Urbanization reduces gut bacterial microbiome diversity in a specialist ground beetle, Carabus convexus.

Urbanization is rapidly shaping and transforming natural environments, creating networks of modified land types. These urbanization-driven modifications lead to local extinctions of several species, but the surviving ones also face numerous novel selection pressures, including exposure to pollutants, habitat alteration, and shifts in food availability and diversity. Based on the assumption that the environmental pool of microorganisms is reduced in urban habitats due to habitat alteration, biodiversity loss, and pollution, we hypothesized that the diversity of bacterial microbiome in digestive tracts of arthropods would be lower in urban than rural habitats. Investigating the gut bacterial communities of a specialist ground beetle, Carabus convexus, in forested rural versus urban habitats by next generation high-throughput sequencing of the bacterial 16S rRNA gene, we identified 3839 bacterial amplicon sequence variants. The composition of gut bacterial samples did not significantly differ by habitat (rural vs. urban), sex (female vs. male), sampling date (early vs. late spring), or their interaction. The microbiome diversity (evaluated by the Rényi diversity function), however, was higher in rural than urban adults. Our findings demonstrate that urbanization significantly reduced the diversity of the gut bacterial microbiome in C. convexus.

Surprising genetic and pathological findings in a patient with giant bilateral periadrenal tumours: PEComas and mutations of PTCH1 in Gorlin-Goltz syndrome.

Gorlin-Goltz syndrome (GGS) or nevoid basal cell carcinoma syndrome is a rare tumour-overgrowth syndrome associated with multiple developmental anomalies and a wide variety of tumours. Here, we describe a case of a man aged 23 years with GGS with bilateral giant tumours adjacent to both adrenals that raised the suspicion of malignancy on imaging. Histological analysis of both surgically resected tumours revealed perivascular epitheloid cell tumours (PEComas) that were independent of the adrenals. Exome sequencing of the patient's blood sample revealed a novel germline heterozygous frameshift mutation in the PTCH1 gene. As a second hit, a somatic five nucleotide long deletion in the PTCH1 gene was demonstrated in the tumour DNA of both PEComas. To the best of our knowledge, this is the first report on PEComa in GGS, and this finding also raises the potential relevance of PTCH1 mutations and altered sonic hedgehog signalling in PEComa pathogenesis. The presence of the same somatic mutation in the bilateral tumours might indicate the possibility of a postzygotic somatic mutation that along with the germline mutation of the same gene could represent an intriguing genetic phenomenon (type 2 segmental mosaicism).

Molecular genetic diagnostics of hypogonadotropic hypogonadism: from panel design towards result interpretation in clinical practice.

Congenital hypogonadotropic hypogonadism (CHH) is a clinically and genetically heterogeneous congenital disease. Symptoms cover a wide spectrum from mild forms to complex phenotypes due to gonadotropin-releasing hormone (GnRH) deficiency. To date, more than 40 genes have been identified as pathogenic cause of CHH. These genes could be grouped into two major categories: genes controlling development and GnRH neuron migration and genes being responsible for neuroendocrine regulation and GnRH neuron function. High-throughput, next-generation sequencing (NGS) allows to analyze numerous gene sequences at the same time. Nowadays, whole exome or whole genome datasets could be investigated in clinical genetic diagnostics due to their favorable cost-benefit. The increasing genetic data generated by NGS reveal novel candidate genes and gene variants with unknown significance (VUSs). To provide clinically valuable genetic results, complex clinical and bioinformatics work are needed. The multifaceted genetics of CHH, the variable mode of inheritance, the incomplete penetrance, variable expressivity and oligogenic characteristics further complicate the interpretation of the genetic variants detected. The objective of this work, apart from reviewing the currently known genes associated with CHH, was to summarize the advantages and disadvantages of the NGS-based platforms and through the authors' own practice to guide through the whole workflow starting from gene panel design, performance analysis and result interpretation. Based on our results, a genetic diagnosis was clearly identified in 21% of cases tested (8/38).

Wnt-Signaling Regulated by Glucocorticoid-Induced miRNAs.

