RTNduals: an R/Bioconductor package for analysis of co-regulation and inference of dual regulons.

MOTIVATION: Transcription factors (TFs) are key regulators of gene expression, and can activate or repress multiple target genes, forming regulatory units, or regulons. Understanding downstream effects of these regulators includes evaluating how TFs cooperate or compete within regulatory networks. Here we present RTNduals, an R/Bioconductor package that implements a general method for analyzing pairs of regulons. RESULTS: RTNduals identifies a dual regulon when the number of targets shared between a pair of regulators is statistically significant. The package extends the RTN (Reconstruction of Transcriptional Networks) package, and uses RTN transcriptional networks to identify significant co-regulatory associations between regulons. The Supplementary Information reports two case studies for TFs using the METABRIC and TCGA breast cancer cohorts. AVAILABILITY AND IMPLEMENTATION: RTNduals is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/RTNduals/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.

Does celiac disease occur in Afro-derived Brazilian populations?

BACKGROUND: Celiac disease is an autoimmune disorder that occurs in genetically susceptible individuals in whom the ingestion of dietary gluten induces intestinal mucosa inflammation. Previous studies suggest that celiac disease may either be very rare or underdiagnosed in African and/or African-derived population. AIM: Determine the prevalence of celiac disease in Sub-Saharan African-derived Brazilian communities using serological screening. SUBJECTS AND METHODS: Inhabitants from 10 African-derived communities from Northeastern of Brazil were screened for celiac disease. All sera were tested for endomysial class IgA antibody using indirect immunofluorescence. RESULTS: No positive test for IgA-endomysial was observed in the 860 individuals tested. CONCLUSION: Our data suggests a low prevalence of celiac disease in African-derived Brazilian populations.

Extracellular vesicle microRNAs in celiac disease patients under a gluten-free diet, and in lactose intolerant individuals.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder triggered by an abnormal immunological response to gluten ingestion and is associated with deregulated expression of cellular microRNAs (miRNAs) of the gut mucosa. It is frequently misdiagnosed as lactose intolerance (LI) due to symptom resemblance. Microvilli loss may be counteracted by a rigorous gluten-free diet (GFD). AIMS: To identify altered extracellular vesicle miRNAs from plasma among CD patients on GFD (n=34), lactose intolerant individuals on restrictive diet (n=14) and controls (n=23), and to predict biological pathways in which these altered miRNAs may play a part. METHODS: Five different small RNA samples of each group were pooled twice and then screened by new-generation sequencing. Four miRNAs were selected to be quantified by RT-qPCR in the entire sample. RESULTS: The levels of four miRNAs - miR-99b-3p, miR-197-3p, miR-223-3p, and miR-374b-5p - differed between CD patients and controls (P<0.05). Apart from miR-223-3p, all these miRNAs tended to have altered levels also between LI and controls (P<0.10). The results for miR-99b-3p and miR-197-3p between CD and controls were confirmed by RT-qPCR, which also indicated different levels of miR-99b-3p and miR-374b-5p between CD-associated LI and LI (P<0.05). CONCLUSIONS: These miRNAs may have targets that affect cell death, cell communication, adhesion, and inflammation modulation pathways. Hence, altered miRNA levels could be associated with CD-related aspects and gut mucosa recovery.

Identification of miRNAs Enriched in Extracellular Vesicles Derived from Serum Samples of Breast Cancer Patients.

MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.

Integrated copy number and miRNA expression analysis in triple negative breast cancer of Latin American patients.

