Identification of Thrombosis-Related Genes in Patients with Advanced Gastric Cancer: Data from AGAMENON-SEOM Registry

Advanced gastric cancer is one of the most thrombogenic neoplasms. However, genetic mechanisms underlying this complication remain obscure, and the molecular and histological heterogeneity of this neoplasm hinder the identification of thrombotic biomarkers. Therefore, our main objective was to identify genes related to thrombosis regardless of Lauren subtypes. Furthermore, in a secondary exploratory study, we seek to discover thrombosis-associated genes that were specific to each TCGA molecular subtype. We designed a nested case-control study using the cohort of the AGAMENON national advanced gastric cancer registry. Ninety-seven patients were selected—48 with and 49 without venous thromboembolism (using propensity score matching to adjust for confounding factors)—and a differential gene expression array stratified by Lauren histopathological subtypes was carried out in primary tumor samples. For the secondary objective, the aforementioned differential expression analysis was conducted for each TCGA group. Fifteen genes were determined to be associated with thrombosis with the same expression trend in both the intestinal and diffuse subtypes. In thrombotic subjects, CRELD1, KCNH8, CRYGN, MAGEB16, SAA1, ARL11, CCDC169, TRMT61A, RIPPLY3 and PLA2G6 were underexpressed (adjusted-p < 0.05), while PRKD3, MIR5683, SDCBP, EPS8 and CDC45 were overexpressed (adjusted-p < 0.05), and correlated, by logistic regression, with lower or higher thrombotic risk, respectively, in the overall cohort. In each TCGA molecular subtype, we identified a series of genes differentially expressed in thrombosis that appear to be subtype-specific. We have identified several genes associated with venous thromboembolism in advanced gastric cancer that are common to Lauren intestinal and diffuse subtypes. Should these genetic factors be validated in the future, they could be complemented with existing clinical models to bolster the ability to predict thrombotic risk in individuals with advanced gastric adenocarcinoma.

The accuracy of absolute differential abundance analysis from relative count data

Concerns have been raised about the use of relative abundance data derived from next generation sequencing as a proxy for absolute abundances. In the differential abundance setting compositional effects are hypothesized to contribute to increased rates of spurious differences (false positives). However in practice, partial reconstruction of total abundance can be imputed through renormalization of observed per-sample abundance. Given the renormalized data differential abundance need not be called on relative counts themselves but on estimates of absolute counts. We use simulated data to explore the consistency of differential abundance calls made on these adjusted relative abundances and find that while overall rates of false positive calls are low substantial error is possible. Conditions consistent with microbial community profiling are the most at risk of error induced by compositional effects. Increasing complexity of composition (i.e. increasing feature number) is generally protective against this effect. In real data sets drawn from 16S metabarcoding, expression array, bulk RNA-seq, and single-cell RNA-seq experiments, results are similar: though median accuracy is high, microbial community profiling and single-cell transcriptomic data sets can have poor outcomes. However, we show that problematic data sets can often be identified by summary characteristics of their relative abundances alone, giving researchers a means of anticipating problems and adjusting analysis strategies where appropriate.

Activation of fibroblast growth factor-inducible 14 in the early phase of childhood IgA nephropathy

IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. Pediatric patients in Japan are diagnosed with IgAN at an early stage of the disease through annual urinary examinations. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) have various roles, including proinflammatory effects, and modulation of several kidney diseases; however, no reports have described their roles in pediatric IgAN. In this study, we performed pathological and immunohistochemical analyses of samples from 14 pediatric IgAN patients. Additionally, gene expression arrays of glomeruli by laser-captured microdissection were performed in hemi-nephrectomized high serum IgA (HIGA) mice, a model of IgA nephropathy, to determine the role of Fn14. Glomeruli with intense Fn14 deposition were observed in 80% of mild IgAN cases; however, most severe cases showed glomeruli with little or no Fn14 deposition. Fn14 deposition was not observed in obvious mesangial proliferation or the crescent region of glomeruli, but was detected strongly in the glomerular tuft, with an intact appearance. In HIGA mice, Fn14 deposition was observed mildly beginning at 11 weeks of age, and stronger Fn14 deposition was detected at 14 weeks of age. Expression array analysis indicated that Fn14 expression was higher in HIGA mice at 6 weeks of age, increased slightly at 11 weeks, and then decreased at 26 weeks when compared with controls at equivalent ages. These findings suggest that Fn14 signaling affects early lesions but not advanced lesions in patients with IgAN. Further study of the TWEAK/Fn14 pathway will contribute to our understanding of the progression of IgAN.

