Visceral Adipose Tissue Inflammatory Factors (TNF-Alpha, SOCS3) in Gestational Diabetes (GDM): Epigenetics as a Clue in GDM Pathophysiology

Gestational diabetes (GDM) is among the most challenging diseases in westernized countries, affecting mother and child, immediately and in later life. Obesity is a major risk factor for GDM. However, the impact visceral obesity and related epigenetics play for GDM etiopathogenesis have hardly been considered so far. Our recent findings within the prospective 'EaCH' cohort study of women with GDM or normal glucose tolerance (NGT), showed the role, critical factors of insulin resistance (i.e., adiponectin, insulin receptor) may have for GDM pathophysiology with epigenetically modified expression in subcutaneous (SAT) and visceral (VAT) adipose tissues. Here we investigated the expression and promoter methylation of key inflammatory candidates, tumor necrosis factor-alpha (TNF-α) and suppressor of cytokine signaling 3 (SOCS3) in maternal adipose tissues collected during caesarian section (GDM, n = 19; NGT, n = 22). The mRNA expression of TNF-α and SOCS3 was significantly increased in VAT, but not in SAT, of GDM patients vs. NGT, accompanied by specific alterations of respective promoter methylation patterns. In conclusion, we propose a critical role of VAT and visceral obesity for the pathogenesis of GDM, with epigenetic alterations of the expression of inflammatory factors as a potential factor

Challenges and (Un)Certainties for DNAm Age Estimation in Future

Age estimation is a paramount issue in criminal, anthropological, and forensic research. Because of this, several areas of research have focused on the establishment of new approaches for age prediction, including bimolecular and anthropological methods. In recent years, DNA methylation (DNAm) has arisen as one of the hottest topics in the field. Many studies have developed age- prediction models (APMs) based on evaluation of DNAm levels of many genes in different tissue types and using different methodological approaches. However, several challenges and confounder factors should be considered before using methylation levels for age estimation in forensic contexts. To provide in-depth knowledge about DNAm age estimation (DNAm age) and to understand why it is not yet a current tool in forensic laboratories, this review encompasses the literature for the most relevant scientific works published from 2015 to 2021 to address the challenges and future directions in the field. More than 60 papers were considered focusing essentially on studies that developed models for age prediction in several sample typesinfo:eu-repo/semantics/publishedVersio

Children's biobehavioral reactivity to challenge predicts DNA methylation in adolescence and emerging adulthood.

A growing body of research has documented associations between adverse childhood environments and DNA methylation, highlighting epigenetic processes as potential mechanisms through which early external contexts influence health across the life course. The present study tested a complementary hypothesis: indicators of children's early internal, biological, and behavioral responses to stressful challenges may also be linked to stable patterns of DNA methylation later in life. Children's autonomic nervous system reactivity, temperament, and mental health symptoms were prospectively assessed from infancy through early childhood, and principal components analysis (PCA) was applied to derive composites of biological and behavioral reactivity. Buccal epithelial cells were collected from participants at 15 and 18 years of age. Findings revealed an association between early life biobehavioral inhibition/disinhibition and DNA methylation across many genes. Notably, reactive, inhibited children were found to have decreased DNA methylation of the DLX5 and IGF2 genes at both time points, as compared to non-reactive, disinhibited children. Results of the present study are provisional but suggest that the gene's profile of DNA methylation may constitute a biomarker of normative or potentially pathological differences in reactivity. Overall, findings provide a foundation for future research to explore relations among epigenetic processes and differences in both individual-level biobehavioral risk and qualities of the early, external childhood environment

