Affymetrix Platform Research Articles

GEDI: An R Package for Integration of Transcriptomic Data from Multiple Platforms for Bioinformatics Applications.

Transcriptomic data is often expensive and difficult to generate in large cohorts relative to genomic data; therefore, it is often important to integrate multiple transcriptomic datasets from both microarray- and next generation sequencing (NGS)-based transcriptomic data across similar experiments or clinical trials to improve analytical power and discovery of novel transcripts and genes. However, transcriptomic data integration presents a few challenges including reannotation and batch effect removal. We developed the Gene Expression Data Integration (GEDI) R package to enable transcriptomic data integration by combining existing R packages. With just four functions, the GEDI R package makes constructing a transcriptomic data integration pipeline straightforward. Together, the functions overcome the complications in transcriptomic data integration by automatically reannotating the data and removing the batch effect. The removal of the batch effect is verified with principal component analysis and the data integration is verified using a logistic regression model with forward stepwise feature selection. To demonstrate the functionalities of the GEDI package, we integrated five bovine endometrial transcriptomic datasets from the NCBI Gene Expression Omnibus. These transcriptomic datasets were from multiple high-throughput platforms, namely, array-based Affymetrix and Agilent platforms, and NGS-based Illumina paired-end RNA-seq platform. Furthermore, we compared the GEDI package to existing tools and found that GEDI is the only tool that provides a full transcriptomic data integration pipeline including verification of both batch effect removal and data integration for downstream genomic and bioinformatics applications. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ReadGE, a function to import gene expression datasets Basic Protocol 2: GEDI, a function to reannotate and merge gene expression datasets Basic Protocol 3: BatchCorrection, a function to remove batch effects from gene expression data Basic Protocol 4: VerifyGEDI, a function to confirm successful integration of gene expression data.

Lnc-216 regulates the miR-143-5p /MMP2 signaling axis aggravates retinal endothelial cell dysfunction

PURPOSE: Diabetic retinopathy (DR) is a serious retinal vascular disease that affects many individuals in their prime working years. The present research aimed at whether and how LOC681216 (LNC-216) is involved in retinal vascular dysfunction under diabetic conditions. METHODS: Rat retinal microvascular endothelial cells (RRMECs) treated with high glucose (HG) were used for functional analysis. Gene expression analysis was conducted using the Clariom D Affymetrix platform. The wound healing, transwell, and vascular tube formation assays were used to identify the migration, invasion, and tube formation capability of RRMECs. The dual-luciferase reporter confirmed the binding interaction between miR-143-5p and LNC-216 or matrix metallopeptidase 2 (MMP2). RESULTS: Lnc-216 was upregulated in RRMECs treated with HG. Lnc-216 knockdown markedly suppressed the tube formation, cell migration, and wound healing of cultured RRMECs under HG conditions. Mechanistically, Lnc-216 acted as a miR-143-5p sponge to affect the biological activity of miR-143-5p, which led to increased expression of matrix metallopeptidase 2 (MMP2). CONCLUSIONS: Lnc-216 attenuates diabetic retinal vascular dysfunction through the miR-143-5p/MMP2 axis, providing a potential therapeutic strategy for DR.

Association of tumor-infiltrating lymphocytes (TILs) and immune signatures with PAM50 intrinsic subtyping hormone receptor-positive (HR+)/HER2-negative (HER2-) early breast cancer (BC): A translational analysis of two multicentric neoadjuvant trials.

558 Background: HR+/HER2− BCs are generally described as immunologically cold, with low levels of TILs and PD-L1. Nevertheless, the addition of immunotherapy (ICI) to chemo has been reported to increase pCR rates in high-risk early HR+/HER2– BCs. Assessing the interaction between immune features and tumor biology may identify of a subset of patients (pts) with HR+/HER2− BC that would be the ideal candidates for the combination of chemo and ICI. Methods: Gene-expression data from two multicentric neoadjuvant phase II trials (GIADA, LETLOB) enrolling stage II-IIIA HR+HER2- BC pts was retrieved (Dieci, CCR 2022; Guarneri, JCO 2014). In the GIADA trial, 758 genes were assessed on baseline tumor samples by nCounter (N=43). In the LETLOB trial, gene-expression data (Affymetrix platform) from baseline core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 predictor. TILs were evaluated following guidelines. Association of TILs and immune signatures with intrinsic subtyping was assessed by Kruskal-Wallis test. Results: PAM50 subtype distribution (N=109) was 44% Luminal B (LumB), 33% Luminal A (LumA), 18% Basal-like, 5% HER2-enriched (HER2-E). TILs were assessed on 101 samples: median 2 (range 0-100). Basal-like BCs showed higher TILs than other subtypes (p=0.008). No significant difference was observed between LumA and LumB BCs (Table). Basal-like tumors showed higher levels of previously published immune signatures associated with CD8 T-cells (p<0.001), Cytotoxic cells (p<0.001), IFN-gamma (p<0.001), and response to ICI in the GeparNuevo trial (p=0.003) (N=109). PD-1 (p=0.002), PD-L1 (p=0.001), and PD-L2 (p=0.001) genes were also more expressed in Basal-like BCs (evaluable in 43 samples). No significant difference was observed between LumA and LumB tumors. A significant weak to moderate positive correlation was observed between continuous TILs and immune signatures and basal-like PAM50 signature (p<0.001, Spearman coefficient=0.372 for TILs, from 0.354 to 0.648 for immune signatures). Conclusions: PAM50 intrinsic subtyping and TILs/immune signatures capture only partially overlapping information on the biology of HR+/HER2- BCs. Pts with HR+/HER2- BCs simultaneously showing features of biological aggressiveness and enhanced immunogenicity may represent the ideal candidates for the combination of ICI and chemo. [Table: see text]

