Epigenetic variation associated with genetic and environmental factors in the aetiology of Type 2 diabetes.

PhDType 2 diabetes, as a complex disease, has a range of genetic and environmental factors that underpin its aetiology. It is hoped that the emerging study of epigenetic processes will provide the necessary mechanistic insight into the genetic and environmental interactions that, to date, are poorly understood. This thesis considers the role of DNA methylation, an epigenetic modification, in the aetiology of type 2 diabetes. A range of different genome-wide and whole genome techniques are applied to a study of established type 2 diabetes and experimental models (human and animal) of fetal programming. Samples from a recent genome-wide association study of type 2 diabetes were used to identify DNA methylation patterns at areas of genetic variation associated with disease risk. Analysis of data from methylated DNA immunoprecipitation and microarray identified a genetic-epigenetic interaction in the FTO gene. At this locus, the presence or absence of a SNP created or abrogated a CpG site capable of methylation and further analyses highlighted possible functional relevance via enhancer activity. Models of fetal programming were then used to identify whether variation in DNA methylation may underlie the ‘programmed’ phenotype of diabetes and related cardiometabolic disease. Pre-existing human models of programming via maternal vitamin B12 deficiency and maternal famine exposure have been used to generate exploratory evidence of such mechanisms. Whole genome-based techniques (Medip-seq and Illumina 450k methylation array) were used to profile DNA methylation in whole blood samples from the offspring born to each of these studies. Custom bioinformatic analysis was performed to identify differences in methylation between offspring exposed versus unexposed to the in utero environmental insult. Technical replication and validation studies are ongoing to confirm or refute the presence of regions of differential methylation. Finally, this thesis considers whether a state of ‘over nutrition’ gestational diabetes, may play a role in fetal programming. This condition is of increasing prevalence across the world and is characterised by maternal hyperglycaemia and insulin resistance, often resulting in fetal overgrowth. A mouse model using an inbred strain (Lepr) of mice induced a programmed phenotype of glucose intolerance and obesity in aged offspring born to mothers with gestational diabetes. Medip-seq performed on the livers of late gestation mouse embryos identified differential methylation in cases vs. controls, located at genomic regions with potential functional relevance. A human cohort of women with gestational diabetes was collected to develop further hypothesis around the multiple environmental factors that could interact in pregnancy. Prevalent nutritional deficiencies of vitamin D, iron and one-carbon metabolites were found in women with and without gestational diabetes recruited from a local antenatal clinic. This thesis presents preliminary findings that variation in DNA methylation may be involved in the genetic and environmental risk of type 2 diabetes. The work presented highlights how future studies must incorporate integrated genetic, epigenetic and functional analysis with sufficient sample size if their results are to be translatable to diverse populations at risk of diabetes

Novel DNA methylation profiles associated with key gene regulation and transcription pathways in blood and placenta of growth-restricted neonates

BB/H012494/1/ Biotechnology and Biological Sciences Research Counci

Contact Hypersensitivity to Oxazolone Provokes Vulvar Mechanical Hyperalgesia in Mice

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition

Rare variant contribution to cholestatic liver disease in a South Asian population in the United Kingdom

This study assessed the contribution of five genes previously known to be involved in cholestatic liver disease in British Bangladeshi and Pakistani people. Five genes (ABCB4, ABCB11, ATP8B1, NR1H4, TJP2) were interrogated by exome sequencing data of 5236 volunteers. Included were non-synonymous or loss of function (LoF) variants with a minor allele frequency < 5%. Variants were filtered, and annotated to perform rare variant burden analysis, protein structure, and modelling analysis in-silico. Out of 314 non-synonymous variants, 180 fulfilled the inclusion criteria and were mostly heterozygous unless specified. 90 were novel and of those variants, 22 were considered likely pathogenic and 9 pathogenic. We identified variants in volunteers with gallstone disease (n = 31), intrahepatic cholestasis of pregnancy (ICP, n = 16), cholangiocarcinoma and cirrhosis (n = 2). Fourteen novel LoF variants were identified: 7 frameshift, 5 introduction of premature stop codon and 2 splice acceptor variants. The rare variant burden was significantly increased in ABCB11. Protein modelling demonstrated variants that appeared to likely cause significant structural alterations. This study highlights the significant genetic burden contributing to cholestatic liver disease. Novel likely pathogenic and pathogenic variants were identified addressing the underrepresentation of diverse ancestry groups in genomic research

