Gene expression profiling and enrichment functional analyses to compare coronary microvessels and cardiomyocytes in patients with hypertrophic cardiomyopathy

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Pisano ◽  
L Le Pera ◽  
M Lombardi ◽  
F Ferre ◽  
R Carletti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Hypertrophic Cardiomyopathy ◽  
Interventricular Septum ◽  
Functional Enrichment ◽  
Public Institution ◽  
Cardiomyocyte Hypertrophy ◽  
Coronary Microvascular Dysfunction ◽  
Funding Source ◽  
Rna Seq

Abstract Background Hypertrophic cardiomyopathy (HCM) is characterized by severe alterations of cardiac architecture and function involving cardiomyocytes (CM) and coronary microvessels (MV). Coronary microvascular dysfunction, cardiomyocyte hypertrophy and disarray, sarcomeric alterations and interstitial fibrosis are HCM features. The transcriptome profile associated with coronary MV and CM in HCM patients is presently unknown. Purpose Aim of this study was to improve knowledge of the molecular and biological pahways involved in HCM. To this purpose, the gene expression profile of coronary MV and CM was investigated. Methods Interventricular septum myectomies from patients with obstructive HCM and donors' hearts (CTR) were collected. Coronary MV (HCM=20, CTR=6) and CM (HCM=10, CTR=5) were laser capture microdissected. RNA-seq was performed by Illumina Nextseq 500, with 76 nt long single-reads. Adapter trimming and quality filtering of the sequenced reads were performed before alignment to the human reference genome. Univocally mapped reads estimated gene expression/sample. Normalized expressed gene levels were quantified. Statistical tests compared HCM and CTR to identify differentially expressed genes (DEG), i.e. up- and down-expressed genes in CM and MV samples. Functional enrichment analysis was performed. Biological categories, i.e. KEGG and Reactome pathways, Gene Ontology terms, protein domains in InterPro database, putative interactors collected in the Intact database and protein annotations in UniProt were considered for inter group comparison of DEGs. Results Transcriptome analysis identified 392 genes significantly up-regulated and 514 down-regulated in CM samples of HCM vs. CTR, while in MV 681 genes were up-regulated and 815 down-regulated. Although some DEGs were shared between MV and CM (26 and 146 are up- and down-expressed in both sample types), the majority of DEGs displayed a sample-specific pattern. A comparative functional analysis of DEGs highlighted some statistically enriched biological categories including an enrichment of phosphoproteins, with down-expressed genes both in CM (490) and MV (314). Other biological categories annotated as “ubiquitin-like protein conjugation” or “acetylation” in Uniprot database were enriched in down-regulated genes, both in MV and CM. Interestingly, “ribosomal protein” and “ribonucleoprotein” categories resulted as enriched up-regulated DEGs in MV. Conversely, the “citrullination” category was specifically present in annotations associated to down-regulated DEGs in MV from HCM compared to CTR. Conclusions Our preliminary results support the suitability of RNA-seq analysis to assess: i. the transcriptome profiles and pathways associated to coronary MV and CM; ii. the possible relationship/interplay of MV and CM profiles and HCM disease. The enrichment functional analysis provides preliminary data on candidate DEGs and target proteins for in vitro studies on HCM-related mechanisms. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Ministry of Health

A chromatin signature by the methyltransferase SETD7 regulates semaphorin-3G transcription and angiogenic response in diabetes: insights for personalized epigenetic therapies

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S.A Mohammed ◽  
S Costantino ◽  
A Akhmedov ◽  
G Karsai ◽  
S Ambrosini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gastrocnemius Muscle ◽  
Tube Formation ◽  
Public Institution ◽  
Epigenetic Therapy ◽  
Great Promise ◽  
Funding Source ◽  
Rna Seq ◽  
Angiogenic Response ◽  
Pharmacological Inhibition