Glucocorticoids (GCs) are pleiotropic hormones which regulate innumerable physiological processes. Their comprehensive effects are due to the diversity of signaling mechanism networks. MiRNAs, small, non-coding RNAs contribute to the fine tuning of signaling pathways and reciprocal regulation between GCs and miRNAs has been suggested. Our aim was to investigate the expressional change and potential function of GC mediated miRNAs. The miRNA expression profile was measured in three models: human adrenocortical adenoma vs. normal tissue, steroid-producing H295R cells and in hormonally inactive HeLa cells before and after dexamethasone treatment. The gene expression profile in 82 control and 57 GC-affected samples was evaluated in GC producing and six different GC target tissue types. Tissue-specific target prediction (TSTP) was applied to identify the most relevant miRNA-mRNA interactions. Glucocorticoid treatment resulted in cell type-dependent miRNA expression changes. However, 19.5% of the influenced signaling pathways were common in all three experiments, of which the Wnt-signaling pathway seemed to be the most affected. Transcriptome data and TSTP showed similar results, as the Wnt pathway was significantly altered in both the GC-producing adrenal gland and all investigated GC target tissue types. In different cell types, different miRNAs led to the regulation of similar pathways. Wnt signaling may be one of the most important signaling pathways affected by hypercortisolism. It is, at least in part, regulated by miRNAs that mediate the glucocorticoid effect. Our findings on GC producing and GC target tissues suggest that the alteration of Wnt signaling (together with other pathways) may be responsible for the leading symptoms observed in Cushing's syndrome.

Expression of GLP-1 receptors in insulin-containing interneurons of rat cerebral cortex.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons. METHODS: We harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5 mmol/l glucose) and hyperglycaemia (10 mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results. RESULTS: Single-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p < 0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development. CONCLUSIONS/INTERPRETATION: Our results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells.

An unexpected, mild phenotype of glucocorticoid resistance associated with glucocorticoid receptor gene mutation case report and review of the literature.

BACKGROUND: Glucocorticoid resistance is a rare, sporadic or familial condition caused by mutation of the gene encoding the glucocorticoid receptor (GR). Clinically it is characterized by symptoms developed due to local, tissue-specific, or generalized partial insensitivity to glucocorticoids. CASE PRESENTATION: A 31-year-old woman was evaluated because of infertility at the Endocrine Unit of the 2nd Department of Medicine, Semmelweis University. During her laboratory investigations, elevated serum and salivary cortisol were observed which failed to be suppressed after administration of 1 mg dexamethasone. 24 h urinary cortisol was increased, but a normal midnight serum cortisol was detected suggesting a maintained circadian rhythm. Plasma dehydroepiandrosterone-sulfate and androstendione levels were also elevated. Repeated plasma ACTH measurements indicated slightly elevated or normal values. Bone mineral density was normal. All laboratory results confirmed the diagnosis of glucocorticoid resistance. Genetic counseling followed by Sanger sequencing of the coding region of the gene encoding human glucocorticoid receptor was performed and a missense mutation (Arg714Gln, R714Q) in a heterozygous form was detected. Following family screening, the same mutation was found in her clinically-healthy 35-year-old sister who had no fertility problems.This variant was not detected in more than 60 patients and controls tested either for glucocorticoid resistance or Cushing's syndrome in our Laboratory and it was absent in Exome Variant Server, HumanGene Mutation Database and ExAC databases. CONCLUSIONS: Our case fulfils the diagnostic criteria of glucocorticoid resistance, also named Chrousos syndrome. The glucocorticoid receptor gene mutation detected in our patient has been already reported in a 2-year-old child with hypoglycaemia, hypokalaemia, hypertension and premature puberty. These distinct phenotypes may suggest that other factors may modify the functional consequences of the R714Q variant of GR.

[Extraadrenal glucocorticoid synthesis]. / Extraadrenalis glükokortikoidszintézis.

