👤 Bart P Leroy

Gene copy number variations have theranostic impact and require reliable methods for their identification. We aimed to evaluate the reliability of combined next-generation sequencing (NGS) and digital droplet PCR (ddPCR) method for gene amplification evaluation. We conducted a retrospective multicentric observational study. 55 patients (9 control, 25 Combined NGS-based script and ddPCR method is reliable and easily feasible for the detection of gene amplifications, providing useful data for guided therapy in cancer. Show less

Thoracic aortic aneurysms (TAA) are a progressive disease characterized by inflammation, smooth muscle cell activation and matrix degradation. We hypothesized that mesenchymal stem cells (MSCs) can immunomodulate vascular inflammation and remodeling via altered microRNA (miRNAs) expression profile to attenuate TAA formation. C57BL/6 mice underwent topical elastase application to form descending TAAs. Mice were also treated with MSCs on days 1 and 5 and aortas were analyzed on day 14 for aortic diameter. Cytokine array was performed in aortic tissue and total RNA was tagged and hybridized for miRNAs microarray analysis. Immunohistochemistry was performed for elastin degradation and leukocyte infiltration. Treatment with MSCs significantly attenuated aortic diameter and TAA formation compared to untreated mice. MSC administration also attenuated T-cell, neutrophil and macrophage infiltration and prevented elastic degradation to mitigate vascular remodeling. MSC treatment also attenuated aortic inflammation by decreasing proinflammatory cytokines (CXCL13, IL-27, CXCL12 and RANTES) and upregulating anti-inflammatory interleukin-10 expression in aortic tissue of elastase-treated mice. TAA formation demonstrated activation of specific miRNAs that are associated with aortic inflammation and vascular remodeling. Our results also demonstrated that MSCs modulate a different set of miRNAs that are associated with decrease leukocyte infiltration and vascular inflammation to attenuate the aortic diameter and TAA formation. These results indicate that MSCs immunomodulate specific miRNAs that are associated with modulating hallmarks of aortic inflammation and vascular remodeling of aortic aneurysms. Targeted therapies designed using MSCs and miRNAs have the potential to regulate the growth and development of TAAs. Show less

Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-β in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-β, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-β are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-β,we show that there is positive correlations between the effect of differentiation and TGF-β on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and β-Catenin, two molecules from TGF-βsignaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-β-induced mechanisms. Show less

Autosomal recessive retinal dystrophies are clinically and genetically heterogeneous, which hampers molecular diagnosis. We evaluated identity-by-descent-guided Sanger sequencing or whole-exome sequencing in 26 families with nonsyndromic (19) or syndromic (7) autosomal recessive retinal dystrophies to identify disease-causing mutations. Patients underwent genome-wide identity-by-descent mapping followed by Sanger sequencing (16) or whole-exome sequencing (10). Whole-exome sequencing data were filtered against identity-by-descent regions and known retinal dystrophy genes. The medical history was reviewed in mutation-positive families. We identified mutations in 14 known retinal dystrophy genes in 20/26 (77%) families: ABCA4, CERKL, CLN3, CNNM4, C2orf71, IQCB1, LRAT, MERTK, NMNAT1, PCDH15, PDE6B, RDH12, RPGRIP1, and USH2A. Whole-exome sequencing in single individuals revealed mutations in either the largest or smaller identity-by-descent regions, and a compound heterozygous genotype in NMNAT1. Moreover, a novel deletion was found in PCDH15. In addition, we identified mutations in CLN3, CNNM4, and IQCB1 in patients initially diagnosed with nonsyndromic retinal dystrophies. Our study emphasized that identity-by-descent-guided mutation analysis and/or whole-exome sequencing are powerful tools for the molecular diagnosis of retinal dystrophy. Our approach uncovered unusual molecular findings and unmasked syndromic retinal dystrophies, guiding future medical management. Finally, elucidating ABCA4, LRAT, and MERTK mutations offers potential gene-specific therapeutic perspectives. Show less

Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution. We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1β and HP1α. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by short hairpin RNA. Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states. Show less

Mitogen-activated protein (MAP) kinases play a central role in controlling a wide range of cellular functions following their activation by a variety of extracellular stimuli. MAP kinase phosphatases (MKPs) represent a subfamily of dual specificity phosphatases, which negatively regulate MAP kinases. Although ERK2 activity is regulated by its phosphorylation state, MKP3 is regulated by physical interaction with ERK2, independent of its enzymatic activity (Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S., (1998) Science 280, 1262-1265; Farooq, A., Chaturvedi, G., Mujtaba, S., Plotnikova, O., Zeng, L., Dhalluin, C., Ashton, R., and Zhou, M. M. (2001), Mol. Cell 7, 387-399; Zhou, B., and Zhang, Z. Y. (1999) J. Biol. Chem. 274, 35526-35534). The interaction of ERK2 and MKP3 allows the reciprocal cross-regulation of their catalytic activity. Indeed, MKP3 acts as a negative regulator on ERK2-MAP kinase signal transduction activity, representing thus a negative feedback for this MAPK pathway. To identify novel proteins able to complex MKP3, we used the yeast two-hybrid system. Here we report that MKP3 and protein kinase CK2 form a protein complex, which can include ERK2. The phosphatase activity of MKP3 is then slightly increased in vitro, whereas in transfected cells, ERK2 dephosphorylation is reduced. In addition, we demonstrated that CK2 selectively phosphorylates MKP3, suggesting cross-regulation between CK2alpha and MKP3, as well as a modulation of ERK2-MAPK signaling by CK2alpha via MKP3. Show less