Single-cell landscape of functionally cured chronic hepatitis B patients reveals activation of innate and altered CD4-CTL-driven adaptive immunity.

BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. METHODS: We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC. RESULTS: We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC. CONCLUSIONS: Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis. IMPACT AND IMPLICATIONS: This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B.

Suppression of tumour growth from transplanted astrocytoma cells transfected with luciferase in mice by bioluminescence mediated, systemic, photodynamic therapy.

BACKGROUND: Grade 4 astrocytomas are usually incurable due to their diffusely infiltrative nature. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is impractical when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources to generate light at cancer cells. In vitro, astrocytoma cells, transfected with firefly luciferase, can be killed by bioluminescence-mediated PDT (bPDT). This study asks if bPDT can suppress tumour growth In vivo, when all components of treatment are administered systemically. METHODS: Transfected astrocytoma cells were injected subcutaneously or intra-cranially in athymic CD1 nu/nu mice. bPDT required ip bolus of mTHPC (photosensitiser) and delivery of the d-luciferin substrate over 7 days via an implanted osmotic pump. Control animals had no treatment, photosensitiser only or d-luciferin only. For subcutaneous tumours, size and BLI (light emitted after d-luciferin bolus) were measured before and every 2 days after PDT. For intracranial tumours, monitoring was weekly BLI. RESULTS: For subcutaneous tumours, there was significant suppression of the tumour growth rate (P<0.05), and absolute tumour size (P<0.01) after bPDT. Proliferation of subcutaneous and intracranial tumours (monitored by BrdU uptake) was significantly reduced in treated mice. (P<0.001) CONCLUSIONS: This study reports bPDT suppression of tumour growth from luciferase transfected astrocytoma cells with all components of treatment given systemically, as required for effective management of recurrent astrocytomas in unknown sites. However, research on systemic bPDT is needed to establish whether effects on non-transfected tumours can be achieved without any unacceptable effects on normal tissues.

A novel approach to risk exposure and epigenetics-the use of multidimensional context to gain insights into the early origins of cardiometabolic and neurocognitive health.

BACKGROUND: Each mother-child dyad represents a unique combination of genetic and environmental factors. This constellation of variables impacts the expression of countless genes. Numerous studies have uncovered changes in DNA methylation (DNAm), a form of epigenetic regulation, in offspring related to maternal risk factors. How these changes work together to link maternal-child risks to childhood cardiometabolic and neurocognitive traits remains unknown. This question is a key research priority as such traits predispose to future non-communicable diseases (NCDs). We propose viewing risk and the genome through a multidimensional lens to identify common DNAm patterns shared among diverse risk profiles. METHODS: We identified multifactorial Maternal Risk Profiles (MRPs) generated from population-based data (n = 15,454, Avon Longitudinal Study of Parents and Children (ALSPAC)). Using cord blood HumanMethylation450 BeadChip data, we identified genome-wide patterns of DNAm that co-vary with these MRPs. We tested the prospective relation of these DNAm patterns (n = 914) to future outcomes using decision tree analysis. We then tested the reproducibility of these patterns in (1) DNAm data at age 7 and 17 years within the same cohort (n = 973 and 974, respectively) and (2) cord DNAm in an independent cohort, the Generation R Study (n = 686). RESULTS: We identified twenty MRP-related DNAm patterns at birth in ALSPAC. Four were prospectively related to cardiometabolic and/or neurocognitive childhood outcomes. These patterns were replicated in DNAm data from blood collected at later ages. Three of these patterns were externally validated in cord DNAm data in Generation R. Compared to previous literature, DNAm patterns exhibited novel spatial distribution across the genome that intersects with chromatin functional and tissue-specific signatures. CONCLUSIONS: To our knowledge, we are the first to leverage multifactorial population-wide data to detect patterns of variability in DNAm. This context-based approach decreases biases stemming from overreliance on specific samples or variables. We discovered molecular patterns demonstrating prospective and replicable relations to complex traits. Moreover, results suggest that patterns harbour a genome-wide organisation specific to chromatin regulation and target tissues. These preliminary findings warrant further investigation to better reflect the reality of human context in molecular studies of NCDs.

The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

Background : Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. Results : Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, but reassuringly were robust to placental processing time. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. Conclusions : This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. Further, we demonstrate that estimating epiphenotype variables from the DNAme data itself, when possible, provides both an independent check of clinically-obtained data and can provide a robust approach to compare variables across different datasets.

Bioluminescence-activated photodynamic therapy for luciferase transfected, grade 4 astrocytoma cells in vitro.

BACKGROUND: . Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells. METHODS: . Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitizers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitizer only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls. RESULTS: . There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P<0.001) using hypericin and 35% (P<0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene. CONCLUSIONS: . bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies.

Gold nanoparticles as cell regulators: beneficial effects of gold nanoparticles on the metabolic profile of mice with pre-existing obesity.