Endogenous glucocorticoids exert a diverse array of physiological processes and play an important role in immune modulatory and anti-inflammatory responses. The secretion of cortisol by the adrenal gland is regulated through two mechanisms. Systemic regulation is substantiating by the hypothalamo-pituitary-adrenal axis. Furthermore, a tissue-specific local regulatory system, containing the 11ß-hydroxysteroid dehydrogenase enzyme responsible for local glucocorticoid synthesis and the glucocorticoid receptor, has also been demonstrated. Based on the recent evidences, an extra-adrenal corticosteroid synthesis exists in various tissues. Steroidogenic enzymes necessary for this de novo corticosteroid synthesis have been observed in the skin, intestine, thymus and possibly in the brain, heart and lung. These locally synthesized steroids most likely act in an autocrine and paracrine manner and their regulation is mediated by local regulatory loops. The importance of this de novo corticosteroid synthesis seems to be important in the regulation of local homeostasis, immune processes and tissue-specific inflammatory reactions. Orv Hetil. 2018; 159(7): 260-268.

[Evolution of molecular genetic methods in the clinical diagnosis of hereditary endocrine tumour syndromes]. / Molekuláris genetikai vizsgálatok az örökletes endokrinológiai tumor szindrómák klinikai diagnosztikájában.

The common features of hereditary endocrine tumour syndromes or multiple endocrine neoplasias (MEN) are the association of various tumours of different endocrine organs in one patient or within the same family. Different types can be distinguished from among which type 1 and type 2 are the most common. The mode of inheritance is autosomal dominant, meaning that there is a 50% chance to inherit the pathogenic alteration. The pathogenic variants of genes responsible for MEN syndromes have also been identified in sporadic endocrine tumours and many cases initially referred to as sporadic have been later categorized as familiar based on genetic analysis. The main role of the molecular genetic analysis in these syndromes is to identify the pathogenic variant, then, after appropriate genetic counseling, to perform the genetic screening of first-degree relatives. Following molecular genetic analysis, the state-of-the-art clinical follow-up of the clinically healthy mutation carriers may decrease or even prevent the morbidity and mortality. Due to technological developments in recent years, the molecular genetic analysis of hereditary tumour syndromes has also been changed. Using next generation based sequencing methods in routine clinical diagnostics, the number of pathogenic genes in endocrine tumours has also increased. The present review focuses on the genetic background of hereditary endocrine tumour syndromes and the recently used molecular biological methods will also be presented. Orv Hetil. 2018; 159(7): 285-292.

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.

[Investigation of CNTF, COMT, DDR1, DISC1, DRD2, DRD3, and DTNBP1 candidate genes in schizophrenia: Results from the Hungarian SCHIZOBANK Consortium]. / CNTF, COMT, DDR1, DISC1, DRD2, DRD3 és DTNBP1 kandidáns gének vizsgálata szkizofréniában: eredmények a Magyar SCHIZOBANK Konzorcium vizsgálatából.

Schizophrenia is a chronic, debilitating psychiatric disorder characterized by heterogeneous clinical symptoms. Although the pathogenesis of this disorder is poorly understood, several lines of evidence support the role of both common and rare genetic variants in the etiology of schizophrenia. Common variants, single nucleotide polymorphisms can be investigated by candidate gene association studies or genome-wide association studies, while rare variants, single nucleotide variants are assessable by means of candidate gene resequencing or whole-exome and genome sequencing using next generation sequencing. In this study we investigated polymorphisms of 7 candidate genes in a Hungarian schizophrenia cohort. Candidate genes were chosen on the basis of previous results and biological plausibility. 390 patients were recruited in 5 centers in the framework of the Hungarian SCHIZOBANK Consortium, the schizophrenia sample was contrasted to 1069 healthy control individuals. In this sample SNPs of DDR1 and DRD2 genes demonstrated significant association with schizophrenia. The role of DDR1 and DRD2 genes in the etiology of schizophrenia warrant further investigation, based on their genomic localization and biological functions.

Autophagy may contribute to the recovery of rat mesothelium following acute inflammation in vivo.