Discovering Promising Biomarkers and Therapeutic Targets for Duchenne Muscular Dystrophy: a Multiomics Meta-Analysis Approach.

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes muscle weakness and degeneration. In this study, we identified potential biomarkers and drug targets for DMD through a comprehensive meta-analysis of mRNA profiles. We conducted an in-depth analysis of three microarray datasets from the GEO database, utilizing the Affymetrix platform. A rigorous data pre-processing pipeline encompassed background correction, normalization, log2 transformation and probe-to-gene symbol mapping. Robust multi-array average method followed by Limma package in R was employed to ensure differential expression analysis within individual datasets, yielding gene-specific p-values. We identified 63 genes exhibiting statistically significant differential expression across the three datasets (p < 0.05) and an absolute log fold change > 1.5. Functional enrichment analyses of these differentially expressed genes were done, followed by pathway analyses. Our results suggested pertinent biological processes, molecular functions and cellular components associated with DMD. Finally, eight hub genes-COL6A3, COL1A1, COL3A1, COL1A2, POSTN, TIMP1, THBS2 and SPP1-were pinpointed as central players in the network. Two differentially expressed genes with substantial absolute log-fold changes, namely, DMD, downregulated and MYH3, upregulated, were identified as potential therapeutic candidates. In light of these findings, our work contributes not only to understanding DMD at the molecular level but also presents potential targets for therapeutic strategies. Finally,our study facilitates the development of therapeuticinterventions that can effectively control and mitigate the impact of DMD.

Comparative Analysis of Transcriptome Profiles in Patients with Thromboangiitis Obliterans.

Thromboangiitis obliterans (TAO) causes vascular insufficiency due to chronic inflammation and abrupt thrombosis of the medium and small arteries of the extremities. In our study, we aimed to determine biomarkers for the diagnosis of TAO by evaluating 15 male TAO patients with Shinoya diagnostic criteria and 5 healthy controls who did not have TAO-related symptoms in their family histories. The Clariom D Affymetrix platform was used to conduct microarray analysis on total RNA extracted from whole blood. A total of 477 genes (FC ≤ 5 or >5) common to the fifteen patient and five control samples were selected using comparative microarray analysis; among them, 79 genes were upregulated and 398 genes were downregulated. According to FC ≤ 10 or >10, in the same TAO patient and control group, 13 genes out of 28 were upregulated, whereas 15 genes were downregulated. The 11 key genes identified according to their mean log2FC values were PLP2, RPL27A, CCL4, FMNL1, EGR1, EIF4A1, RPL9, LAMP2, RNF149, EIF4G2, and DGKZ. The genes were ranked according to their relative expression as follows: FMNL1 > RNF149 > RPL27A > EIF4G2 > EIF4A1 > LAMP2 > EGR1 > PLP2 > DGKZ > RPL9 > CCL4. Using protein-protein interaction network analysis, RPL9, RPL27A, and RPL32 were found to be closely related to EIF4G2 and EIF4A1. The Reactome pathway found pathways linked to 28 genes. These pathways included the immune system, cellular responses to stress, cytokine signaling in the immune system, and signaling by ROBO receptors. By figuring out the protein expression levels of the genes that have been found to explain how TAO disease works at the molecular level, it will be possible to figure out how well these chosen transcripts can diagnose and predict the disease.

Nondisjunction in Trisomy 21: Origin and Mechanisms

Down’s Syndrome is a chromosomal abnormality with a gain of a third copy of chromosome 21, characterized by craniofacial abnormalities, intellectual developmental delay and growth retardation. In the present study, one-day-old neonate and a patient with an aborted fetus were presented to rule out genetic aberrations via CMA screening. DNA was extracted using from the case samples and evaluated by CMA Affymetrix platform. Chromosomal abnormalities in the fetus such as trisomy emerges due to several factors including maternal age, genetic mutational events, non-disjunction of chromosomes and epigenetic changes. Genetic Cause of trisomy 21 is chromosomal aneuploidy and gain of three copies of chromosome 21. The Trisomy 21 can occur due to Robertsonian translocation, Mosaicisms or duplication of critical region of chromosome 21. The trisomy 21 is the result of nondisjunction of homologous chromosomes 21 during gametogenesis at the time of embryo development. CMA is an effective molecular diagnostic tool for predicting and diagnosing chromosomal abnormalities which needs to be promoted in India for healthy pregnancy outcomes. We report two cases of confirmed post-natal Trisomy 21. The main aim of this study is to focus on genetic diagnosis and its awareness which is still lacking with 100% coverage, in developing countries to reduce the burden of genetic diseases and associated emotional and economic consequences.

Abstract P4-02-13: Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials

Abstract Background: Hormone-receptor (HR)+/HER2- breast cancer (BC) is a biologically heterogeneous disease. Homologous recombination deficiency (HRD) and BRCA mutations have been previously reported to be associated with worse outcomes in HR+/HER2- metastatic BC patients receiving CDK4/6 inhibitors and endocrine therapy. Here, we assess the relation between HRD and RB-loss signatures, intrinsic subtyping, the PAM50-based chemo-endocrine score, and response to chemotherapy-based therapy and endocrine treatment in HR+/HER2- early BC. Methods: GIADA is a multicentric neoadjuvant phase II trial that treated premenopausal patients with Luminal B (LumB)-like BC (HR-positive, HER2-negative, with Ki67&amp;gt;20% and/or histologic Grade 3) with a combination of chemotherapy, immunotherapy and endocrine treatment. Expression of 758 genes on baseline tumor samples from all 43 patients was quantified by nCounter platform. The LETLOB phase II trial randomized postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available from 66 out of 92 pts enrolled. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. A published HRD signature (Peng, Nat Commun 2014) and a signature of RB loss (RBsig), previously reported to potentially predict resistance to CDK4/6 inhibitors in HR+/HER2- BC (Malorni, Oncotarget 2016) were computed. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate chemosensitivity. Association between genomic signatures was assessed through Pearson’s correlation coefficient. Association of genomic signatures with pCR was assessed through logistic regression and association with PEPI scores was assessed through Kruskal-Wallis test. Results: HRD signature levels were significantly higher in non-luminal (Basal-like and HER2-enriched) tumors as compared to Luminal (A or B) tumors (p&amp;gt;0.001 in the GIADA trial, p=0.021 in the LETLOB trial). Moreover, higher levels of HRD signature were associated with higher levels of RB-loss signature (Pearson correlation 0.355, p=0.020 in the GIADA trial; Pearson correlation 0.942, p&amp;lt; 0.001 in the LETLOB trial), higher levels of Basal-like signature (Pearson correlation 0.502, p&amp;lt; 0.001 in the GIADA trial; Pearson correlation 0.373, p=0.002 in the LETLOB trial) and lower levels of CES (Pearson correlation -0.422, p=0.005 in the GIADA trial; Pearson correlation -0.763, p&amp;lt; 0.001 in the LETLOB trial), indicative of higher chemosensitivity. In the GIADA trial, higher levels of HRD signature (p=0.018) and RBloss signature (p=0.073) and lower levels of CES (p=0.007) were associated with higher pCR rates after chemo, endocrine and immunotherapy. In the LETLOB trial, lower levels of HRD signature (p=0.068) and RBloss signature (p=0.042) and higher levels of CES (p=0.050) were associated with higher sensitivity to endocrine treatment (lower PEPI scores, 0 vs 1-3 vs 4 or more, after neoadjuvant letrozole). Conclusions: In HR+/HER2- early BC, HRD gene signatures, RB-loss gene signatures and non-luminal (especially Basal-like) intrinsic subtyping are associated with each other and associated with higher sensitivity to chemotherapy-based therapy and lower sensitivity to endocrine treatment. These observations might help correctly tailor systemic therapy, including biologic agents, in patients with HR+/HER2- early and advanced BC. Citation Format: Gaia Griguolo, Federica Miglietta, Laia Paré, Daniele G. Generali, Antonio Frassoldati, Antonino Musolino, Simon Spazzapan, Grazia Vernaci, Tommaso Giarratano, Marcello Lo Mele, Giancarlo Bisagni, Federico Piacentini, Enrico Tagliafico, Katia Cagossi, Francesca Schiavi, Claudia Pinato, Aleix Prat, Valentina Guarneri, Maria Vittoria Dieci. Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-13.

Genome-wide association study (GWAS) of circulating vitamin D outcomes among individuals of African ancestry

BackgroundVitamin D deficiency is more common among African-ancestry individuals and may be associated with adverse health outcomes. Vitamin D binding protein (VDBP) regulates concentrations of biologically active vitamin D. ObjectiveWe conducted genome-wide association study (GWAS) of VDBP and 25-hydroxyvitamin D among African-ancestry individuals. MethodsData were collected from 2,602 African American adults from the Southern Community Cohort Study (SCCS) and 6,934 African- or Caribbean-ancestry adults from the UK Biobank. Serum VDBP concentrations were available only in the SCCS and were measured by using the Polyclonal Human VDBP ELISA kit. Serum 25-hydroxyvitamin D concentrations for both study samples were measured by using Diasorin Liason, a chemiluminescent immunoassay. Participants were genotyped for single nucleotide polymorphisms (SNPs) with genome-wide coverage by using Illumina or Affymetrix platforms. Fine-mapping analysis was performed by using forward stepwise linear regression models including all variants with P value < 5 × 10−8 and within 250 kbps of a lead SNP. ResultsWe identified 4 loci notably associated with VDBP concentrations in the SCCS population: rs7041 (per allele β = 0.61 μg/mL, SE = 0.05, P = 1.4 × 10−48) and rs842998 (per allele β = 0.39 μg/mL, SE = 0.03, P = 4.0 × 10−31) in GC, rs8427873 (per allele β = 0.31 μg/mL, SE = 0.04, P = 3.0 × 10−14) near GC and rs11731496 (per allele β = 0.21 μg/mL, SE = 0.03, P = 3.6 × 10−11) in between GC and NPFFR2. In conditional analyses, which included the above-mentioned SNPs, only rs7041 remained notable (P = 4.1 × 10−21). SNP rs4588 in GC was the only GWAS-identified SNP associated with 25-hydroxyvitamin D concentration. Among UK Biobank participants: per allele β = −0.11 μg/mL, SE = 0.01, P = 1.5 × 10−13; in the SCCS: per allele β = −0.12 μg/mL, SE = 0.06, P = 2.8 × 10−02). rs7041 and rs4588 are functional SNPs that influence the binding affinity of VDBP to 25-hydroxyvitamin D. ConclusionsOur results were in line with previous studies conducted in European-ancestry populations, showing that GC, the gene that directly encodes for VDBP, would be important for VDBP and 25-hydroxyvitamin D concentrations. The current study extends our knowledge of the genetics of vitamin D in diverse populations.

MAPEAMENTO ASSOCIATIVO PARA PRODUTIVIDADE DE GRÃOS EM GERMPLASMA EXÓTICO DE SOJA SOB ALTAS TEMPERATURAS

ABSTRACT Soybeans are among the world’s main crops because they are excellent sources of proteins, micronutrients, and oil. Considering that abiotic stress affects agribusiness, resulting in losses, the grain yield of the crop must be maintained even at high temperatures. In this context, the objective of this study was to select markers related to soybean yield assessed under high temperatures, using associative mapping. The mapping population included 80 soybean PIs and 15 controls. For phenotyping, genotypes were evaluated at high temperatures in an experiment conducted in Teresina (in the state of Piauí) and four characters of interest for agronomy were evaluated: height of the plant when mature, agronomic value, 100-seed weight, and grain yield. Genotyping was carried out using the Affymetrix Platform (180 K Axiom® Soybean Genotyping Array), and the imbalance in the connection between pairs of markers was calculated through the coefficient of determination using the fast permutation test. The analysis of the association between markers and the phenotype of interest was carried out using a generalized linear model approach, including phenotyping data, SNP markers, and information on population structure. The results revealed that 34.06% of loci showed a significant linkage disequilibrium (p &lt; 0.001), and 16 significant associations were found for the four characters related to heat tolerance. These associations can aid breeders that aim to incorporate high temperature tolerance in programs of soybean genetic improvement via selection assisted by markers.

Abstract 4018: Long-term response to Trastuzumab in patients with advanced gastric or gastroesophageal adenocarcinoma - A retrospective study

Abstract Since 2010, Trastuzumab combined with chemotherapy is the standard treatment for patients with HER2 (ERBB2) positive advanced or metastatic gastric and gastroesophageal junction cancer (GGEJ). While few patients show long-term response to the treatment, most suffer from rapid disease progression. Several studies revealed that genomic alterations, gene expression changes and altered HER2 signaling activity contribute to impaired therapy response. However, the underlying mechanism remains unclear. The aim of this research project is to analyze differences between patients with long and short progression-free survival by utilizing combined genetic, histologic, clinical and gene expression data. We collected a retrospective German patient cohort (n=20) including patients with HER2 positive advanced GGEJ who received Trastuzumab combined with chemotherapy. Using archival samples that were obtained prior to Trastuzumab treatment, we created a dataset including clinical information, histologic assessment, immunostaining, target amplicon sequencing (TAS; 409 gene panel) and human transcriptome data using the Affymetrix platform. To analyze the TAS sequencing data, we generated an automated analysis pipeline, which detects single nucleotide variants (SNV) and copy number alterations (CNA). Information about genomic alteration will be correlated with pathway and gene set enrichment analysis (GSEA) from the Affymetrix data. Furthermore, the data will be supported by investigating the functional status of HER2 in patient samples via electron microscopy. The results of this study could give insights on how genetic and transcriptomic alterations are connected to long-term Trastuzumab therapy response in HER2 positive GGEJ patients. This could potentially support therapy decision in personalized medicine. Citation Format: Isabel Porth, Philip Weidner, Sylvie Lorenzen, Wilko Weichert, Thorsten Oliver Götze, Sven Perner, Kim Luley, Daniela Hirsch, Ralf-Dieter Hofheinz, Diana B. Peckys, Zahra Mostajeran, Niels de Jonge, Timo Gaiser. Long-term response to Trastuzumab in patients with advanced gastric or gastroesophageal adenocarcinoma - A retrospective study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4018.

Exploring the Molecular Mechanism of lncRNA-miRNA-mRNA Networks in Non-Syndromic Cleft Lip with or without Cleft Palate.