BACKGROUND: We have previously shown that intraperitoneal injection of gold nanoparticles (AuNPs, 20-30 nm) into mice, decreases high-fat diet (HFD) induced weight gain and glucose intolerance, via suppression of inflammatory responses in both fat and liver tissues. This study investigates whether AuNPs provide similar benefit to mice with pre-existing obesity. Male C57BL/6 mice were fed a HFD for 15 weeks. AuNPs (OB-EAu 0.0785 µg/g/day, OB-LAu 0.785 µg/g/day, OB-HAu7.85 µg/g/day, ip) were administered to subgroups of HFD-fed mice over the last 5 weeks. Control group was fed standard chow and administered vehicle injection. RESULTS: Only the OB-LAu group demonstrated significant weight loss (12%), while all AuNP treated groups showed improved glycaemic control and reduced blood lipid levels. In the fat tissue, mRNA expression of pro-inflammatory markers were unchanged following AuNP treatment, while glucose and lipid metabolic markers were improved in OB-LAu and OB-HAu mice. In the liver, AuNP treatment downregulated inflammatory markers and improved lipid metabolic markers, with marked effects in OB-EAu and OB-LAu groups. CONCLUSIONS: AuNP treatment can improve glucose and fat metabolism in mice with long-term obesity, however weight loss was only observed in a single specific dose regime. AuNP therapy is a promising new technology for managing metabolic disorders in the obese.

Cognitive outcome in childhood of birth weight discordant monochorionic twins: the long-term effects of fetal growth restriction.

AIM: Intrauterine growth restriction (IUGR) is associated with poorer outcomes in later life. We used a monochorionic twin model with IUGR in one twin to determine its impact on growth and neurocognitive outcomes. METHODS: Monochorionic twins with ≥20% birth weight discordance born in the north of England were eligible. Cognitive function was assessed using the British Ability Scales. The Strength and Difficulties Questionnaire was used to identify behavioural problems. Auxological measurements were collected. Generalised estimating equations were used to determine the effects of birth weight on cognition. RESULTS: Fifty-one monochorionic twin pairs were assessed at a mean age of 6.3 years. Mean birth weight difference was 664 g at a mean gestation of 34.7 weeks. The lighter twin had a General Conceptual Ability (GCA) score that was three points lower (TwinL -105.4 vs TwinH -108.4, 95% CI -0.9 to -5.0), and there was a significant positive association (B 0.59) of within-pair birth weight differences and GCA scores. Mathematics and memory skills showed the largest differences. The lighter twin at school age was shorter (mean difference 2.1 cm±0.7) and lighter (mean difference 1.9 kg±0.6). Equal numbers of lighter and heavier twins were reported to have behavioural issues. CONCLUSIONS: In a monochorionic twin cohort, fetal growth restriction results in lower neurocognitive scores in early childhood, and there remain significant differences in size. Longer term follow-up will be required to determine whether growth or cognitive differences persist in later child or adulthood, and whether there are any associated longer term metabolic sequelae.

Gold nanoparticles improve metabolic profile of mice fed a high-fat diet.

BACKGROUND: Obesity is a high risk for multiple metabolic disorders due to excessive influx of energy, glucose and lipid, often from a western based diet. Low-grade inflammation plays a key role in the progression of such metabolic disorders. The anti-inflammatory property of gold compounds has been used in treating rheumatoid arthritis in the clinic. Previously we found that pure gold nanoparticles (AuNPs, 21 nm) also possess anti-inflammatory effects on the retroperitoneal fat tissue following intraperitoneal injection, by downregulating tumor necrosis factor (TNF) α. However, whether such an effect can change the risk of metabolic disorders in the obese has not been well studied. The study employed C57BL/6 mice fed a pellet high fat diet (HFD, 43% as fat) that were treated daily with AuNPs [low (HFD-LAu) or high (HFD-HAu) dose] via intraperitoneal injection for 9 weeks. In the in vitro study, RAW264.7 macrophages and 3T3-L1 adipocytes were cultured with low and high concentrations of AuNPs alone or together. RESULTS: The HFD-fed mice showed a significant increase in fat mass, glucose intolerance, dyslipidemia, and liver steatosis. The HFD-LAu group showed an 8% reduction in body weight, ameliorated hyperlipidemia, and normal glucose tolerance; while the HFD-HAu group had a 5% reduction in body weight with significant improvement in their glucose intolerance and hyperlipidemia. The underlying mechanism may be attributed to a reduction in adipose and hepatic local proinflammatory cytokine production, e.g. TNFα. In vitro studies of co-cultured murine RAW264.7 macrophage and 3T3-L1 adipocytes supported this proposed mechanism. CONCLUSION: AuNPs demonstrate a promising profile for potential management of obesity related glucose and lipid disorders and are useful as a research tool for the study of biological mechanisms.

Allostatic Load and Preterm Birth.