Abstract Background Despite advances in revascularization strategies, type 2 diabetic (T2D) patients with peripheral artery disease (PAD) continue to have a high risk of limb amputation. Modulation of blood vessel growth holds great promise for the treatment of PAD patients. Epigenetic modifications, namely histone post-translational modifications, have shown to regulate transcriptional programs implicated in the pathogenesis of cardiovascular disease. Aim To investigate the role of chromatin changes in regulating post-ischemic vascularization in experimental diabetes as well as in patients with T2D. Methods Experiments were performed in primary human aortic endothelial cells (HAECs), double-mutant leptin deficient mice (Lepdb/db) carrying a genetic deletion of the methyltransferase SETD7 (Setd7−/−Lepdb/db) as well as in gastrocnemius muscle samples from T2D patients with PAD and age-matched non-diabetic controls. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). Pharmacological blockade of SETD7 was performed by using the selective inhibitor (R)-PFI-2. Scratch and tube formation assays were performed to investigate the impact of SETD7 on angiogenic response. Results RNA-seq in high glucose-treated HAECs revealed a profound upregulation of the methyltransferase SETD7 (fold change 2.8, p<0.001), an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and Chromatin Immunoprecipitation (ChIP) assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Increased SEMA-3G transcript was associated with enhanced secretion from HAECs. Co-immunofluorescence experiments showed that SEMA-3G blunts the angiogenic response by competing with VEGF receptors VEGFR/Neuropillin2. Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. SETD7 and SEMA-3G were significantly upregulated in endothelial cells from Lepdb/db mice, whereas SEMA-3G transcription was blunted in Setd7−/−Lepdb/db animals. Consistently, endothelial sprouting was defective in aortas from Lepdb/db as compared to WT mice, whereas Setd7−/−Lepdb/db mice displayed a preserved angiogenic response. Of clinical relevance, SETD7/SEMA-3G axis was upregulated in gastrocnemius muscle specimens from T2D patients with PAD as compared with non-diabetic controls. Conclusion In HAECs, genetically modified mice and T2D patients we show that SETD7-dependent chromatin changes regulate SEMA-3G transcription and angiogenic response. Pharmacological inhibition of SETD7 may represent a novel epigenetic therapy to boost neovascularization in T2D patients with PAD. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Zurich/Universitätsspital Zürich

Role of angiotensin-signalling in anthracycline-induced cardiotoxicity

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Findlay ◽  
J.H Gill ◽  
R Plummer ◽  
C.J Plummer
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Late Onset ◽  
Left Ventricular Systolic Dysfunction ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Funding Source ◽  
Study Results

Abstract   Anthracycline chemotherapy remains a key component of cancer treatment regimens in both paediatric and adult patients. A significant issue with their use is the development of anthracycline-induced cardiotoxicity (AIC), with subclinical AIC and clinical heart failure observed in 13.8% and 3.1% of patients, respectively. The major clinical complication of AIC is the development of late-onset cardiotoxicity, occurring several years after drug administration, presenting as life-threatening heart failure (HF). Determining the relationship between subclinical AIC and late-onset HF, strategies for mitigation of AIC, and impacts upon the cancer survivor population remains a complex challenge. Administration of drugs targeting the angiotensin system, specifically angiotensin converting enzyme inhibitors (ACEi), have been reported to reduce AIC in the clinic. Whilst the therapeutic effect of ACEi in management of left ventricular systolic dysfunction and consequent HF is principally through optimisation of cardiac haemodynamics, the mechanism involved with mitigation of late-onset AIC several years after anthracycline exposure are currently unknown. Using a variety of human cardiomyocyte in vitro models we have previously demonstrated induction of cardiomyocyte hypertrophy by angiotensin II and anthracyclines. Importantly, selective blockade of the angiotensin II receptor 1 (ATR1) on cardiomyocytes mitigated the anthracycline-induced hypertrophic response, implicating synergism between AIC and angiotensin signalling in cardiomyocytes. Adult human ventricular cardiac myocyte AC10 cell-line were treated in vitro with a range of clinically relevant doxorubicin doses for clinically appropriate durations, with AT1 receptor gene expression evaluated using semi-quantitative PCR. Our results confirm a positive correlation between clinically-relevant concentration of doxorubicin and induction of genetic expression of ATR1 in AC10 cells, with up to 200% increases in ATR1 expression observed. Maximal doxorubicin-induced gene expression being observed at 8 and 24-hours, respectively. These preliminary results agreeing with clinical exposure parameters for this drug with protein expression studies being optimised to support these gene expression study results. Our preliminary studies also imply patients developing AIC carry a deleted polymorphism within intron 16 of the ACE gene and increased systemic levels of the ACE product angiotensin II, both with a known association to hypertrophic cardiomyopathy. Taken together, these data support our mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes, involving structural cellular changes and asymptomatic cardiac hypertrophy. An elevation in angiotensin II levels, potentially through polymorphisms in ACE, could thereby exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced HF. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK funded PhD