Following Freund's adjuvant-induced acute inflammation, the regeneration of rat mesothelium is accompanied by the reduction of cell organelles. The aim of the present study is to test whether autophagy may play a role in the recovery process of mesothelial cells by eliminating accumulated cell organelles and also to investigate the presence of potential inducers and molecular transmitters of the process. Control and treated (from day 2 to day 11; D2-D11) mesothelial cells (n = 16 samples/group) obtained from male rats were isolated and phenotypically characterized. Morphological studies included light and electron microscopy. Biochemical studies performed on tissue samples as well as isolated cells were used to evaluate the dynamics of autophagy and also to detect the expression levels of TNF-α, LC3B, estrogen receptors (ER-α and GPR30) and Erk1/2. Gene expression was measured by individual Taqman assays on quantitative RT-PCR. Protein expression study was performed by Western blotting and immunolabeling. Estradiol concentration was measured both in peritoneal fluid and plasma samples in control and treated animals (n = 3-10 animals per group). Our conventional electron microscopic and morphometric results showed a progressive autophagosome formation with a peak by the termination of inflammation (D5). Subsequently, autophagolysosome formation dominated between D6 and D8 with a concomitant expression of LC3B proved by immunoblotting. We further observed the reduction of cell compartments by D11 parallel with the morphological restitution of mesothelium. Estradiol showed a sustained level in the peritoneal fluid but not in plasma samples between D3 and D11 compared to levels obtained from untreated animals. The mRNA expression of TNF-α was increased between D2 and D11 compared to control. Western blot analysis showed a constitutive expression of GPR30, while ER-α could not be detected between D6 and D11. Erk1/2 was activated by phosphorylation with a peak at D6. Considering our present in vivo results, we hypothesize that the facilitated autophagy might play an important role in the removal of cytoplasmic organelles during the recovery of mesothelium, while our results also suggest that the detected peritoneal estradiol as well as TNF-α may contribute to this process.

[Novel methods and their applicability in the evaluation of the genetic background of endocrine system tumours]. / Új módszertani lehetoségek és ezek alkalmazása a hormonális rendszer daganatainak genetikai kivizsgálásában.

The technical developments leading to revolution in clinical genetic testing offer new approaches for patients with cancer. From one mutation or one gene approach the scale of genetic testing moved to whole exome or whole genome scale. It is well known that many tumours are genetically determined and they are part of familial tumour syndromes. In addition, some mutations indicate specific molecular targeted therapies. Although sampling and sample preparation are different for testing germline and somatic mutations, the technical background of the analysis is the same. The aim of clinical genetic testing is to identify patients who are carriers of disease-causing mutations or to test tumour tissue for the presence of genetic alterations which may be targets for therapeutic approaches. In this review the authors summarize novel possibilities offered by next-generation sequencing in clinical genetic testing of patients with endocrine tumours. In addition, the authors review recent guidelines on technical and ethical issues related to these novel methods.

DNA methylome, R-loop and clinical exome profiling of patients with sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes. The above datasets were combined to create a comprehensive repository of genetic and epigenetic changes associated with the ALS cases studied. This repository is well-suited to unveil new correlations within individual patients and across the entire patient cohort. The molecular attributes described here are expected to guide further mechanistic studies on ALS, shedding light on the underlying genetic causes and facilitating the development of new epigenetic therapies to combat this life-threatening disease.

[Importance of the 11ß-hydroxysteroid dehydrogenase enzyme in clinical disorders]. / A 11-ß-hidroxi-szteroid-dehidrogenáz enzim jelentosége klinikai kórképekben.

Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11ß-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11ß-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

Context-Dependent Role of Glucocorticoid Receptor Alpha and Beta in Breast Cancer Cell Behaviour.

Background. The dual role of GCs has been observed in breast cancer; however, due to many concomitant factors, GR action in cancer biology is still ambiguous. In this study, we aimed to unravel the context-dependent action of GR in breast cancer. Methods. GR expression was characterized in multiple cohorts: (1) 24,256 breast cancer specimens on the RNA level, 220 samples on the protein level and correlated with clinicopathological data; (2) oestrogen receptor (ER)-positive and -negative cell lines were used to test for the presence of ER and ligand, and the effect of the GRß isoform following GRα and GRß overexpression on GR action, by in vitro functional assays. Results. We found that GR expression was higher in ER- breast cancer cells compared to ER+ ones, and GR-transactivated genes were implicated mainly in cell migration. Immunohistochemistry showed mostly cytoplasmic but heterogenous staining irrespective of ER status. GRα increased cell proliferation, viability, and the migration of ER- cells. GRß had a similar effect on breast cancer cell viability, proliferation, and migration. However, the GRß isoform had the opposite effect depending on the presence of ER: an increased dead cell ratio was found in ER+ breast cancer cells compared to ER- ones. Interestingly, GRα and GRß action did not depend on the presence of the ligand, suggesting the role of the "intrinsic", ligand-independent action of GR in breast cancer. Conclusions. Staining differences using different GR antibodies may be the reason behind controversial findings in the literature regarding the expression of GR protein and clinicopathological data. Therefore, caution in the interpretation of immunohistochemistry should be applied. By dissecting the effects of GRα and GRß, we found that the presence of the GR in the context of ER had a different effect on cancer cell behaviour, but independently of ligand availability. Additionally, GR-transactivated genes are mostly involved in cell migration, which raises GR's importance in disease progression.