Preterm birth is a universal health problem that is one of the largest unmet medical needs contributing to the global burden of disease. Adding to its complexity is that there are no means to predict who is at risk when pregnancy begins or when women will actually deliver. Until these problems are addressed, there will be no interventions to reduce the risk because those who should be treated will not be known. Considerable evidence now exists that chronic life, generational or accumulated stress is a risk factor for preterm delivery in animal models and in women. This wear and tear on the body and mind is called allostatic load. This review explores the evidence that chronic stress contributes to preterm birth and other adverse pregnancy outcomes in animal and human studies. It explores how allostatic load can be used to, firstly, model stress and preterm birth in animal models and, secondly, how it can be used to develop a predictive model to assess relative risk among women in early pregnancy. Once care providers know who is in the highest risk group, interventions can be developed and applied to mitigate their risk.

Ancestral experience as a game changer in stress vulnerability and disease outcomes.

Stress is one of the most powerful experiences to influence health and disease. Through epigenetic mechanisms, stress may generate a footprint that propagates to subsequent generations. Programming by prenatal stress or adverse experience in parents, grandparents, or earlier generations may thus be a critical determinant of lifetime health trajectories. Changes in regulation of microRNAs (miRNAs) by stress may enhance the vulnerability to certain pathogenic factors. This review explores the hypothesis that miRNAs represent stress-responsive elements in epigenetic regulation that are potentially heritable. Recent findings suggest that miRNAs are key players linking adverse early environments or ancestral stress with disease risk, thus they represent useful predictive disease biomarkers. Since miRNA signatures of disease are potentially heritable, big data management platforms will be vital to harness multi-generational information and capture succinct yet potent biomarkers capable of directing preventative treatments. This feature would offer a unique window of opportunity to advance personalized medicine.

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

Hypermethylation and down-regulation of DLEU2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-1.

BACKGROUND: Acute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML. METHODS: We interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p <0.01 and Δß >0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons. RESULTS: We identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis. CONCLUSIONS: The DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology.

Stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance.

BACKGROUND: Patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response. RESULTS: We found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson's correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness. CONCLUSIONS: The findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia.

BACKGROUND: DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. METHODS: We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. RESULTS: Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000-5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. CONCLUSIONS: Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.

Epigenetic deregulation in pediatric acute lymphoblastic leukemia.

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (> 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes.

Prenatal maternal depression symptoms and nutrition, and child cognitive function.

BACKGROUND: Little is currently known about how maternal depression symptoms and unhealthy nutrition during pregnancy may developmentally interrelate to negatively affect child cognitive function. AIMS: To test whether prenatal maternal depression symptoms predict poor prenatal nutrition, and whether this in turn prospectively associates with reduced postnatal child cognitive function. METHOD: In 6979 mother-offspring pairs participating in the Avon Longitudinal Study of Parents and Children (ALSPAC) in the UK, maternal depression symptoms were assessed five times between 18 weeks gestation and 33 months old. Maternal reports of the nutritional environment were assessed at 32 weeks gestation and 47 months old, and child cognitive function was assessed at age 8 years. RESULTS: During gestation, higher depressive symptoms were related to lower levels of healthy nutrition and higher levels of unhealthy nutrition, each of which in turn was prospectively associated with reduced cognitive function. These results were robust to postnatal depression symptoms and nutrition, as well as a range of potential prenatal and postnatal confounds (i.e. poverty, teenage mother, low maternal education, parity, birth complications, substance use, criminal lifestyle, partner cruelty towards mother). CONCLUSIONS: Prenatal interventions aimed at the well-being of children of parents with depression should consider targeting the nutritional environment.

Exploring the utility of human DNA methylation arrays for profiling mouse genomic DNA.

Illumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species.

Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells.

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age," given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.

Genetic variation in the 15q25 nicotinic acetylcholine receptor gene cluster (CHRNA5-CHRNA3-CHRNB4) interacts with maternal self-reported smoking status during pregnancy to influence birth weight.

Maternal smoking during pregnancy is associated with low birth weight. Common variation at rs1051730 is robustly associated with smoking quantity and was recently shown to influence smoking cessation during pregnancy, but its influence on birth weight is not clear. We aimed to investigate the association between this variant and birth weight of term, singleton offspring in a well-powered meta-analysis. We stratified 26 241 European origin study participants by smoking status (women who smoked during pregnancy versus women who did not smoke during pregnancy) and, in each stratum, analysed the association between maternal rs1051730 genotype and offspring birth weight. There was evidence of interaction between genotype and smoking (P = 0.007). In women who smoked during pregnancy, each additional smoking-related T-allele was associated with a 20 g [95% confidence interval (95% CI): 4-36 g] lower birth weight (P = 0.014). However, in women who did not smoke during pregnancy, the effect size estimate was 5 g per T-allele (95% CI: -4 to 14 g; P = 0.268). To conclude, smoking status during pregnancy modifies the association between maternal rs1051730 genotype and offspring birth weight. This strengthens the evidence that smoking during pregnancy is causally related to lower offspring birth weight and suggests that population interventions that effectively reduce smoking in pregnant women would result in a reduced prevalence of low birth weight.

The role of longitudinal cohort studies in epigenetic epidemiology: challenges and opportunities.

Longitudinal cohort studies are ideal for investigating how epigenetic patterns change over time and relate to changing exposure patterns and the development of disease. We highlight the challenges and opportunities in this approach.