scholarly journals Genome-wide methylation and expression analyses reveal the epigenetic landscape of immune-related diseases for tobacco smoking

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Ying Mao ◽  
Peng Huang ◽  
Yan Wang ◽  
Maiqiu Wang ◽  
Ming D. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cigarette Smoke ◽  
Tobacco Smoking ◽  
Molecular Mechanisms ◽  
Functional Enrichment ◽  
Chronic Obstructive ◽  
Rna Seq ◽  
Immune System Diseases ◽  
Genome Wide ◽  
A Genome

Abstract Background Smoking is a major causal risk factor for lung cancer, chronic obstructive pulmonary disease (COPD), cardiovascular disease (CVD), and is the main preventable cause of deaths in the world. The components of cigarette smoke are involved in immune and inflammatory processes, which may increase the prevalence of cigarette smoke-related diseases. However, the underlying molecular mechanisms linking smoking and diseases have not been well explored. This study was aimed to depict a global map of DNA methylation and gene expression changes induced by tobacco smoking and to explore the molecular mechanisms between smoking and human diseases through whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). Results We performed WGBS on 72 samples (36 smokers and 36 nonsmokers) and RNA-seq on 75 samples (38 smokers and 37 nonsmokers), and cytokine immunoassay on plasma from 22 males (9 smokers and 13 nonsmokers) who were recruited from the city of Jincheng in China. By comparing the data of the two groups, we discovered a genome-wide methylation landscape of differentially methylated regions (DMRs) associated with smoking. Functional enrichment analyses revealed that both smoking-related hyper-DMR genes (DMGs) and hypo-DMGs were related to synapse-related pathways, whereas the hypo-DMGs were specifically related to cancer and addiction. The differentially expressed genes (DEGs) revealed by RNA-seq analysis were significantly enriched in the “immunosuppression” pathway. Correlation analysis of DMRs with their corresponding gene expression showed that genes affected by tobacco smoking were mostly related to immune system diseases. Finally, by comparing cytokine concentrations between smokers and nonsmokers, we found that vascular endothelial growth factor (VEGF) was significantly upregulated in smokers. Conclusions In sum, we found that smoking-induced DMRs have different distribution patterns in hypermethylated and hypomethylated areas between smokers and nonsmokers. We further identified and verified smoking-related DMGs and DEGs through multi-omics integration analysis of DNA methylome and transcriptome data. These findings provide us a comprehensive genomic map of the molecular changes induced by smoking which would enhance our understanding of the harms of smoking and its relationship with diseases.

scholarly journals Acute and chronic cholinergic activity in the inflammatory and lipid regulation of epicardial stromal cells from patients with and without atrial fibrillation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M Couselo Seijas ◽  
X Fu ◽  
J.N Lopez-Cano ◽  
A Rozados-Luis ◽  
A.L Fernandez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Atrial Fibrillation ◽  
Stromal Cells ◽  
Lipid Accumulation ◽  
Plasma Levels ◽  
Calcium Mobilization ◽  
Public Institution ◽  
Epicardial Fat ◽  
Funding Source ◽  
Cholinergic Activity