Triple negative breast cancer (TNBC), a clinically aggressive breast cancer subtype, affects 15-35% of women from Latin America. Using an approach of direct integration of copy number and global miRNA profiling data, performed simultaneously in the same tumor specimens, we identified a panel of 17 miRNAs specifically associated with TNBC of ancestrally characterized patients from Latin America, Brazil. This panel was differentially expressed between the TNBC and non-TNBC subtypes studied (p ≤ 0.05, FDR ≤ 0.25), with their expression levels concordant with the patterns of copy number alterations (CNAs), present mostly frequent at 8q21.3-q24.3, 3q24-29, 6p25.3-p12.2, 1q21.1-q44, 5q11.1-q22.1, 11p13-p11.2, 13q12.11-q14.3, 17q24.2-q25.3 and Xp22.33-p11.21. The combined 17 miRNAs presented a high power (AUC = 0.953 (0.78-0.99);95% CI) in discriminating between the TNBC and non-TNBC subtypes of the patients studied. In addition, the expression of 14 and 15 of the 17miRNAs was significantly associated with tumor subtype when adjusted for tumor stage and grade, respectively. In conclusion, the panel of miRNAs identified demonstrated the impact of CNAs in miRNA expression levels and identified miRNA target genes potentially affected by both CNAs and miRNA deregulation. These targets, involved in critical signaling pathways and biological functions associated specifically with the TNBC transcriptome of Latina patients, can provide biological insights into the observed differences in the TNBC clinical outcome among racial/ethnic groups, taking into consideration their genetic ancestry.

Integrative Analysis Identifies Genetic Variants Associated With Autoimmune Diseases Affecting Putative MicroRNA Binding Sites.

Genome-wide and fine mapping studies have shown that more than 90% of genetic variants associated with autoimmune diseases (AID) are located in non-coding regions of the human genome and especially in regulatory sequences, including microRNAs (miRNA) target sites. MiRNAs are small endogenous noncoding RNAs that modulate gene expression at the post-transcriptional level. Single nucleotide polymorphisms (SNPs) located within the 3' untranslated region of their target mRNAs (miRSNP) can alter miRNA binding sites. Yet, little is known about their effect on regulation by miRNA and the consequences for AID. Conversely, it is well known that two or more AID may share part of their genetic background. Here, we hypothesized that miRSNPs could be associated with more than one AID. To identify miRSNPs associated with AID, we integrated results from three different prediction tools (Polymirts, miRSNP, and miRSNPscore) using a naïve Bayes classifier approach to identify miRSNPs predicted to affect binding sites of miRNAs. Further, to detect miRSNPs associated with two or more AID, we integrated predictions with summary statistics from 12 AID studies. In addition, to prioritize miRSNPs, miRNAs and AID-associated target genes, we used public expression quantitative trait locus (eQTL) data and mRNA-seq and small RNA-seq data. We identified 34 miRNSPs associated with at least two AID. Furthermore, we found 86 miRNAs predicted to target 18 of the associated gene's mRNAs. Our integrative approach revealed new insights into miRNAs and AID associated target genes. Thus, it helped to prioritize AID noncoding risk SNPs that might be involved in the causal mechanisms, providing valuable information for further functional studies.

Small RNA sequencing reveals a comprehensive miRNA signature of BRCA1-associated high-grade serous ovarian cancer.

AIMS: BRCA1 mutation carriers are at increased risk of developing high-grade serous ovarian cancer (HGSOC), a malignancy that originates from fallopian tube epithelium. We aimed to identify differentially expressed known and novel miRNAs in BRCA1-associated HGSOC. METHODS: Small RNA sequencing was performed on eight normal tubal and five HGSOC samples of BRCA1 carriers. Differential expression of a subset of known and novel miRNAs was validated by qRT-PCR on the samples used for small RNA sequencing and a second sample cohort comprising normal and HGSOC tissue of matched BRCA1 and non-BRCA carriers. Data from The Cancer Genome Atlas were used to determine the clinical relevance of the validated differentially expressed miRNAs. RESULTS: 59 known and 20 novel miRNAs showed a significant >fourfold expression difference between normal tubal tissue and HGSOC. qRT-PCR validation confirmed a significant difference in expression levels for 10 out of 11 known miRNAs. Upregulation of two novel miRNAs could not be confirmed. Interestingly, for seven miRNAs a significant increase in expression was observed when comparing normal tubal tissue of postmenopausal women with premenopausal women. Expression levels of miR-145-5p significantly increased with International Federation of Gynecology and Obstetrics stage, while the expression levels of the other nine validated miRNAs were not associated with clinical characteristics. CONCLUSIONS: We report a comprehensive expression signature including both known and novel miRNAs of BRCA1-associated HGSOC. Comparison with previous profiling studies showed a good overlap and a large number of miRNAs not reported to be differentially expressed in HGSOC before underscoring the importance of this study.