Early life risk factors and epigenetic biomarkers of obesity across the life course

Ph. D. Thesis.Obesity prevalence continues to rise and can be partially attributed to the obesogenic environment. However, there is increasing evidence that environmental exposures in early development can influence later-life disease, known as the Developmental Origins of Health and Disease hypothesis. Whilst some early life exposures have been associated with later-life adiposity, the underlying mechanism is less understood. One hypothesised mechanism is through epigenetic changes, such as DNA methylation. Multiple longitudinal cohorts were used to investigate the hypothesis; DNA methylation is a mediating mechanism between early life events and subsequent obesity. The Newcastle Thousand Families (NTFS) and Gateshead Millennium (GMS) studies (established 1947 and 2000 respectively), were used to investigate the impact of early life exposures (i.e. socioeconomic status, growth, adversity) on childhood (GMS) and adult (NTFS) obesity. Using both cohorts provided an opportunity to investigate regional temporal changes on childhood obesity, and the impact of obesogenic environments. The Avon Longitudinal study of Parents and Children (ALSPAC), which has methylation data (Illumina 450K array), was used to investigate associations between early life exposures and DNA methylation (in childhood and late adolescence) at CpG loci. Early life rapid weight gain (RWG) was consistently associated with childhood body composition in both local cohorts over time. In ALSPAC, RWG was significantly associated with a 1% increase in childhood methylation (age 7, n=116) at an individual CpG locus (CG11531579). Furthermore, the highest levels of methylation (+2%) were in those with RWG who were subsequently overweight/obese (OWOB, age 17). The CG11531579 loci was investigated further in NTFS adults (age 50, n=134) to examine whether the epigenetic marks persist. RWG was also associated with methylation changes in adults, although this was a decrease in methylation (-2%, age 50). These findings suggest that RWG in infancy is associated with small, dynamic variations in methylation at this locus.BBSR

Determination of qPCR Reference Genes Suitable for Normalizing Gene Expression in a Canine Model of Duchenne Muscular Dystrophy

Background:Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy. Appropriate normalization of expression data via carefully selected reference genes is consequently essential for accurate quantitative assessment. Unlike the expression profile of healthy skeletal muscle, the dystrophic muscle environment is highly dynamic: transcriptional profiles of dystrophic muscle might alter with age, disease progression, disease severity, genetic background and between muscle groups. Objectives:The aim of this work was to identify reference genes suitable for normalizing gene expression in healthy and dystrophic dogs under various comparative scenarios. Methods:Using the delta-E50 MD canine model of DMD, we assessed a panel of candidate reference genes for stability of expression across healthy and dystrophic animals, at different ages and in different muscle groups. Results:We show that the genes HPRT1, SDHA and RPL13a appear universally suitable for normalizing gene expression in healthy and dystrophic canine muscle, while other putative reference genes are exceptionally poor, and in the case of B2M, actively disease-correlated. Conclusions:Our findings suggest consistent cross-sample normalization is possible even throughout the dynamic progression of dystrophic pathology, and furthermore highlight the importance of empirical determination of suitable reference genes for neuromuscular diseases

DNA methylation patterns of LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 μg/day) and vitamin B12 (500 μg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD

Impact of in Utero Folate Exposure on Dna Methylation and Its Potential Relevance for Later‐Life Health – Evidence from Mouse Models Translated to Human Cohorts

Scope: Persistent DNA methylation changes may mediate the effects of early-life exposures on later-life health. However, the human lifespan is challenging for prospective studies, therefore data from longitudinal studies are limited. Projecting data from mouse models of early-life exposure to existing human studies offers a potential tool to address this challenge. Methods and Results: C57BL/6J mice were fed low or normal folate diets before and during pregnancy and lactation. Genome-wide promoter methylation was measured in male offspring livers at 17.5 days gestation and 28 weeks. Eight promoters were concurrently hypermethylated by folate depletion in fetuses and adults (>1.10 fold-change;p<0.05). Processes/pathways potentially influenced by global changes, and function of these 8 genes, suggest neurocognitive effects. Human observational and randomized controlled trial data were interrogated for translational findings. Methylation at birth was inversely associated with maternal plasma folate in 6 of the genes (-1.15% to-0.16%/nmol/l;p<0.05), whilst maternal folic acid supplementation was associated with differential methylation of 4 of these genes in adulthood. Three CpGs were persistently hypermethylated with lower maternal folate (p = 0.04). Conclusion: Some persistent folate-induced methylation changes observed in mice were mirrored in humans. This demonstrates utility of mouse data in identifying human loci for interrogation as biomarkers of later-life health

Anti-cancer therapy is associated with long-term epigenomic changes in childhood cancer survivors

Childhood cancer survivors (CCS) exhibit significantly increased chronic diseases and premature death. Abnormalities in DNA methylation are associated with development of chronic diseases and reduced life expectancy. We investigated the hypothesis that anti-cancer treatments are associated with long-term DNA methylation changes that could be key drivers of adverse late health effects. Genome-wide DNA methylation was assessed using MethylationEPIC arrays in paired samples (before/after therapy) from 32 childhood cancer patients. Separately, methylation was determined in 32 samples from different adult CCS (mean 22-years post-diagnosis) and compared with cancer-free controls (n = 284). Widespread DNA methylation changes were identified post-treatment in childhood cancer patients, including 146 differentially methylated regions (DMRs), which were consistently altered in the 32 post-treatment samples. Analysis of adult CCS identified matching methylation changes at 107/146 of the DMRs, suggesting potential long-term retention of post-therapy changes. Adult survivors also exhibited epigenetic age acceleration, independent of DMR methylation. Furthermore, altered methylation at the DUSP6 DMR was significantly associated with early mortality, suggesting altered methylation may be prognostic for some late adverse health effects in CCS. These novel methylation changes could serve as biomarkers for assessing normal cell toxicity in ongoing treatments and predicting long-term health outcomes in CCS. [Abstract copyright: © 2022. The Author(s).

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

The Biological and Social Determinants of Childhood Obesity:Comparison of 2 Cohorts 50 Years Apart

Objective: To determine whether the same relationships between early-life risk factors and socioeconomic status (SES) with childhood body mass index (BMI) are observed in a modern cohort (2000) compared with a historic cohort (1947). Study design: The relationships between early-life factors and SES with childhood BMI were examined in 2 prospective birth cohorts from the same region, born 50 years apart: 711 children in the 1947 Newcastle Thousand Families Study (NTFS) and 475 from the 2000 Gateshead Millennium Study (GMS). The associations between birth weight, breastfeeding, rapid infancy growth (0-12 months), early-life adversity (0-12 months), and parental SES (birth and childhood) with childhood BMI z-scores and whether overweight/obese (BMI >91st percentile using UK 1990 reference) aged 9 years were examined using linear regression, path analyses, and logistic regression. Results: In the NTFS, the most advantaged children were taller than the least (+0.91 height z-score, P = .001), whereas in GMS they had lower odds of overweight/obese than the least (0.35 [95% CI 0.14-0.86]). Rapid infancy growth was associated with increased BMI z-scores in both cohorts, and with increased likelihood of overweight/obese in GMS. Conclusions: This study suggests that children exposed to socioeconomic disadvantage or who have rapid infancy growth in modern environments are now at lower risk of growth restriction but greater risk of overweight