Understanding the molecular and functional consequences of epigenome dynamics in cell fate, aging, and disease

DNA replication plays an important part in allowing cells to proliferate and develop into complex tissues. The advent of multicellular organisms, however, has been theorized to be intertwined with the tradeoff of aging and disease. These events are highly associated with drastic changes in gene expression across a cell population, often regulated by the epigenome. The set of heritable modifications that make up the epigenetic landscape are known to be altered by cell fate, aging, and disease. However, the dynamic processes by which the changes in the epigenome, and subsequently transcriptome, lead to these modified cell states are not clearly understood. In this dissertation, we demonstrate that DNA replication leads to a transient window of epigenetic entropy, providing the first evidence of a molecular link between cell fate, aging, and disease. In order to elucidate this link, we made use of replication-associated bisulfite sequencing (Repli-BS) and replication-associated assay for transposase-accessible chromatin sequencing (Repli-ATAC) datasets in human embryonic stem cells (hESCs). Our results suggest that the temporality of this window for both the chromatin architecture and DNA methylation differs across the genome. Specifically, we identified that the regions with the most prolonged window of epigenetic entropy are located at regulatory features, associate with expression variability, and are susceptible to age- and disease-related epigenetic drift. Additionally, this dissertation explores the impact of individual LMNA mutations on the epigenome that lead to unique disease outcomes of dilated cardiomyopathy (DCM) and brachydactyly using patient-derived fibroblasts and induced pluripotent stem cells (iPSCs). Analyses combining multiple epigenetic features and transcriptomic data suggest that differentially methylated regions (DMRs) are associated with the misregulation of regulatory elements, and that, in combination with chromatin remodeling, could lead to gene dysregulation ending in DCM. Ultimately, our results provide evidence that somatic and reprogrammed patient cells could serve as models to understand the mechanism behind which disease-related regulatory abnormalities lead to laminopathies like DCM and brachydactyly

Prevalence and direct costs of potentially inappropriate prescriptions in France: a population-based study

BACKGROUND: Potentially inappropriate prescriptions (PIPs) in the older population remain a growing public health concern due to the many associated adverse events increasing healthcare service use and health costs. This study aimed to assess the prevalence and direct costs of PIPs in older adults aged ≥65 years in France. METHODS: A population-based cross-sectional study was conducted in 2017 using a representative sample of the French national healthcare reimbursement system database. PIPs were defined using the French REMEDI[e]S tool. Overall reimbursed direct costs and by PIP category were extrapolated to the French older population. RESULTS: The overall PIP prevalence was estimated at 56.7% (95% CI: 56.4-57.0). Medications with an unfavorable benefit/risk ratio had the highest prevalence (34.0%, 95% CI: 33.7-34.3). Direct costs associated with PIPs represented 6.3% of the total reimbursed medication costs in 2017 (€507 million). Drug duplications were the main contributors to these costs (39.2% of the total reimbursed PIP costs, €199 million) and among all PIPs, proton pump inhibitors (>8 weeks) were the most expensive PIPs (€152 million). CONCLUSIONS: PIP prevalence is still high among French older adults, with substantial direct costs. Large-scale interventions targeting the most prevalent and/or costly PIPs are needed to reduce their clinical and economic impacts

DNA methylation analysis reveals epimutation hotspots in patients with dilated cardiomyopathy-associated laminopathies.

BackgroundMutations in LMNA, encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly.ResultsFamily-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA-related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development.ConclusionsOur results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages

Simultaneous determination of eight β-lactam antibiotics in human plasma and cerebrospinal fluid by liquid chromatography coupled to tandem mass spectrometry

International audienceTherapeutic drug monitoring of β-lactam antibiotics is increasingly used for dose optimization in the individual patient to increase efficacy and reduce the risk of toxicity. The objective of this work is to develop and validate a fast and reliable method using liquid chromatography coupled to tandem mass spectrometric detection to quantify simultaneously amoxicillin, cloxacillin, cefazolin, cefotaxime, ceftazidime, cefepime, meropenem and piperacillin in plasma and cerebrospinal fluid (CSF). Sample clean-up included protein precipitation with acetonitrile followed by evaporation of the supernatant and reconstitution of the residue with mobile phase solvents. Eight deuterated β-lactam antibiotics were used as internal standards. Chromatographic separation was performed on a C18 column (50 mm x 2.1 mm) using a binary gradient elution of water and acetonitrile both containing 0.1% (v/v) formic acid. The total run time was 8 min. The method was then used to perform therapeutic drug monitoring on 2221 patient plasma samples. 32 CSF samples were also analyzed. This method, with its simple sample preparation provides sensitive, accurate and precise quantification of the plasma and cerebrospinal fluid concentration of β-lactam antibiotics and can be used for therapeutic drug monitoring

Examining age-dependent DNA methylation patterns and gene expression in the male and female mouse hippocampus.

DNA methylation is a well-characterized epigenetic modification involved in numerous molecular and cellular functions. Methylation patterns have also been associated with aging mechanisms. However, how DNA methylation patterns change within key brain regions involved in memory formation in an age- and sex-specific manner remains unclear. Here, we performed reduced representation bisulfite sequencing (RRBS) from mouse dorsal hippocampus - which is necessary for the formation and consolidation of specific types of memories - in young and aging mice of both sexes. Overall, our findings demonstrate that methylation levels within the dorsal hippocampus are divergent between sexes during aging in genomic features correlating to mRNA functionality, transcription factor binding sites, and gene regulatory elements. These results define age-related changes in the methylome across genomic features and build a foundation for investigating potential target genes regulated by DNA methylation in an age- and sex-specific manner

Stochastic modeling reveals kinetic heterogeneity in post-replication DNA methylation.

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer.

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression