globally. Yet the aetiology and pathophysiology of this noncommunicable, polygenic disease, is poorly understood. Lifestyle factors, such as poor dietary intake, lack of exercise, and abnormal glycaemia, are purported to play a role in disease onset and progression, and these environmental factors may disrupt specific epigenetic mechanisms, leading to a reprogramming of gene transcription. The hyperglycaemic cell per se, alters epigenetics through chemical modifications to DNA and histones via metabolic intermediates such as succinate, α-ketoglutarate and O-GlcNAc. To illustrate, α-ketoglutarate is considered a salient co-factor in the activation of the ten-eleven translocation (TET) dioxygenases, which drives DNA demethylation. On the contrary, succinate and other mitochondrial tricarboxylic acid cycle intermediates, inhibit TET activity predisposing to a state of hypermethylation. Hyperglycaemia depletes intracellular ascorbic acid, and damages DNA by enhancing the production of reactive oxygen species (ROS); this compromised cell milieu exacerbates the oxidation of 5-methylcytosine alongside a destabilisation of TET. These metabolic connections may regulate DNA methylation, affecting gene transcription and pancreatic islet ß-cell function in T2DM. This complex interrelationship between metabolism and epigenetic alterations may provide a conceptual foundation for understanding how pathologic stimuli modify and control the intricacies of T2DM. As such, this narrative review will comprehensively evaluate and detail the interplay between metabolism and epigenetic modifications in T2DM.

DNA methylation of hypertension-related genes and effect of riboflavin supplementation in adults stratified by genotype for the MTHFR C677T polymorphism.

BACKGROUND: The interaction between genetic, epigenetic and environmental factors plays an important role in the aetiology of hypertension. GWAS and observational studies link the C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR) with hypertension, while riboflavin, the MTHFR cofactor, has been shown to reduce blood pressure and global DNA methylation in homozygous (TT genotype) individuals. It is currently unclear whether riboflavin modulates DNA methylation of other hypertension-related genes. OBJECTIVES: To compare DNA methylation of hypertension-related genes in adults stratified by MTHFR genotype and effect of riboflavin intervention in adults with the variant MTHFR 677TT genotype. METHOD: Pyrosequencing was carried out for hypertension-related genes (ACE, AGTR1, GCK, GNA12, IGF2, MMP9 and NOS3) in blood samples from participants in previous trials (CC, n = 40; TT, n = 40). The effect of intervention with riboflavin (1.6 mg/d for16 weeks) or placebo on DNA methylation was investigated in adults with the variant MTHFR 677TT genotype (n = 80). RESULTS: Individuals with the MTHFR 677TT v CC genotype had significantly higher average DNA methylation at NOS3 (+1.66%, P = 0.044). In response to riboflavin supplementation in TT individuals, there was an increase in average DNA methylation at IGF2 (+1.09%, P = 0.019) and a decrease at ACE (-0.44%, P = 0.021) in females only. Specific CpG sites were hypomethylated in GNA12 and hypermethylated in AGTR1. CONCLUSION: This study provides the first RCT evidence that riboflavin alters DNA methylation of hypertension-related genes in adults with the MTHFR 677TT genotype, providing some insight into mechanisms linking hypertension with the genotype-specific response of BP to riboflavin.

Depression, anxiety and suicidal behaviour among college students: Comparisons pre-COVID-19 and during the pandemic.

BACKGROUND: Many students struggle with psychological problems during their college years. These problems may be even more apparent during the COVID-19 pandemic with the accompanying restrictions and transition to an online learning environment, but few longitudinal studies have been conducted to date. The aim of this study was to compare symptoms of depression, anxiety and suicidality prior to and during the pandemic, and identify stressors. METHODS: This study was conducted among students attending Ulster University, Northern Ireland (NI) and LYIT, Republic of Ireland (ROI), as part of the World Mental Health International College Student Initiative (WMH-ICS). Data was collected from first year students in September 2019. The completed response rate was 25.22% (NI) and 41.9% (ROI) in relation to the number of first-year students registered. A follow up study was conducted in Autumn 2020, with 884 students fully completing the online survey in both years, equating to just under half of those who completed initially. RESULTS: High levels of mental health problems were found in year 1, especially in the ROI. Levels of depression increased significantly in year 2, particularly among students in NI, however, levels of anxiety decreased. No significant variations were found for suicidal behaviour. Several stressors were revealed, including increased social isolation, and worrying about loved ones. LIMITATIONS: The findings may not be generalised to other student populations. CONCLUSIONS: This study reveals variation in symptoms of depression and anxiety since the onset of the pandemic. In particular, the large increase in students with depression is of concern.

Nutritional Epigenomics and Age-Related Disease.

Recent advances in epigenetic research have enabled the development of epigenetic clocks, which have greatly enhanced our ability to investigate molecular processes that contribute to aging and age-related disease. These biomarkers offer the potential to measure the effect of environmental exposures linked to dynamic changes in DNA methylation, including nutrients, as factors in age-related disease. They also offer a compelling insight into how imbalances in the supply of nutrients, particularly B-vitamins, or polymorphisms in regulatory enzymes involved in 1-carbon metabolism, the key pathway that supplies methyl groups for epigenetic reactions, may influence epigenetic age and interindividual disease susceptibility. Evidence from recent studies is critically reviewed, focusing on the significant contribution of the epigenetic clock to nutritional epigenomics and its impact on health outcomes and age-related disease. Further longitudinal studies and randomized nutritional interventions are required to advance the field.