Epigenetic age estimation in saliva and in buccal cells

Age estimation based on epigenetic markers is a DNA intelligence tool with the potential to provide relevant information for criminal investigations, as well as to improve the inference of age-dependent physical characteristics such as male pattern baldness or hair color. Age prediction models have been developed based on different tissues, including saliva and buccal cells, which show different methylation patterns as they are composed of different cell populations. On many occasions in a criminal investigation, the origin of a sample or the proportion of tissues is not known with certainty, for example the provenance of cigarette butts, so use of combined models can provide lower prediction errors. In the present study, two tissue-specific and seven age-correlated CpG sites were selected from publicly available data from the Illumina HumanMethylation 450 BeadChip and bibliographic searches, to help build a tissue-dependent, and an age-prediction model, respectively. For the development of both models, a total of 184 samples (N = 91 saliva and N = 93 buccal cells) ranging from 21 to 86 years old were used. Validation of the models was performed using either k-fold cross-validation and an additional set of 184 samples (N = 93 saliva and N = 91 buccal cells, 21–86 years old). The tissue prediction model was developed using two CpG sites (HUNK and RUNX1) based on logistic regression that produced a correct classification rate for saliva and buccal swab samples of 88.59 % for the training set, and 83.69 % for the testing set. Despite these high success rates, a combined age prediction model was developed covering both saliva and buccal cells, using seven CpG sites (cg10501210, LHFPL4, ELOVL2, PDE4C, HOXC4, OTUD7A and EDARADD) based on multivariate quantile regression giving a median absolute error (MAE): ± 3.54 years and a correct classification rate ( %CP±PI) of 76.08 % for the training set, and an MAE of ± 3.66 years and a %CP±PI of 71.19 % for the testing set. The addition of tissue-of origin as a co-variate to the model was assessed, but no improvement was detected in age predictions. Finally, considering the limitations usually faced by forensic DNA analyses, the robustness of the model and the minimum recommended amount of input DNA for bisulfite conversion were evaluated, considering up to 10 ng of genomic DNA for reproducible results. The final multivariate quantile regression age predictor based on the models we developed has been placed in the open-access Snipper forensic classification websiteThis project was funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (Modalidade B, ED481B 2018/010) by a postdoctorate grant awarded to AFA. MVL is supported by the Ministerio de Educación, Cultura y Ciencia, Spain (PID2019-107876RB-I00).M.d.l.P. is supported by a post-doctorate grant funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (ED481D-2021-008). J.R. is supported by the “Programa de axudas á etapa predoutoral” funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (ED481A-2020/039)S

Omic characterisation of placental development and phenotype

Gene expression is influenced by precise epigenetic mechanisms. In the context of pregnancy proper placental development and pregnancy outcome are dependent upon these mechanisms. These are poorly understood in the placenta and historically have not been investigated. In many biomedical research fields epigenetic modifications such as DNA methylation have been proven to be an effective biomarker. However, this has yet to be shown in the reproduction research field. The overall aim of this thesis was to investigate new epigenetic mechanisms in placental development and to identify novel biomarkers for phenotype prediction. This thesis firstly focuses on sex-biased gene expression in multiple human tissues to identify targets of sexual dimorphism. Secondly, it investigates novel transcripts in the placenta and finally focuses on using DNA methylation as a biomarker. Firstly, the research has identified potential new gene targets and mechanisms which may explain sexual dimorphism in many phenotypic traits and diseases. These results suggest that sex-biased gene expression is dynamic and tissue specific. It also highlights the need to consider sex as a biological variable in biomedical research and to address the lack of female representation in many studies. Secondly, by performing a de novo transcript analysis on the placenta this thesis has identified new non-coding RNAs. These placental transcripts were also found to be specific to the placenta and were differentially expressed across gestation and in preeclampsia compared to uncomplicated pregnancies. This suggests these transcripts may be involved in placental development and may have roles in the pathogenesis of preeclampsia. Identifying novel placenta specific transcripts has uncovered new research opportunities involving the placenta. There are potentially hundreds of other unannotated transcripts in the placenta which may have roles in placental development and may be crucial to a successful pregnancy outcome. Thirdly, using DNA methylation as a biomarker has led to the development of two key prediction models. The first one used the level of methylation at 62 cytosine-phosphate-guanosine (CpG) sites to determine the gestational age of a placenta. This computational tool was also used to identify placental aging in placentas from women with early onset preeclampsia. This tool points to potential mechanisms underpinning placental aging which may have an impact on pregnancy complications. The second prediction tool has identified 84 methylated sites in the methylome of maternal circulating leukocytes which can distinguish five pregnancy outcomes. This tool has potential clinical application to identify women at risk of a pregnancy complication. This would enable clinicians to intervene and potentially prevent or reduce morbidity and mortality for mother and child. In summary, this thesis has focused on sex differences in gene expression and DNA methylation in placental development. It has also shown that DNA methylation has potential as an effective biomarker in the field of reproduction research.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 201

FRA2A is a CGG repeat expansion associated with silencing of AFF3

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5-12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship