Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans
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ABSTRACT: DNA methylation likely plays a role in the regulation of human stress reactivity. In a genome-wide analysis of blood DNA methylation in 85 healthy individuals a locus in the Kit ligand (KITLG) gene (cg27512205) had the strongest association with cortisol stress reactivity (p=5.8x10-6). Replication was obtained in two independent samples, one using blood (N=45, p=0.001) and the other using buccal cells (N=255,p=0.004). KITLG methylation strongly mediated the relationship between childhood trauma and cortisol stress reactivity (32% mediation). Its genomic location (CpG island shore within an H3K27ac enhancer mark) provide further evidence that KITLG methylation is functionally relevant for the programming of stress reactivity. Our results extend preclinical evidence for epigenetic regulation of stress reactivity to humans and provide leads to enhance our understanding of the neurobiological pathways underlying stress vulnerability. Bisulphite converted DNA from whole blood of 85 healthy controls exposed to psychosocial stress task (TSST-G) was hybridised to the Illumina Infinium 450k Human Methylation Beadchip The DOI for this paper will be 10.1038/NCOMMS10967.
Project description:Juvenile myelomonocytic leukemia (JMML) is a very rare and aggressive stem cell disease that mainly occurs in young children. RAS activation constitutes the core component of oncogenic signaling. In addition, the leukemic blasts of a quarter of JMML patients present with monosomy 7 (-7), whereas more than half of the patients show enhanced age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care. This results in an event-free survival of 50 - 60%, indicating that novel molecular driven therapeutic options are urgently needed. Using gene expression profiling in an extensive series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. Interestingly, LIN28B overexpression was significantly correlated with higher HbF levels whereas patients with -7 seldom showed enhanced LIN28B expression. In line with LIN28Bâ??s role as mediator of fetal hematopoiesis, this explains the biology behind the observation that patients with -7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Finally, high LIN28B expression was associated with poor clinical outcome in our JMML patient series, but not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified LIN28B as a crucial molecular player at the heart of a novel fetal-like subgroup in JMML. Gene expression was measured on Affymetrix in 38 JMML patients and 9 healthy donors in a validation cohort.
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
Project description:Background and Aims Small intestinal neuroendocrine tumours (SINETs) are the commonest malignancy of the small intestine; however underlying pathogenic mechanisms remain poorly characterised. Whole genome and exome sequencing has demonstrated that SINETs are mutationally quiet with the most frequent known mutation in the cyclin dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ~8% of tumours, suggesting that alternative mechanisms may drive tumourigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumour type. Methods Here we present data from integrated molecular analysis of SINETs (n=97) including whole exome or targeted CDKN1B sequencing (n=29), HumanMethylation450 BeadChip (Illumina) array profiling (n=69), methylated DNA immunoprecipitation sequencing (n=16), copy number variance analysis (n=47) and Whole Genome-DASL (Illumina) expression array profiling (n=43). Results Based on molecular profiling SINETs can be classified in to three groups which demonstrate significantly different progression-free survival after resection of primary tumour (not reached at 10 years vs 56 months vs 21 months, p=0.04). Epimutations were found at a recurrence rate of up to 85% and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%) and GIPR (74%). Conclusions This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumours are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression free survival. Background and Aims Small intestinal neuroendocrine tumours (SINETs) are the commonest malignancy of the small intestine; however underlying pathogenic mechanisms remain poorly characterised. Whole genome and exome sequencing has demonstrated that SINETs are mutationally quiet with the most frequent known mutation in the cyclin dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ~8% of tumours, suggesting that alternative mechanisms may drive tumourigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumour type. Methods Here we present data from integrated molecular analysis of SINETs (n=97) including whole exome or targeted CDKN1B sequencing (n=29), HumanMethylation450 BeadChip (Illumina) array profiling (n=69), methylated DNA immunoprecipitation sequencing (n=16), copy number variance analysis (n=47) and Whole Genome-DASL (Illumina) expression array profiling (n=43). Results Based on molecular profiling SINETs can be classified in to three groups which demonstrate significantly different progression-free survival after resection of primary tumour (not reached at 10 years vs 56 months vs 21 months, p=0.04). Epimutations were found at a recurrence rate of up to 85% and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%) and GIPR (74%). Conclusions This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumours are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression free survival. This study included 97 tumour samples from 85 individuals, this included both primary and metastatic tumour samples. 25 normal small intestinal samples were analysed.
Project description:miRNA expression profiles were evaluated in a series of 64 prostate clinical specimens, including 32 cancer and 32 non-neoplastic tissues. For 26 individuals, paired cancer and non-neplastic tissue was available. Tumor and non-neoplastic tissues were obtained, with appropriate informed consent and Institutional Review Board approval, from untreated prostate cancer patients subjected to radical prostatectomy. Freshly frozen surgical blocks were carefully dissected by the pathologist using H&E-stained sections as a template to identify areas containing at least 70% of tumor or normal cells. The total series comprises 64 clinical specimens, of which 32 from cancer areas and 32 from benign areas. For 26 patients, matched tumor and normal samples were available.
Project description:To detect genetic variations affecting gene expression in mouse lung tumor tissue, we carried out a genetic linkage study on the transcriptome of lung tumors induced by urethane in an (A/J x C57BL/6)F4 intercross population, whose individual lung tumor multiplicity (Nlung) is linked to the genotype at the pulmonary adenoma susceptibility 1 (Pas1) locus (containing the Kras-37-bp marker) . In particular, susceptible animals carry the A/J-derived version of Pas1 in homozygousity or in heterozygousity, while tumor-resistant animals carry the C57BL/6-derived version. We found that expression levels of 1,179 and 1,579 genes are modulated by an expression quantitative trait locus (eQTL) in cis and in trans, respectively (LOD score > 5). Of note, the genomic area surrounding and including the Pas1 locus regulated 14 genes in cis and 857 genes in trans. In lung tumors of the same (A/J x C57BL/6)F4 mice, we found 1,124 genes whose transcript levels associated with Nlung (FDR < 0.001). The expression levels of about a third of these genes (n = 401) were regulated by the genotype at the Pas1 locus. Pathway analysis of the sets of genes associated with Nlung and regulated by Pas1 revealed a set of 14 recurrently represented genes that are components or targets of the RasâErk and Pi3kâAkt signaling pathways. In conclusion, our results illustrate the architecture of germline control of gene expression in mouse lung cancer: they highlight the importance of Pas1 as a tumor-modifier locus, attribute to it a novel role as a major regulator of transcription in lung tumor nodules and strengthen the candidacy of the Kras gene as the effector of this locus. Female animals of the lung tumor-susceptible A/J strain (A) were mated with males of the lung tumor-resistant C57BL/6 strain (B), and offspring were bred to the fourth generation (ABF4). At 4 weeks of age, 143 male ABF4 mice were treated with a single intraperitoneal injection of the chemical carcinogen urethane to induce the development of lung tumors. When the animals were 40 weeks of age, they were killed, the lungs were removed for tumor counting (values of lung tumor multiplicity [Nlung] varied substantially among individuals, ranging from 1 to 33 in the animals that developed tumors), and a single lung tumor, 1â1.5 mm in diameter, was excised from each for RNA extraction.
Project description:Using a dataset of 54 pregnant and 113 age/stage-matched non-pregnant breast cancer patients with complete clinical and survival data; we evaluated the pattern of hot spot somatic mutations and performed transcriptomic profiling using Sequenom® and Affymetrix®, respectively. Breast cancer molecular subtypes were defined using PAM50 and 3-Gene classifiers. We performed Gene set enrichment analysis (GSEA) to evaluate pathways associated with diagnosis during pregnancy. We investigated the differential expression of cancer-related genes and published gene sets according to pregnancy. We finally investigated genes associated with disease-free survival. We identified 65 patients who were diagnosed with breast cancer during pregnancy. Moreover, for each case, two breast cancer patients who were not diagnosed during pregnancy or lactation, but who were matched according to age (±2 years), tumors size, nodal status, year of surgery (±2 years) and whether neoadjuvant chemotherapy was received were selected. Messenger RNA from tumor tissue has been extracted and hybridized on Affymetrix microarrays. However, due to experimental issues, 54 pregnant and 113 non-pregnant patients were remaining and used in our study.
Project description:We investigated changes in the human immune system following vaccination against H1N1 swine flu, to define the healthy immue response to vaccination, and to correlate it to cases of non-response and adverse events. Gene expression was measured twice before and twice after vaccination (week before/on the day of vaccination/one day and week after). Responsiveness to the vaccine was defined as fourfold increase in HAI (hemagglutination inhibition) and NM (neuraminidase inhibition) titers and adverse reaction as a sum of patient reported symptoms, measured n a Likert scale.
Project description:Scientific evidence indicates that genetic factors may contribute to differences in lung cancer risk for individuals with similar levels of tobacco exposure, which is the main environmental risk factor of lung cancer. Moreover, lung cancer patients show large differences in clinical staging and survival; these differences seem to be attributable, at least partially, to the genetic background. The analysis of the molecular properties (e.g., germline variations and genome-wide expression levels) of non-involved tissue from lung cancer patients may contribute in the identification of genetic factors involved in the development and progression of this pathology. To this aim, we analyzed two series (discovery series, n = 204, and validation series, n = 78) of non-tumor lung tissue samples from smokers that underwent surgical lobectomy for adenocarcinoma in Milan, Italy. First, we examined the whole transcriptome of these two series to define the candidate genes and pathways associated with either lung cancer risk or prognosis in this cohort. Moreover, as sex and age are known to strongly influence the pathophysiology of human lungs, we used transcriptome data from the same samples to identify sex- and age-related transcriptional differences in lung. Samples of non-involved (apparently normal) lung parenchyma excised from patients who underwent lobectomy for lung adenocarcinoma in the area around Milan, Italy. The samples were analyzed in two sets: a discovery series (n = 204) and a validation series (n = 78).
Project description:We present a meta-dataset comprising of a total of 178 samples including both primary tumors and tumor-free pancreatic tissues from four independent GEO datasets. To minimise inter-platform variation, only datasets generated from the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array) were processed to develop the meta-dataset. Using multiple open source R packages implemented in our previously developed bioinformatics pipeline, each dataset has been preprocessed with RMA normalisation, merged, and batch effect-corrected via Combat method. With increased sample size, the present meta-dataset serves an excellent 'discovery cohort' for discovering differentially expressed in diseased phenotype.