Desensitization of human breast progenitors by a transient exposure to pregnancy levels of estrogen

Full term pregnancy at an early age is the only factor known to consistently protect against breast cancer. Because hormone receptor positive progenitors in the human breast relay endocrine signaling, we here sought to determine whether an experimental mimicry of the third trimester surge of hormones would change their susceptibility to growth stimulation. Hormone receptor positive, reduction mammoplasty-derived human breast epithelial progenitors were exposed to a short-term, pregnancy-level of estradiol, and their subsequent response to estradiol stimulation was analyzed. Exposure to pregnancy-level of estradiol results in subsequent lower sensitivity to estrogen-induced proliferation. Expression array and immunoblotting reveal upregulation of S100A7 and down-regulation of p27, both associated with parity and epithelial differentiation. Notably, we find that the epithelial differentiation is accompanied by upregulation of E-cadherin and down-regulation of vimentin as well as by diminished migration and more mature luminal epithelial differentiation in a mouse transplantation model. Our findings are in support of a de-sensitization mechanism for pregnancy-induced prevention against breast cancer.

Large-scale integration of DNA methylation and gene expression array platforms

Epigenome-wide association studies (EWAS) aim to provide evidence that marks of DNA methylation (DNAm) have downstream consequences that can result in the development of human diseases. Although these methods have been successful in identifying DNAm patterns associated with disease states, any further characterization of etiologic mechanisms remains elusive. This knowledge gap does not originate from a lack of DNAm-trait associations, but rather stems from study design issues that affect the interpretability of EWAS results. Despite known limitations in predicting the function of a particular CpG site, most EWAS maintain the broad assumption that altered DNAm results in a concomitant change of transcription at the most proximal gene. This study integrated DNAm and gene expression (GE) measurements in two cohorts, the Adolescent and Young Adult Twin Study (AYATS) and the Pregnancy, Race, Environment, Genes (PREG) study, to improve the understanding of epigenomic regulatory mechanisms. CpG sites associated with GE in cis were enriched in areas of transcription factor binding and areas of intermediate-to-low CpG density. CpG sites associated with trans GE were also enriched in areas of known regulatory significance, including enhancer regions. These results highlight issues with restricting DNAm-transcript annotations to small genomic intervals and question the validity of assuming a canonical cis DNAm-GE pathway. Based on these findings, the interpretation of EWAS results is limited in studies without multi-omic support and further research should identify genomic regions in which GE-associated DNAm is overrepresented.

Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

N6-Methylandenosine-Related lncRNAs in Tumor Microenvironment Are Potential Prognostic Biomarkers in Colon Cancer

BackgroundLncRNA dysregulation and the tumor microenvironment (TME) have been shown to play a vital role in the progression and prognosis of colon cancer (CC). We aim to reveal the potential molecular mechanism from the perspective of lncRNA in the TME and provide the candidate biomarkers for CC prognosis.MethodsESTIMATE analysis was used to divide the CC patients into high and low immune or stromal score groups. The expression array of lncRNA was re-annotated by Seqmap. Microenvironment-associated lncRNAs were filtered through differential analysis. The m6A-associated lncRNAs were screened by Pearson correlation analysis. Lasso Cox regression analyses were performed to construct the m6A- and tumor microenvironment-related lncRNA prognostic model (m6A-TME-LM). Survival analysis was used to assess the prognostic efficacy of candidate lncRNAs. Enrichment analyses annotated the candidate genes’ functions.ResultsWe obtained 25 common differentially expressed lncRNAs (DELs) associated with immune microenvironment and m6A-related genes for subsequent lasso analysis. Four out of these DELs were selected for the m6A-TME-LM. All the four lncRNAs were related to overall survival, and a test set testified the result. Further stratification analysis of the m6A-TME-LM retained its ability to predict OS for male and chemotherapy adjuvant patients and performed an excellent prognostic efficacy in the TNM stage III and IV subgroups. Network analysis also found the four lncRNAs mediated co-expression network was associated with tumor development.ConclusionWe constructed the m6A-TME-LM, which could provide a better prognostic prediction of CC.