BackgroundNon-syndromic cleft lip with or without cleft palate (NSCL/P) is a common craniofacial birth defect. Growing evidence has demonstrated the competing endogenous RNA (ceRNA) hypothesis has played a role in the pathogenesis of NSCL/P. Here, we identified the important lncRNAs in NSCL/P and constructed a ceRNA regulatory network to predict their underlying functional mechanism.MethodsTotal RNA isolated from the peripheral blood samples were analyzed by the Human Clariom D Affymetrix platform and differentially expressed genes (DEGs) were identified. Using the limma package in R software, DEGs in the expression profile of GSE42589 were identified from Gene Expression Omnibus (GEO) database. Co-differentially expressed lncRNAs (co-DElncRNAs) were used to predict the microRNAs that may bind to them. Co-differentially expressed mRNAs (co-DEmRNAs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The hub genes were screened using the cytohubba plug-in in Cytoscape. A ceRNA network was built to investigate the molecular mechanism underlying the etiology of NSCL/P. The expression levels of lncRNAs, miRNAs, and mRNAs in the network were assessed by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsWe found 116 DElncRNAs and 2955 DEmRNAs from the GSE42589 dataset, and 2626 DElncRNAs and 2771 DEmRNAs from the Human Clariom D gene chip. A network of co-DEmRNAs containing 3712 edges and 621 nodes were identified by PPI analysis. A ceRNA regulatory network comprising lncRNA USP17L6P, hsa-miR-449c-5p, and MYC was established. qRT-PCR results revealed significantly lower expression levels of lncRNA USP17L6P and c-Myc in NSCL/P tissues, while the expression level of hsa-miR-449c-5p was higher as compared to control samples (p < 0.05).ConclusionThe identified lncRNAs and the established ceRNA regulatory network provide novel insight into the pathogenesis of NSCL/P, therefore hold great promise in NSCL/P management in clinical practice.

MicroRNAs as disease specific diagnostic biomarkers for neoplastic aetiology-related and inflammatory-related pericardial fluid effusion

Abstract Background and aim Malignant involvement of the pericardium is seen in 1 to 20 percent of autopsies in patients with cancer. The most common metastatic tumor involving the pericardium is lung cancer [1]. We aimed to distinguish the origin of the pericardial fluid effusion (i.e. pericarditis vs cancer) based on miRNAs expression in peripheral blood plasma. Method 8 patients hospitalized for collection of pericardial fluid with pericardial effusion of neoplastic aetiology (lung cancer). Control group includes 8 patients with effusion of inflammatory aetiology. Plasma RNA was extracted by mirVANAPARISKit and quality of RNA was assessed by fluorometric assay. GEP analysis was performed using the Clariom D pico chips, analysed on the Affymetrix platform. Statistical analysis by TAC software. Additional analyses were performed in and R using Signal information obtained from the TAC output. We performed the following tests using log2 transformed data and all comparison groups (A-F). Additional FDR correction, logistic regression, Mann-whitney t-test was used depending of the variables. We calculated Area under the curve using ROCp R package. Scores were ranging from 0 to 1. Co-expression analysis to identify genes authentically expressed was performed using Spearman correlation (cutoff = 0.9, Rpval = 0.05). In order to identify the targets of DE miRNAs we used our wizbionet R package and previously developed pipelines [2,3]. We performed target screening using multimiR package, selecting top 20% predictions from all available databases. Results We analyzed targets for all mature versions, and if DE miR was identified as pre-miR we generated -3p and -5p version for it. We also screened DisgeNet database for genes associated with cancer and pericarditis, we identified 2823 and 157 such genes. After identification of the targets of DE miRNAs we performed data aggregation, summarization and obtained information how many targets overall and targets associated with IS are regulated by each DE miRNA. Additionally we identified top targets regulated by the top miRNAs. MiR-5695, miR-4446-5p, miR-572, miR-3131 and miR-4784 were found the most significantly differentially expressed miRNAs in blood plasma for patients with malignancy compared to pericarditis. MiR-22-3p, miR-642a, miR-6771, miR-140-3p, and miR660-5p were found the most significantly differentially expressed miRNAs in pericardial fluid plasma for patients with malignancy compared to pericarditis. Importantly, miR-500b, miR-5188, miR-490, miR-24-3p, miR-383-3p were found the most promising differentially diagnostic biomarkers for malignancy and inflammatory-related pericardial fluid effusion. Conclusions For the first time our results indicate the differentially diagnostic power of miRNAs based on comparison of circulating in peripheral blood and pericardial fluid in patients having excessive pericardial fluid effusion due to different etiologies. Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

MicroRNA-223 might be a predictive biomarker for major adverse cardiovascular events prognosis in patients undergoing transcatheter aortic valve implantation procedure