A common genetic variation in GZMB may associate with cancer risk in patients with Lynch syndrome.

Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is a common genetic predisposition to cancer due to germline mutations in genes affecting DNA mismatch repair. Due to mismatch repair deficiency, developing tumors are characterized by microsatellite instability (MSI-H), high frequency of expressed neoantigens and good clinical response to immune checkpoint inhibitors. Granzyme B (GrB) is the most abundant serine protease in the granules of cytotoxic T-cells and natural killer cells, mediating anti-tumor immunity. However, recent results confirm a diverse range of physiological functions of GrB including that in extracellular matrix remodelling, inflammation and fibrosis. In the present study, our aim was to investigate whether a frequent genetic variation of GZMB, the gene encoding GrB, constituted by three missense single nucleotide polymorphisms (rs2236338, rs11539752 and rs8192917) has any association with cancer risk in individuals with LS. In silico analysis and genotype calls from whole exome sequencing data in the Hungarian population confirmed that these SNPs are closely linked. Genotyping results of rs8192917 on a cohort of 145 individuals with LS demonstrated an association of the CC genotype with lower cancer risk. In silico prediction proposed likely GrB cleavage sites in a high proportion of shared neontigens in MSI-H tumors. Our results propose the CC genotype of rs8192917 as a potential disease-modifying genetic factor in LS.

Whole genome sequencing resolves 10 years diagnostic odyssey in familiar myxoma.

Carney complex (CNC) is an ultrarare disorder causing cutaneous and cardiac myxomas, primary pigmented nodular adrenocortical disease, hypophyseal adenoma, and gonadal tumours. Genetic alterations are often missed under routine genetic testing. Pathogenic variants in PRKAR1A are identified in most cases, while large exonic or chromosomal deletions have only been reported in a few cases. Our aim was to identify the causal genetic alteration in our kindred with a clinical diagnosis of CNC and prove its pathogenic role by functional investigation. Targeted testing of PRKAR1A gene, whole exome and whole genome sequencing (WGS) were performed in the proband, one clinically affected and one unaffected relative. WGS identified a novel, large, 10,662 bp (10.6 kbp; LRG_514t1:c.-10403_-7 + 265del; hg19, chr17:g.66498293_66508954del) deletion in the promoter of PRKAR1A in heterozygous form in the affected family members. The exact breakpoints and the increased enzyme activity in deletion carriers compared to wild type carrier were proved. Segregation analysis and functional evaluation of PKA activity confirmed the pathogenic role of this alteration. A novel deletion upstream of the PRKAR1A gene was proved to be the cause of CNC. Our study underlines the need for WGS in molecular genetic testing of patients with monogenic disorders where conventional genetic analysis fails.

Meta-analysis of gene expression profiles associated with histological classification and survival in 829 ovarian cancer samples.

Transcriptomic analysis of global gene expression in ovarian carcinoma can identify dysregulated genes capable to serve as molecular markers for histology subtypes and survival. The aim of our study was to validate previous candidate signatures in an independent setting and to identify single genes capable to serve as biomarkers for ovarian cancer progression. As several datasets are available in the GEO today, we were able to perform a true meta-analysis. First, 829 samples (11 datasets) were downloaded, and the predictive power of 16 previously published gene sets was assessed. Of these, eight were capable to discriminate histology subtypes, and none was capable to predict survival. To overcome the differences in previous studies, we used the 829 samples to identify new predictors. Then, we collected 64 ovarian cancer samples (median relapse-free survival 24.5 months) and performed TaqMan Real Time Polimerase Chain Reaction (RT-PCR) analysis for the best 40 genes associated with histology subtypes and survival. Over 90% of subtype-associated genes were confirmed. Overall survival was effectively predicted by hormone receptors (PGR and ESR2) and by TSPAN8. Relapse-free survival was predicted by MAPT and SNCG. In summary, we successfully validated several gene sets in a meta-analysis in large datasets of ovarian samples. Additionally, several individual genes identified were validated in a clinical cohort.