Ordering of Omics Features Using Beta Distributions on Montecarlo p-Values

The current trend in genetic research is the study of omics data as a whole, either combining studies or omics techniques. This raises the need for new robust statistical methods that can integrate and order the relevant biological information. A good way to approach the problem is to order the features studied according to the different kinds of data so a key point is to associate good values to the features that permit us a good sorting of them. These values are usually the p-values corresponding to a hypothesis which has been tested for each feature studied. The Montecarlo method is certainly one of the most robust methods for hypothesis testing. However, a large number of simulations is needed to obtain a reliable p-value, so the method becomes computationally infeasible in many situations. We propose a new way to order genes according to their differential features by using a score defined from a beta distribution fitted to the generated p-values. Our approach has been tested using simulated data and colorectal cancer datasets from Infinium methylationEPIC array, Affymetrix gene expression array and Illumina RNA-seq platforms. The results show that this approach allows a proper ordering of genes using a number of simulations much lower than with the Montecarlo method. Furthermore, the score can be interpreted as an estimated p-value and compared with Montecarlo and other approaches like the p-value of the moderated t-tests. We have also identified a new expression pattern of eighteen genes common to all colorectal cancer microarrays, i.e., 21 datasets. Thus, the proposed method is effective for obtaining biological results using different datasets. Our score shows a slightly smaller type I error for small sizes than the Montecarlo p-value. The type II error of Montecarlo p-value is lower than the one obtained with the proposed score and with a moderated p-value, but these differences are highly reduced for larger sample sizes and higher false discovery rates. Similar performances from type I and II errors and the score enable a clear ordering of the features being evaluated.