CandiMeth: Powerful yet simple visualization and quantification of DNA methylation at candidate genes.

BACKGROUND: DNA methylation microarrays are widely used in clinical epigenetics and are often processed using R packages such as ChAMP or RnBeads by trained bioinformaticians. However, looking at specific genes requires bespoke coding for which wet-lab biologists or clinicians are not trained. This leads to high demands on bioinformaticians, who may lack insight into the specific biological problem. To bridge this gap, we developed a tool for mapping and quantification of methylation differences at candidate genomic features of interest, without using coding. FINDINGS: We generated the workflow "CandiMeth" (Candidate Methylation) in the web-based environment Galaxy. CandiMeth takes as input any table listing differences in methylation generated by either ChAMP or RnBeads and maps these to the human genome. A simple interface then allows the user to query the data using lists of gene names. CandiMeth generates (i) tracks in the popular UCSC Genome Browser with an intuitive visual indicator of where differences in methylation occur between samples or groups of samples and (ii) tables containing quantitative data on the candidate regions, allowing interpretation of significance. In addition to genes and promoters, CandiMeth can analyse methylation differences at long and short interspersed nuclear elements. Cross-comparison to other open-resource genomic data at UCSC facilitates interpretation of the biological significance of the data and the design of wet-lab assays to further explore methylation changes and their consequences for the candidate genes. CONCLUSIONS: CandiMeth (RRID:SCR_017974; Biotools: CandiMeth) allows rapid, quantitative analysis of methylation at user-specified features without the need for coding and is freely available at https://github.com/sjthursby/CandiMeth.

Methylome profiling of young adults with depression supports a link with immune response and psoriasis.

BACKGROUND: Currently the leading cause of global disability, clinical depression is a heterogeneous condition characterised by low mood, anhedonia and cognitive impairments. Its growing incidence among young people, often co-occurring with self-harm, is of particular concern. We recently reported very high rates of depression among first year university students in Northern Ireland, with over 25% meeting the clinical criteria, based on DSM IV. However, the causes of depression in such groups remain unclear, and diagnosis is hampered by a lack of biological markers. The aim of this exploratory study was to examine DNA methylation patterns in saliva samples from individuals with a history of depression and matched healthy controls. RESULTS: From our student subjects who showed evidence of a total lifetime major depressive event (MDE, n = 186) we identified a small but distinct subgroup (n = 30) with higher risk scores on the basis of co-occurrence of self-harm and attempted suicide. Factors conferring elevated risk included being female or non-heterosexual, and intrinsic factors such as emotional suppression and impulsiveness. Saliva samples were collected and a closely matched set of high-risk cases (n = 16) and healthy controls (n = 16) similar in age, gender and smoking status were compared. These showed substantial differences in DNA methylation marks across the genome, specifically in the late cornified envelope (LCE) gene cluster. Gene ontology analysis showed highly significant enrichment for immune response, and in particular genes associated with the inflammatory skin condition psoriasis, which we confirmed using a second bioinformatics approach. We then verified methylation gains at the LCE gene cluster at the epidermal differentiation complex and at MIR4520A/B in our cases in the laboratory, using pyrosequencing. Additionally, we found loss of methylation at the PSORSC13 locus on chromosome 6 by array and pyrosequencing, validating recent findings in brain tissue from people who had died by suicide. Finally, we could show that similar changes in immune gene methylation preceded the onset of depression in an independent cohort of adolescent females. CONCLUSIONS: Our data suggests an immune component to the aetiology of depression in at least a small subgroup of cases, consistent with the accumulating evidence supporting a relationship between inflammation and depression. Additionally, DNA methylation changes at key loci, detected in saliva, may represent a valuable tool for identifying at-risk subjects.

Riboflavin supplementation alters global and gene-specific DNA methylation in adults with the MTHFR 677 TT genotype.

DNA methylation is important in regulating gene expression and genomic stability while aberrant DNA methylation is associated with disease. Riboflavin (FAD) is a cofactor for methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate recycling, which generates methyl groups for homocysteine remethylation to methionine, the pre-cursor to the universal methyl donor S-adenosylmethionine (SAM). A polymorphism (C677T) in MTHFR results in decreased MTHFR activity and increased homocysteine concentration. Previous studies demonstrated that riboflavin modulates this phenotype in homozygous adults (MTHFR 677 TT genotype), however, DNA methylation was not considered. This study examined DNA methylation, globally and at key MTHFR regulatory sites, in adults stratified by MTHFR genotype and the effect of riboflavin supplementation on DNA methylation in individuals with the 677 TT genotype. Samples were accessed from participants, screened for the MTHFR C677T polymorphism, who participated in observational (n = 80) and targeted riboflavin (1.6 mg/day) RCTs (n = 80). DNA methylation at LINE-1 and key regulatory regions of the MTHFR locus were analysed by pyrosequencing in peripheral blood leukocytes. LINE-1 (+1.6%; p = 0.011) and MTHFR south shelf (+4.7%, p < 0.001) were significantly hypermethylated in individuals with the MTHFR 677 TT compared to CC genotype. Riboflavin supplementation resulted in decreased global methylation, albeit only significant at one CpG. A significant reduction in DNA methylation at the MTHFR north shore (-1.2%, p < 0.001) was also observed in TT adults following intervention with riboflavin. This provides the first RCT evidence that DNA methylation may be modulated by riboflavin in adults with the MTHFR 677 TT genotype.