Menopause leads to elevated expression of macrophage-associated genes in the aging frontal cortex: rat and human studies identify strikingly similar changes.

BACKGROUND: The intricate interactions between the immune, endocrine and central nervous systems shape the innate immune response of the brain. We have previously shown that estradiol suppresses expression of immune genes in the frontal cortex of middle-aged ovariectomized rats, but not in young ones reflecting elevated expression of these genes in middle-aged, ovarian hormone deficient animals. Here, we explored the impact of menopause on the microglia phenotype capitalizing on the differential expression of macrophage-associated genes in quiescent and activated microglia. METHODS: We selected twenty-three genes encoding phagocytic and recognition receptors expressed primarily in microglia, and eleven proinflammatory genes and followed their expression in the rat frontal cortex by real-time PCR. We used young, middle-aged and middle-aged ovariectomized rats to reveal age- and ovariectomy-related alterations. We analyzed the expression of the same set of genes in the postcentral and superior frontal gyrus of pre- and postmenopausal women using raw microarray data from our previous study. RESULTS: Ovariectomy caused up-regulation of four classic microglia reactivity marker genes including Cd11b, Cd18, Cd45 and Cd86. The change was reversible since estradiol attenuated transcriptional activation of the four marker genes. Expression of genes encoding phagocytic and toll-like receptors such as Cd11b, Cd18, C3, Cd32, Msr2 and Tlr4 increased, whereas scavenger receptor Cd36 decreased following ovariectomy. Ovarian hormone deprivation altered the expression of major components of estrogen and neuronal inhibitory signaling which are involved in the control of microglia reactivity. Strikingly similar changes took place in the postcentral and superior frontal gyrus of postmenopausal women. CONCLUSIONS: Based on the overlapping results of rat and human studies we propose that the microglia phenotype shifts from the resting toward the reactive state which can be characterized by up-regulation of CD11b, CD14, CD18, CD45, CD74, CD86, TLR4, down-regulation of CD36 and unchanged CD40 expression. As a result of this shift, microglial cells have lower threshold for subsequent activation in the forebrain of postmenopausal women.

Exploratory Circular RNA Profiling in Adrenocortical Tumors.

Differentiation of adrenocortical adenoma (ACA) and carcinoma (ACC) is often challenging even in the histological analysis. Circular RNAs (circRNAs) belonging to the group of non-coding RNAs have been implicated as relevant factors in tumorigenesis. Our aim was to explore circRNA expression profiles in adrenocortical tumors by next-generation sequencing followed by RT-qPCR validation. Archived FFPE (formalin-fixed, paraffin embedded) including 8 ACC, 8 ACA and 8 normal adrenal cortices (NAC) were used in the discovery cohort. For de novo and known circRNA expression profiling, a next-generation sequencing platform was used. CIRI2, CircExplorer2, AutoCirc bioinformatics tools were used for the discovery of circRNAs. The top five most differentially circRNAs were measured by RT-qPCR in an independent validation cohort (10 ACC, 8 ACA, 8 NAC). In silico predicted, interacting microRNAs potentially sponged by differentially expressed circRNAs were studied by individual RT-qPCR assays. We focused on overexpressed circRNAs here. Significantly differentially expressed circRNAs have been revealed between the cohorts by NGS. Only circPHC3 could be confirmed to be significantly overexpressed in ACC, ACA vs. NAC samples by RT-qPCR. We could not observe microRNA expression changes fully corresponding to our sponging hypothesis. To the best of our knowledge, our study is the first to investigate circRNAs in adrenocortical tumors. Further studies are warranted to explore their biological and diagnostic relevance.