POS0375 CHAPERONE-MEDIATED AUTOPHAGY IS A HALLMARK OF JOINT DISEASE IN OSTEOARTHRITIC PATIENTS

Background:In Osteoarthritis (OA), defects in macroautophagy (autophagy) are evident and precede joint damage. Indeed, pharmacological activation of autophagy protects against joint damage.Objectives:Therefore, identifying hallmarks associated with specific autophagy subtypes could shed light to fundamental mechanisms of joint disease.Methods:A comparative analysis of 35 autophagy genes was performed from blood from the Prospective OA Cohort of A Coruña (PROCOAC). Non-OA subjects (Age:61,44±1,16 years; BMI:25,25±0,52; Females, n=18) and Knee OA subjects (Age:65,50±1,05 years; BMI:29,55±0,67; Females, n=18, OA grade III-IV) were profiled using an autophagy gene expression array by SYBR green qPCR. Confirmatory studies were performed in blood from Non-OA subjects (Age:60,13±1,12 years; BMI:24,85±0,59; Females; n=30) and Knee-OA subjects (Age:68,4±1,11 years; BMI:29,65±0,55; Females; n=30, OA grade III-IV) by Taqman qPCR. The candidate gene was evaluated in human knee joint tissues (cartilage, meniscus, ligaments, synovium) with different KL grades (Age: KL0=28,3±4,50; KL2=77±6,08; KL4=62,3±3,05, n=3) and in both spontaneous aging (2, 6, 12, 18, and 30 months old, n=3) and surgically-induced OA (10 weeks after surgery, n=4) in mice by IHC. The functional consequences were studied in T/C28a2 and primary human OA chondrocytes. Autophagy, FOXO, Chaperone-mediated autophagy (CMA), inflammation, and cellular senescence were analyzing by gene and protein expression. Moreover, oxidative stress and cell death were evaluated by FACS. The contribution of CMA to chondrocyte homeostasis was evaluated by studying the capacity of CMA to restore proteostasis upon autophagy deficiency by siATG5.Results:15 autophagy-related genes were significantly downregulated in blood from knee OA patients compared to non-OA patients. No significant upregulation was found for any studied gene, although a trend towards upregulation was found in genes involved in the mTOR pathway. Four key autophagy-related genes, including ATG16L2, ATG12, ATG4B and MAP1LC3B were found downregulated in knee OA patients. Interestingly, HSP90AA1 and HSPA8, CMA markers involved in stress response and protein folding, were downregulated. Confirmatory studies showed a significant downregulation of MAP1LC3B and HSP90AA1 in blood from knee OA patients. Remarkably, HSP90A was found reduced in femoral cartilage (medial and lateral), meniscus and ACL. Moreover, this reduction was higher in medial cartilage compared to lateral cartilage and meniscus, while in synovial membrane, HSP90A expression was found increased. This expression signature was dependent on OA grade severity. In addition, we observed a decrease of HSP90A with aging and OA in mice. The functional consequences of HSP90AA1 gene silencing are related to an increase in NFκB, MMP13, and p16 expression. Interestingly, LAMP2A, a key CMA mediator, HSPA8, MAP1LC3B and FoxO1 expression were upregulated in chondrocytes with HSP90AA1 deficiency, which might indicate an early response to maintain homeostasis. On the other hand, LAMP2A protein is decreased upon HSP90AA1 deficiency, while LC3II and p62 were increased, indicating a failure in the autophagy flux that leads to impaired lysosomal degradation.Moreover, p21, p16 and prbS6 were increased upon HSP90AA1 deficiency, besides increasing mitochondrial ROS production and apoptosis. ATG5 silencing blocks autophagy by reducing LC3II and increasing prbs6, p62, p16 and p21. Interestingly, LAMP2A and HSP90A were found increased, indicating a possible compensative activation of CMA in response to autophagy defects. These results support that HSP90A has an important role in chondrocyte homeostasis by participating in the cross-talk between CMA and autophagy.Conclusion:Taking together, we identified HSP90A, a CMA regulator, as critical in chondrocyte homeostasis. These disease mechanisms are relevant in OA and constitute hallmarks potentially useful to prevent OA progression.References:[1]Caramés B, et al. Arthritis Rheum. 2010, 2015;[2]Caramés B, et al. Ann Rheum Dis. 2012.Disclosure of Interests:None declared

Multiomics Analysis Reveals Molecular Abnormalities in Granulosa Cells of Women With Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is the most common complex endocrine and metabolic disease in women of reproductive age. It is characterized by anovulatory infertility, hormone disorders, and polycystic ovarian morphology. Regarding the importance of granulosa cells (GCs) in the pathogenesis of PCOS, few studies have investigated the etiology at a single “omics” level, such as with an mRNA expression array or methylation profiling assay, but this can provide only limited insights into the biological mechanisms. Here, genome-wide DNA methylation together with lncRNA-miRNA-mRNA profiles were simultaneously detected in GCs of PCOS cases and controls. A total of 3579 lncRNAs, 49 miRNAs, 669 mRNAs, and 890 differentially methylated regions (DMR)-associated genes were differentially expressed between PCOS cases and controls. Pathway analysis indicated that these differentially expressed genes were commonly associated with steroid biosynthesis and metabolism-related signaling, such as glycolysis/gluconeogenesis. In addition, we constructed ceRNA networks and identified some known ceRNA axes, such as lncRNAs-miR-628-5p-CYP11A1/HSD17B7. We also identified many new ceRNA axes, such as lncRNAs-miR-483-5p-GOT2. Interestingly, most ceRNA axes were also closely related to steroid biosynthesis and metabolic pathways. These findings suggest that it is important to systematically consider the role of reproductive and metabolic genes in the pathogenesis of PCOS.