Abstract Background Transcatheter aortic valve implantation (TAVI) is an emerging invasive therapeutic strategy for patients with severe aortic stenosis (AS) significantly enhancing not only quality-of-life measures but also improving short-and long-term survival rates. Purpose For these purposes we aimed to analyze correlation between expression levels of platelet-derived microRNAs in patients with heart failure (HF) due to AS who underwent TAVI procedure and assess their association with primary major adverse cardiac events (MACE) defined by all cause mortality and secondary MACE defined as cardiovascular mortality, ischemic stroke, and non-fatal myocardial infarction. Methods 61 patients before and after the TAVI procedure were included. PlasmaRNA was extracted by mirVANA PARIS Kit and quality of RNA was assessed by fluorometric assay. GEP analysis was performed using the Clariom D pico chips, analyzed on the Affymetrix platform. RT-PCR was performed in order to validate the miRNAs in 61 patients by using the Taqman advanced protocol. MiRNA related to platelet function/antiplatelet treatment were chosen among those with the most relevant modulation between the groups. Wilcoxon test was performed for miRNAs comparison before and after TAVI. Calculations were performed using SPSS version 22.0, p&amp;lt;0.05 (IBM Corporation, Chicago, USA). Results We have found that miR-223, miR-125b and miR-125a were significantly increased in patients after the TAVI procedure. ROC analysis showed that increased miR-223 expression after the TAVI procedure might slightly have protective value against MACE outcome. Conclusions Our analysis showed alteration of circulating miRNAs after the TAVI procedure and miR-223 might have a predictive value for MACE prognosis in patients with HF due to AS who underwent TAVI procedure. Acknowledgment I-COMET research team Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

Fingerprint of novel circulating microRNAs identify patients with stroke-embolic stroke of undetermined source

Abstract Background Stroke is the second-most common cause of death worldwide. Circulating levels of selected microRNAs (miRNAs) were found to be modulated both in animal experimental models and in patients with stroke, opening up new avenues for the identification of more effective and specific biomarkers to identify and risk-stratify stroke patients. Aim of the present study is to identify all circulating miRNAs that are modulated in patients with stroke, to select specific miRNAs to be used as disease biomarkers to improve prognosis. Methods 48 patients with stroke- ESUS were involved in the study. We have divided the patient groups based on patients who had a second stroke or TIA and did not have (safety vs safety control). Total RNA was extracted from plasma samples quality of extracted material was assessed using a fluorometric electrophoretic assay. MiRNA profiling was performed using the Affymetrix platform using. Statistical analysis was performed in TAC software. Additional analyses were performed in and R using Signal information obtained from the TAC output. We performed the following tests using log2 transformed data and all comparison groups (A-F). We performed additional FDR correction, logistic regression, Mann-whitney test t-test depending if variances were equal or differing. We calculated Area under the curve using ROCp R package. Scores were ranging from 0–1. Co-expression analysis to identify genes authentically expressed was performed using Spearman correlation (cutoff=0.9, Rpval=0.05). In order to identify the targets of DE miRNAs we used our wizbionet R package and previously developed pipelines [1,2]. We performed target screening using multimiR package, selecting top 20% predictions from all available databases. Results MiR-4786, miR-1205, miR-548ar-3p and miR-518e-3p were found the most differentially expressed miRNAs between the groups. So far, miR-4786 was studied only in patients with acute leukemia [3]. Several studies showed the importance of miR-1205 in cell carcinoma and ovarian cancer progression [4]. Moreover, so far only one study showed the regulation of miR-548ar-3p in breast cancer [5]. Finally only one study showed the alteration of miR-518e-3p in Parkinsons disease patients [6]. Besides, our enrichment analysis showed Interleukin-2 signaling pathway, Lipid and lipoprotein metabolism, BDNF signaling pathway, MAPK signaling pathway, Intellectual Disability, Alzheimer's Disease are significantly related to ESUS- patients. Conclusions Any of those miRNAs were never studied in stroke before, our results identified several novel circulating prognostic biomarkers miRNAs that are down- of up-regulated in ESUS-stroke patients (who had only one vs multiple stroke). Among those several miRNAs were identified that are known to play a role in the pathophysiology of neurovascular diseases, paving the way to a new class of smart pathophysiology-based biomarkers in stroke. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Polish National Science Center OPUS Figure 1Figure 2

Arabidopsis Coexpression Tool: a tool for gene coexpression analysis in Arabidopsis thaliana

SummaryGene coexpression analysis refers to the discovery of sets of genes which exhibit similar expression patterns across multiple transcriptomic data sets, such as microarray experiment data of public repositories. Arabidopsis Coexpression Tool (ACT), a gene coexpression analysis web tool for Arabidopsis thaliana, identifies genes which are correlated to a driver gene. Primary microarray data from ATH1 Affymetrix platform were processed with Single-Channel Array Normalization algorithm and combined to produce a coexpression tree which contains ∼21,000 A. thaliana genes. ACT was developed to present subclades of coexpressed genes, as well as to perform gene set enrichment analysis, being unique in revealing enriched transcription factors targeting coexpressed genes. ACT offers a simple and user-friendly interface producing working hypotheses which can be experimentally verified for the discovery of gene partnership, pathway membership, and transcriptional regulation. ACT analyses have been successful in identifying not only genes with coordinated ubiquitous expressions but also genes with tissue-specific expressions.