Abstract Aims Acetylcholine (ACh) released modulation by botulinum toxin injection into epicardial fat diminish atrial fibrillation (AF) recurrence. These results suggest an interaction between autonomic imbalance and epicardial fat as risk factors of AF. Our aim was to study the inflammatory and lipid profile of epicardial stroma from AF patients and their regulation by high cholinergic activity. Methods and results We performed in vitro assays with primary cultures from paired subcutaneous and epicardial stromal cells from 33 patients. We analysed ACh effect on gene expression, intracellular calcium mobilization and neutrophil migration. Plasma protein regulation by parasympathetic denervation was performed in vagotomised rats. Acute ACh treatment up-regulated MCP1 levels on epicardial stromal cells and suggested a neutrophil infiltration enhancement. Patients with AF had a greater FABP4 gene expression (1.54±0.01 vs 1.47±0.01, p=0.005). Its plasma levels were pronouncedly declined on vagotomised rats (2.02±0.21 ng/mL vs 0.65±0.23 ng/mL, p<0.001). Additionally, chronic ACh treatment improved lipid accumulation within epicardial stromal cells (60.50% [22.82–85.13] vs 13.85% [6.17–23.16], p<0.001). Conclusions Acute ACh activity up-regulates MCP1 and calcium mobilization on epicardial stromal cells. Longer ACh treatment enhanced lipid accumulation. In this line, epicardial stroma from patients with permanent AF contains higher FABP4 expression levels. Thus, modulate cholinergic activity might reduce FABP4 since vagus nerve denervation is associated with a sharply decrease in FABP4 plasma levels. FABP4 in human AF and vagotomised rats Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Carlos III Health Institute; Health Research Institute of Santiago de Compostela

scholarly journals GeneQC: A quality control tool for gene expression estimation based on RNA-sequencing reads mapping

2018 ◽  
Author(s):  
Adam McDermaid ◽  
Xin Chen ◽  
Yiran Zhang ◽  
Juan Xie ◽  
Cankun Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Model Fitting ◽  
Functional Enrichment ◽  
Supplementary Information ◽  
Rna Seq ◽  
Quality Control Tool ◽  
Differential Gene ◽  
Supplementary Material ◽  
The Impact

AbstractMotivationOne of the main benefits of using modern RNA-sequencing (RNA-Seq) technology is the more accurate gene expression estimations compared with previous generations of expression data, such as the microarray. However, numerous issues can result in the possibility that an RNA-Seq read can be mapped to multiple locations on the reference genome with the same alignment scores, which occurs in plant, animal, and metagenome samples. Such a read is so-called a multiple-mapping read (MMR). The impact of these MMRs is reflected in gene expression estimation and all downstream analyses, including differential gene expression, functional enrichment, etc. Current analysis pipelines lack the tools to effectively test the reliability of gene expression estimations, thus are incapable of ensuring the validity of all downstream analyses.ResultsOur investigation into 95 RNA-Seq datasets from seven species (totaling 1,951GB) indicates an average of roughly 22% of all reads are MMRs for plant and animal species. Here we present a tool called GeneQC (Gene expression Quality Control), which can accurately estimate the reliability of each gene’s expression level. The underlying algorithm is designed based on extracted genomic and transcriptomic features, which are then combined using elastic-net regularization and mixture model fitting to provide a clearer picture of mapping uncertainty for each gene. GeneQC allows researchers to determine reliable expression estimations and conduct further analysis on the gene expression that is of sufficient quality. This tool also enables researchers to investigate continued re-alignment methods to determine more accurate gene expression estimates for those with low reliability.AvailabilityGeneQC is freely available at http://bmbl.sdstate.edu/GeneQC/[email protected] informationSupplementary data are available at Bioinformatics online.

scholarly journals Making sense of RNA-Seq data: from low-level processing to functional analysis

2014 ◽  
Author(s):  
Oleg Moskvin ◽  
Sean McIlwain ◽  
Irene Ong
Keyword(s):  
Functional Analysis ◽  
Differential Expression ◽  
Biological Response ◽  
Ground Truth ◽  
Functional Enrichment ◽  
Rna Seq ◽  
Biological Factors ◽  
Processing Stage ◽  
Low Level ◽  
The Impact