An evaluation of a pilot of daily testing of SARS-CoV-2 contacts in acute hospital and ambulance trusts in England

OBJECTIVES: Healthcare worker (HCW) SARS-CoV-2 contacts in England have been required to quarantine, creating staff shortages. We piloted daily contact testing (DCT) to assess its feasibility as an alternative. STUDY DESIGN: Observational service evaluation. METHODS: We conducted an observational service evaluation of seven-day daily contact testing using antigen lateral flow devices, (LFDs) at four acute hospital trusts and one ambulance trust in England. Mixed methods were employed, utilising aggregate and individual-level test monitoring data, semi-structured interviews, and a survey of eligible contacts. RESULTS: In total, 138 HCWs were identified as contacts of a confirmed SARS-CoV-2 case. Of these, 111 (80%) consented to daily LFD testing, of whom 82 (74%) completed the required programme without interruption, and 12 (11%) completed with interruption. Fifty-eight (52%) participants and two (7·4%) non-participants completed the survey. In total, 28 interviews were conducted with participants, site and infection control leads, and union representatives. One participant tested positive on LFD and polymerase chain reaction (PCR) test. Three participants tested positive on PCR but not LFD. DCT was well-accepted by trusts and staff. Participants reported no relaxation of their infection prevention and control behaviours. No incidents of transmission were detected. An estimated 729 potential days of work absence were averted. CONCLUSIONS: DCT can be acceptably operated in a healthcare setting, averting quarantine-related work absences in HCW SARS-CoV-2 contacts

Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases

Socio-cultural influences on the behaviour of South Asian women with diabetes in pregnancy: qualitative study using a multi-level theoretical approach

BACKGROUND: Diabetes in pregnancy is common in South Asians, especially those from low-income backgrounds, and leads to short-term morbidity and longer-term metabolic programming in mother and offspring. We sought to understand the multiple influences on behaviour (hence risks to metabolic health) of South Asian mothers and their unborn child, theorise how these influences interact and build over time, and inform the design of culturally congruent, multi-level interventions. METHODS: Our sample for this qualitative study was 45 women of Bangladeshi, Indian, Sri Lankan, or Pakistani origin aged 21-45 years with a history of diabetes in pregnancy, recruited from diabetes and antenatal services in two deprived London boroughs. Overall, 17 women shared their experiences of diabetes, pregnancy, and health services in group discussions and 28 women gave individual narrative interviews, facilitated by multilingual researchers, audiotaped, translated, and transcribed. Data were analysed using the constant comparative method, drawing on sociological and narrative theories. RESULTS: Key storylines (over-arching narratives) recurred across all ethnic groups studied. Short-term storylines depicted the experience of diabetic pregnancy as stressful, difficult to control, and associated with negative symptoms, especially tiredness. Taking exercise and restricting diet often worsened these symptoms and conflicted with advice from relatives and peers. Many women believed that exercise in pregnancy would damage the fetus and drain the mother's strength, and that eating would be strength-giving for mother and fetus. These short-term storylines were nested within medium-term storylines about family life, especially the cultural, practical, and material constraints of the traditional South Asian wife and mother role and past experiences of illness and healthcare, and within longer-term storylines about genetic, cultural, and material heritage - including migration, acculturation, and family memories of food insecurity. While peer advice was familiar, meaningful, and morally resonant, health education advice from clinicians was usually unfamiliar and devoid of cultural meaning. CONCLUSIONS: 'Behaviour change' interventions aimed at preventing and managing diabetes in South Asian women before and during pregnancy are likely to be ineffective if delivered in a socio-cultural vacuum. Individual education should be supplemented with community-level interventions to address the socio-material constraints and cultural frames within which behavioural 'choices' are made

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 x 10(-8)) with GDM, mapping to/near MTNR1B (P = 4.3 x 10(-54)), TCF7L2 (P = 4.0 x 10(-16)), CDKAL1 (P = 1.6 x 10(-4)), CDKN2A-CDKN2B (P = 4.1 x 10(-9)) and HKDC1 (P = 2.9 x 10(-8)). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.Peer reviewe