Identification of Differentially Expressed Genes in Cervical Cancer Patients by Comparative Transcriptome Analysis

Cervical cancer is one of the most malignant reproductive diseases seen in women worldwide. The identification of dysregulated genes in clinical samples of cervical cancer may pave the way for development of better prognostic markers and therapeutic targets. To identify the dysregulated genes (DEGs), we have retrospectively collected 10 biopsies, seven from cervical cancer patients and three from normal subjects who underwent a hysterectomy. Total RNA isolated from biopsies was subjected to microarray analysis using the human Clariom D Affymetrix platform. Based on the results of principal component analysis (PCA), only eight samples are qualified for further studies; GO and KEGG were used to identify the key genes and were compared with TCGA and GEO datasets. Identified genes were further validated by quantitative real-time PCR and receiver operating characteristic (ROC) curves, and the highest Youden index was calculated in order to evaluate cutoff points (COPs) that allowed distinguishing of tissue samples of cervical squamous carcinoma patients from those of healthy individuals. By comparative microarray analysis, a total of 108 genes common across the six patients' samples were chosen; among these, 78 genes were upregulated and 26 genes were downregulated. The key genes identified were SPP1, LYN, ARRB2, COL6A3, FOXM1, CCL21, TTK, and MELK. Based on their relative expression, the genes were ordered as follows: TTK > ARRB2 > SPP1 > FOXM1 > LYN > MELK > CCL21 > COL6A3; this generated data is in sync with the TCGA datasets, except for ARRB2. Protein-protein interaction network analysis revealed that TTK and MELK are closely associated with SMC4, AURKA, PLK4, and KIF18A. The candidate genes SPP1, FOXM1, LYN, COL6A3, CCL21, TTK and MELK at mRNA level, emerge as promising candidate markers for cervical cancer prognosis and also emerge as potential therapeutic drug targets.

Abstract PS5-14: Gene-expression profiling and response to neoadjuvant endocrine treatment in the phase II LETLOB trial

Abstract Background: Hormone receptor positive (HR+)/HER2- breast cancer (BC) is a clinically and biologically heterogeneous disease, encompassing all BC molecular intrinsic subtypes. We here explore the association between gene-expression profiles and response to neoadjuvant endocrine-based treatment in early HR+/HER2- BC. Methods: The LETLOB phase II trial randomized 92 postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to receive neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months. Objective response was the primary endpoint, as defined by study protocol and previously published (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate higher chemosensitivity. Results: Intrinsic subtype distribution was as follows: Luminal A 39% (N=25), Luminal B 36% (N=24), HER2-enriched 8% (N=5), basal-like 18% (N=12). Non-luminal tumors (basal-like and HER2-enriched) were characterized by less differentiated histological grades (Grade 3 65% vs 33%, p=0.037) and higher Ki67 expression at baseline as compared to luminal tumors (median 20% vs 15%, p=0.013). Considering both treatment arms combined, non-luminal tumors showed a significantly lower objective response rate as compared to luminal tumors (47% vs 78%, p=0.031). This difference was statistically significant in the lapatinib arm (43% vs 90%, p=0.021), but not in the placebo arm (50% vs 68%, p=0.449; interaction test p=0.169). After treatment, non-luminal tumors had significantly higher Ki67 levels than luminal ones (post-treatment median 10% and 7%, respectively, p=0.004). Median CES level at baseline was -0.03 (range: -1.11, +1.12). Each unit increase in CES levels at baseline was numerically associated with higher probability of achieving an objective response (Odds Ratio 1.95, 95% CI 0.74-5.18, p=0.178, both arms combined). Conclusions: In HR+/HER2- early BC patients enrolled in the LET-LOB trial, patients with non-luminal tumor or low CES score showed reduced sensitivity to endocrine-based treatment. The highest response rate (90%) was observed in luminal patients treated with letrozole + lapatinib. Gene-expression profiling could potentially be used to identify patients with low sensitivity to endocrine treatment for which neoadjuvant chemotherapy should be preferred. Citation Format: Gaia Griguolo, Maria Vittoria Dieci, Daniele Giulio Generali, Laia Paré, Antonio Frassoldati, Luigi Cavanna, Enrico Tagliafico, Katia Cagossi, Giancarlo Bisagni, Federico Piacentini, Federica Miglietta, Guido Ficarra, Aleix Prat, PierFranco Conte, Valentina Guarneri. Gene-expression profiling and response to neoadjuvant endocrine treatment in the phase II LETLOB trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-14.

Concordance rate in cattle and sheep between genotypes differing in Illumina GenCall quality score.

Proper quality control of data prior to downstream analyses is fundamental to ensure integrity of results; quality control of genomic data is no exception. While many metrics of quality control of genomic data exist, the objective of the present study was to quantify the genotype and allele concordance rate between called single nucleotide polymorphism (SNP) genotypes differing in GenCall (GC) score; the GC score is a confidence measure assigned to each Illumina genotype call. This objective was achieved using Illumina beadchip genotype data from 771 cattle (12428767 genotypes in total post-editing) and 80 sheep (1557360 SNPs genotypes in total post-editing) each genotyped in duplicate. The called genotype with the lowest associated GC score was compared to the genotype called for the same SNP in the same duplicated animal sample but with a GC score of >0.90 (assumed to represent the true genotype). The mean genotype concordance rate for a GC score of <0.300, 0.300-0.549, and ≥0.550 in the cattle (sheep in parenthesis) was 0.9467 (0.9864), 0.9707 (0.9953), and 0.9994 (0.99997) respectively; the respective allele concordance rate was 0.9730 (0.9930), 0.9849 (0.9976), and 0.9997 (0.99998). Hence, concordance eroded as the GC score of the called genotype reduced, albeit the impact was not dramatic and was not very noticeable until a GC score of <0.55. Moreover, the impact was greater and more consistent in the cattle population than in the sheep population. Furthermore, an impact of GC score on genotype concordance rate existed even for the same SNP GenTrain value; the GenTrain value is a statistical score that depicts the shape of the genotype clusters and the relative distance between the called genotype clusters.