Numerous methods of RNA-Seq data analysis have been developed, and there are more under active development. In this paper, our focus is on evaluating the impact of each processing stage; from pre-processing of sequencing reads to alignment/counting to count normalization to differential expression testing to downstream functional analysis, on the inferred functional pattern of biological response. We assess the impact of 6,912 combinations of technical and biological factors on the resulting signature of transcriptomic functional response. Given the absence of the ground truth, we use two complementary evaluation criteria: a) consistency of the functional patterns identified in two similar comparisons, namely effects of a naturally-toxic medium and a medium with artificially reconstituted toxicity, and b) consistency of results in RNA-Seq and microarray versions of the same study. Our results show that despite high variability at the low-level processing stage (read pre-processing, alignment and counting) and the differential expression calling stage, their impact on the inferred pattern of biological response was surprisingly low; they were instead overshadowed by the choice of the functional enrichment method. The latter have an impact comparable in magnitude to the impact of biological factors per se.

scholarly journals Characterization of kinase gene expression and splicing profile in prostate cancer with RNA-Seq data

2016 ◽  
Author(s):  
Huijuan Feng ◽  
Tingting Li ◽  
Xuegong Zhang
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Alternative Splicing ◽  
Differential Expression ◽  
Functional Enrichment ◽  
Cancer Development ◽  
Rna Seq ◽  
Differential Splicing ◽  
Kinase Gene ◽  
Isoform Switching

AbstractBackgroundAlternative splicing is a ubiquitous post-transcriptional process in most eukaryotic genes. Aberrant splicing isoforms and abnormal isoform ratios can contribute to cancer development. Kinase genes are key regulators of various cellular processes. Many kinases are found to be oncogenic and have been intensively investigated in the study of cancer and drugs. RNA-Seq provides a powerful technology for genome-wide study of alternative splicing in cancer besides the conventional gene expression profiling. But this potential has not been fully demonstrated yet.MethodsHere we characterized the transcriptome profile of prostate cancer using RNA-Seq data from viewpoints of both differential expression and differential splicing, with an emphasis on kinase genes and their splicing variations. We built up a pipeline to conduct differential expression and differential splicing analysis. Further functional enrichment analysis was performed to explore functional interpretation of the genes. With focus on kinase genes, we performed kinase domain analysis to identify the functionally important candidate kinase gene in prostate cancer. We further calculated the expression level of isoforms to explore the function of isoform switching of kinase genes in prostate cancer.ResultsWe identified distinct gene groups from differential expression and splicing analysis, which suggested that alternative splicing adds another level to gene expression regulation. Enriched GO terms of differentially expressed and spliced kinase genes were found to play different roles in regulation of cellular metabolism. Function analysis on differentially spliced kinase genes showed that differentially spliced exons of these genes are significantly enriched in protein kinase domains. Among them, we found that gene CDK5 has isoform switching between prostate cancer and benign tissues, which may affect cancer development by changing androgen receptor (AR) phosphorylation. The observation was validated in another RNA-Seq dataset of prostate cancer cell lines.ConclusionsOur work characterized the expression and splicing profile of kinase genes in prostate cancer and proposed a hypothetical model on isoform switching of CDK5 and AR phosphorylation in prostate cancer. These findings bring new understanding to the role of alternatively spliced kinases in prostate cancer and demonstrate the use of RNA-Seq data in studying alternative splicing in cancer.

scholarly journals Transcriptome analysis provides a blueprint of coral egg and sperm functions

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9739
Author(s):  
Julia Van Etten ◽  
Alexander Shumaker ◽  
Tali Mass ◽  
Hollie M. Putnam ◽  
Debashish Bhattacharya
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Coral Species ◽  
Functional Enrichment ◽  
Ion Binding ◽  
Rna Seq ◽  
Missing Information ◽  
Broadcast Spawning ◽  
Gamete Recognition ◽  
Sperm Functions

Background Reproductive biology and the evolutionary constraints acting on dispersal stages are poorly understood in many stony coral species. A key piece of missing information is egg and sperm gene expression. This is critical for broadcast spawning corals, such as our model, the Hawaiian species Montipora capitata, because eggs and sperm are exposed to environmental stressors during dispersal. Furthermore, parental effects such as transcriptome investment may provide a means for cross- or trans-generational plasticity and be apparent in egg and sperm transcriptome data. Methods Here, we analyzed M. capitata egg and sperm transcriptomic data to address three questions: (1) Which pathways and functions are actively transcribed in these gametes? (2) How does sperm and egg gene expression differ from adult tissues? (3) Does gene expression differ between these gametes? Results We show that egg and sperm display surprisingly similar levels of gene expression and overlapping functional enrichment patterns. These results may reflect similar environmental constraints faced by these motile gametes. We find significant differences in differential expression of egg vs. adult and sperm vs. adult RNA-seq data, in contrast to very few examples of differential expression when comparing egg vs. sperm transcriptomes. Lastly, using gene ontology and KEGG orthology data we show that both egg and sperm have markedly repressed transcription and translation machinery compared to the adult, suggesting a dependence on parental transcripts. We speculate that cell motility and calcium ion binding genes may be involved in gamete to gamete recognition in the water column and thus, fertilization.

Transcriptomic responses associated with kidney injury and repair in acute decompensated heart failure

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Pilbrow ◽  
M.T Rademaker ◽  
L.J Ellmers ◽  
S.C Palmer ◽  
T Davidson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
New Zealand ◽  
Renal Injury ◽  
Acute Decompensated Heart Failure ◽  
Kidney Injury ◽  
Decompensated Heart Failure ◽  
Public Institution ◽  
Funding Source ◽  
Injury And Repair

Abstract Background The discovery of new markers for acute kidney injury (AKI) in acute decompensated heart failure (ADHF) has been hampered by an incomplete understanding of the pathological processes underlying AKI in ADHF. Purpose In a sheep model of ADHF, we investigated changes in kidney gene expression in response to the development of, and recovery from, ADHF. Methods We collected serial kidney biopsies from 6 sheep prior to rapid cardiac pacing (day 0), after development of ADHF (pacing @220bpm for 14 days), and at the end of a 25-day (non-pacing) recovery period. Serial biopsies were supplemented with kidney samples collected post-mortem from animals undergoing a similar pacing/recovery protocol, giving a total of 11 “baseline” (B), 13 “heart failure” (HF) and 8 “recovery” (R) samples. We prepared RNA-Sequencing libraries using total RNA and Illumina TruSeq stranded mRNA library kits. Hormonal, haemodynamic, biochemical and urine measurements were also performed in all sheep before, during, and after development of ADHF. The study followed the principles of laboratory animal care and was approved by our institution's Animal Ethics Committee. Results We observed profound changes in hormonal, haemodynamic, biochemical and urine measures of cardio-renal injury in all sheep, confirming simulation of the peripheral consequences of ADHF, including clinically-relevant kidney dysfunction. This occurred in conjunction with altered kidney expression of 982 genes during ADHF development and 1,807 genes during ADHF recovery (p adj.<0.05, Fig 1). During ADHF development, changes in kidney gene expression were associated with activation of the pro-inflammatory p38 MAPK pathway and repression of several anti-inflammatory and reno-protective pathways, including eNOS signalling (all p adj.<0.001). In contrast, during ADHF recovery, changes in kidney gene expression were associated with reactivation of reno-protective pathways repressed during ADHF development, activation of anti-fibrotic pathways (including PTEN signalling) and repression of pathways that mediate inflammation and renal injury (including NF-kB signalling, all p adj.<0.001). Among 431 ADHF “responsive” genes (i.e. those that increased during ADHF development and decreased during ADHF recovery, or vice versa, Fig. 1), 37 genes encoded proteins detectable in plasma or urine and may represent markers of kidney repair in ADHF. Although most gene expression changes were transient, 192 genes remained altered after 4-weeks recovery (p adj.<0.05, Fig 1). Of these, 13 genes were predicted to encode proteins detectable in plasma or urine and may represent persistent markers of kidney injury in ADHF. Conclusion Our data provide the first insight into the gene pathways associated with kidney injury and repair in ADHF, in an established ovine model. Understanding the pathological processes underlying AKI in ADHF may enable discovery of novel markers for monitoring kidney injury and repair in ADHF. Figure 1. Genes altered in the kidney in ADHF Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Health Research Council of New Zealand, Heart Foundation of New Zealand