78 Effects of invitro production on the epigenome and transcriptome of bovine embryos determined through a multi-omics data integration approach

In cattle, ovarian superovulation followed by invivo embryo collection and transfer (MOET), and the invitro production (IVP) of embryos are used all over the world to improve animal genetics. Application of MOET has resulted in the production of billions of healthy animals during the past 40 years, and IVP has evolved and given rise to significant numbers of calves during the past 10 years. Nevertheless, the use of MOET and IVP can affect the embryo epigenome, and therefore its transcriptome, before and after elongation, as shown by different studies. The integration of publicly available epigenome-transcriptome datasets generated by these studies could lead to a robust characterisation of the impacts of the application of MOET and IVP. The goal of this study was to integrate all publicly available data about MOET and IVP embryos to determine temporally differentially methylated regions (DMRs) and differentially expressed genes (DEGs) from blastocyst to elongation between IVP and MOET embryos. Datasets were downloaded from the Gene Expression Omnibus (GEO) database. Accession numbers were (1) for epigenomics: GSE69173, GSE97517, and GSE101895, plus one provided dataset from O’Doherty et al. (2018 BMC Genomics, 19, 438; https://doi.org/10.1186/s12864-018-4818-3), all hybridized to the EDMA platform GPL18384; (2) for transcriptomics: GSE12327, GSE21030, GSE24596, GSE24936, GSE27817, and GSE40101, all hybridized to the Affymetrix platform GPL2112. Both types of data were analysed with the limma package for R software, and functional enrichment analysis was done with the DAVID database. For DMRs, comparisons between IVP and MOET were made from spherical blastocysts (n=16 per group) on Day 7, to embryos on Day 15, specifically in the trophectoderm (TE) or embryonic disc (ED) regions (n=4 per region and per group). For DEGs, comparisons between IVP and MOET were made from spherical blastocysts (n=9 per group) to elongated blastocysts on Day 13 and embryos undergoing gastrulation on Day 16 (n=6 per group). Considering a P-value &amp;lt;0.05 and fold-change &amp;gt;2, there were 16 672 (TE) and 26 264 (ED) DMRs and 2236 DEGs that temporally differed between IVP and MOET. Most of the identified DMRs were found in intronic regions (around 36%) rather than exonic regions (8%). However, DMRs that were more methylated at IVP compared with MOET contained exons encoding for genes that enriched the Wnt signalling Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway in the ED, and focal adhesion and ECM-receptor interaction KEGG pathways (P&amp;lt;0.05) in the TE. Accordingly, DEGs with lower expression in elongated embryos (Day 13 and Day 16) at IVP as opposed to MOET were mainly associated with these three pathways. In conclusion, this multi-omics analysis demonstrates that even when embryos are produced under different conditions and experiments, the main changes imposed by IVP affected genes involved in embryonic development and adhesion to the endometrium, which could explain the lower survival rates at IVP compared with MOET.

Transcriptional Profiling and Biological Pathway(s) Analysis of Type 2 Diabetes Mellitus in a Pakistani Population.

The epidemic of type 2 diabetes mellitus (T2DM) is an important global health concern. Our earlier epidemiological investigation in Pakistan prompted us to conduct a molecular investigation to decipher the differential genetic pathways of this health condition in relation to non-diabetic controls. Our microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their canonical pathways. High-throughput qRT-PCR TaqMan Low Density Array (TLDA) was performed to validate the selected differentially expressed genes of our interest, viz., ARNT, LEPR, MYC, RRAD, CYP2D6, TP53, APOC1, APOC2, CYP1B1, SLC2A13, and SLC33A1 using a small population validation sample (n = 15 cases and their corresponding matched controls). Overall, our small pilot study revealed a discrete gene expression profile in cases compared to controls. The disease pathways included: Insulin Receptor Signaling, Type II Diabetes Mellitus Signaling, Apoptosis Signaling, Aryl Hydrocarbon Receptor Signaling, p53 Signaling, Mitochondrial Dysfunction, Chronic Myeloid Leukemia Signaling, Parkinson’s Signaling, Molecular Mechanism of Cancer, and Cell Cycle G1/S Checkpoint Regulation, GABA Receptor Signaling, Neuroinflammation Signaling Pathway, Dopamine Receptor Signaling, Sirtuin Signaling Pathway, Oxidative Phosphorylation, LXR/RXR Activation, and Mitochondrial Dysfunction, strongly consistent with the evidence from epidemiological studies. These gene fingerprints could lead to the development of biomarkers for the identification of subgroups at high risk for future disease well ahead of time, before the actual disease becomes visible.