P–292 Transcriptome signature of receptive endometrium is not affected by the presence of mild adenomyosis

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Prasnikar ◽  
T Kunej ◽  
M Gorenjak ◽  
P Uroš ◽  
B Kovačič ◽  
...  
Keyword(s):  
Gene Expression ◽  
Significant Influence ◽  
Gene Expression Signature ◽  
Study Groups ◽  
Endometrial Receptivity ◽  
Functional Enrichment ◽  
Control Group ◽  
P Value ◽  
Rna Seq ◽  
Expression Signature

Abstract Study question Does the presence of mild adenomyosis, a common acquired uterine anomaly, affect the endometrial gene expression levels during window of receptivity? Summary answer Mild adenomyosis has no significant influence on gene expression signature in the window of implantation (WOI). What is known already The improvements in imaging techniques have led to frequent detection of adenomyosis in women undergoing investigations for infertility. Although the data are conflicting, some clinical studies have shown that the presence of adenomyosis may interfere with embryo implantation and lead to poor pregnancy outcomes. The knowledge of molecular background that would lead to the phenomenon of altered endometrial receptivity in women with adenomyosis is limited and mainly demonstrated by selected candidate genes. Next-generation sequencing platforms enable genome-wide transcriptomic profiling of desired tissue samples and present a powerful tool to identify differentially expressed genes (DEGs) between women with adenomyosis and controls. Study design, size, duration We designed a prospective case-control study comparing women with sonographic evidence of mild adenomyosis (n = 10) and women with normal uteri seeking assisted reproduction due to male factor infertility as the control group (n = 10). All eligible women underwent infertility treatment at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between years 2018 and 2020. For the present study, they were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests. Participants/materials, setting, methods Each endometrial biopsy was obtained in the presumed window of implantation (WOI) on days LH + 7 to LH + 9 after LH surge (LH + 0). Isolated total RNA was applied for mRNA + lncRNA sequencing (RNA-seq) by Illumina Novaseq 6000. An aliquot of RNA samples was used to verify the WOI by the endometrial receptivity test “beREADY” (CCHT, Estonia). Gene Ontology and Reactome pathway enrichment analyses were conducted in ClueGO bioinformatics tool to study biological role behind obtained DEGs. Main results and the role of chance The R program language and Bioconductor packages were used to align generated RNA-seq reads on the human reference genome assembly (hg19) and to calculate gene expression differences between study groups using normalized counts per million (CPM)>10 in at least 10 samples. A total 233 DEGs (p < 0.05) was identified of which 126 genes were up- and 107 were down-regulated in adenomyosis compared to the control group. However, there was no significantly DEG according to the adjusted p-value. According to the beREADY test, all 20 samples were in receptive phase, however two samples were early-receptive and five were late-receptive. In a sensitivity analysis, all border receptive samples were removed and RNA-seq data sets were re-analysed only by 8 adenomyosis cases and 5 controls. A total of 382 DEGs (p < 0.05) were detected in adenomyosis group (216 up- and 166 down-regulated genes), again with no statistical difference between both groups after adjustment. Functional enrichment analyses of 233 and 382 DEGs identified pathways (adjusted p-value< 0.05) associated with positive regulation of exosomal secretion and expression of IFN-induced genes, respectively. The comparison of 233 and 382 DEGs revealed 28 common genes that may present stronger candidate of adenomyosis-related markers associated with endometrial receptivity. Limitations, reasons for caution Only mild adenomyosis was considered in this study, which is most commonly detected in women. The results could differ in women in severe cases of adenomyosis. Multicellular whole-tissue endometrial samples that were used for RNA isolation could mask gene expression differences of specific cell types between study groups. Wider implications of the findings: According to our results of transcriptome analysis, the presence of mild adenomyosis has no significant influence on the gene expression signature during endometrial receptivity in natural menstrual cycle. Women being investigated for infertility can be reassured that this common acquired anomaly does not significantly influence the chances of successful conception. Trial registration number 0120–259/2018/16

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