Laureats

The high mortality rate in septic shock patients is likely due to environmental and genetic factors, which influence the host response to infection. Two genome-wide association studies (GWAS) on 832 septic shock patients were performed. We used integrative bioinformatic approaches to annotate and prioritize the sepsis-associated single nucleotide polymorphisms (SNPs). An association of 139 SNPs with death based on a false discovery rate of 5% was detected. The most significant SNPs were within the CISH gene involved in cytokine regulation. Among the 139 SNPs associated with death and the 1311 SNPs in strong linkage disequilibrium with them, we investigated 1439 SNPs within non-coding regions to identify regulatory variants. The highest integrative weighted score (IW-score) was obtained for rs143356980, indicating that this SNP is a robust regulatory candidate. The rs143356980 region is located in a non-coding region close to the CISH gene. A CRISPR-Cas9-mediated deletion of this region and specific luciferase assays in K562 cells showed that rs143356980 modulates the enhancer activity in K562 cells. These analyses allowed us to identify several genes associated with death in patients with septic shock. They suggest that genetic variations in key genes, such as CISH, perturb relevant pathways, increasing the risk of death in sepsis patients.

Keywords:

sepsis; GWAS; SNPs; non coding region; CISH; enhancer; CRISPR-Cas9; luciferase assay

ReMap (https://remap.univ-amu.fr) aims to provide manually curated, high-quality catalogs of regulatory regions resulting from a large-scale integrative analysis of DNA-binding experiments in Human, Mouse, Fly and Arabidopsis thaliana for hundreds of transcription factors and regulators. In this 2022 update, we have uniformly processed >11 000 DNA-binding sequencing datasets from public sources across four species. The updated Human regulatory atlas includes 8103 datasets covering a total of 1210 transcriptional regulators (TRs) with a catalog of 182 million (M) peaks, while the updated Arabidopsis atlas reaches 4.8M peaks, 423 TRs across 694 datasets. Also, this ReMap release is enriched by two new regulatory catalogs for Mus musculus and Drosophila melanogaster. First, the Mouse regulatory catalog consists of 123M peaks across 648 TRs as a result of the integration and validation of 5503 ChIP-seq datasets. Second, the Drosophila melanogaster catalog contains 16.6M peaks across 550 TRs from the integration of 1205 datasets. The four regulatory catalogs are browsable through track hubs at UCSC, Ensembl and NCBI genome browsers. Finally, ReMap 2022 comes with a new Cis Regulatory Module identification method, improved quality controls, faster search results, and better user experience with an interactive tour and video tutorials on browsing and filtering ReMap catalogs.

Gene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response.

Abstract

Background: Facioscapulohumeral dystrophy (FSHD) is a late-onset autosomal dominant form of muscular dystrophy involving specific groups of muscles with variable weakness that precedes inflammatory response, fat infiltration, and muscle atrophy. As there is currently no cure for this disease, understanding and modelling the typical muscle weakness in FSHD remains a major milestone towards deciphering the disease pathogenesis as it will pave the way to therapeutic strategies aimed at correcting the functional muscular defect in patients.

Methods: To gain further insights into the specificity of the muscle alteration in this disease, we derived induced pluripotent stem cells from patients affected with Types 1 and 2 FSHD but also from patients affected with Bosma arhinia and microphthalmia. We differentiated these cells into contractile innervated muscle fibres and analysed their transcriptome by RNA Seq in comparison with cells derived from healthy donors. To uncover biological pathways altered in the disease, we applied MOGAMUN, a multi-objective genetic algorithm that integrates multiplex complex networks of biological interactions (protein-protein interactions, co-expression, and biological pathways) and RNA Seq expression data to identify active modules.

Results: We identified 132 differentially expressed genes that are specific to FSHD cells (false discovery rate < 0.05). In FSHD, the vast majority of active modules retrieved with MOGAMUN converges towards a decreased expression of genes encoding proteins involved in sarcomere organization (P value 2.63e-12 ), actin cytoskeleton (P value 9.4e-5 ), myofibril (P value 2.19e-12 ), actin-myosin sliding, and calcium handling (with P values ranging from 7.9e-35 to 7.9e-21 ). Combined with in vivo validations and functional investigations, our data emphasize a reduction in fibre contraction (P value < 0.0001) indicating that the muscle weakness that is typical of FSHD clinical spectrum might be associated with dysfunction of calcium release (P value < 0.0001), actin-myosin interactions, motor activity, mechano-transduction, and dysfunctional sarcomere contractility.

Conclusions: Identification of biomarkers of FSHD muscle remain critical for understanding the process leading to the pathology but also for the definition of readouts to be used for drug design, outcome measures, and monitoring of therapies. The different pathways identified through a system biology approach have been largely overlooked in the disease. Overall, our work opens new perspectives in the definition of biomarkers able to define the muscle alteration but also in the development of novel strategies to improve muscle function as it provides functional parameters for active molecule screening.

Keywords: Facioscapulohumeral dystrophy; Induced pluripotent stem cells; Muscle contraction; Muscle weakening; Pathophysiology; Sarcomere; System biology.
 

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes’ mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Oxytocin is an important regulator of the social brain. In some animal models of autism, notably in Magel2tm1.1Mus-deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms, focusing on social memory performance. Using in situ hybridization (RNAscope), we have established that Magel2 and oxytocin receptor are co-expressed in the dentate gyrus and CA2/CA3 hippocampal regions involved in the circuitry underlying social memory. Then, we have shown that Magel2tm1.1Mus-deficient mice, evaluated in a three-chamber test, present a deficit in social memory. Next, in hippocampus, we conducted neuroanatomical and functional studies using immunostaining, oxytocin-binding experiments, ex vivo electrophysiological recordings, calcium imaging and biochemical studies. We demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin receptors and of somatostatin interneurons in both DG and CA2/CA3 regions. We also revealed a delay in the GABAergic development sequence in Magel2tm1.1Mus-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring hippocampal alterations and social memory at adulthood. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.

Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

Background

Segmental progeroid syndromes are a heterogeneous group of rare and often severe genetic disorders that have been studied since the twentieth century. These progeroid syndromes are defined as segmental because only some of the features observed during natural aging are accelerated.

Methods

Since 2015, the Molecular Genetics Laboratory in Marseille La Timone Hospital proposes molecular diagnosis of premature aging syndromes including laminopathies and related disorders upon NGS sequencing of a panel of 82 genes involved in these syndromes.

We analyzed the results obtained in 4 years on 66 patients issued from France and abroad.

Results

Globally, pathogenic or likely pathogenic variants (ACMG class 5 or 4) were identified in about 1/4 of the cases; among these, 9 pathogenic variants were novel. On the other hand, the diagnostic yield of our panel was over 60% when the patients were addressed upon a nosologically specific clinical suspicion, excepted for connective tissue disorders, for which clinical diagnosis may be more challenging. Prenatal testing was proposed to 3 families. We additionally detected 16 variants of uncertain significance and reclassified 3 of them as benign upon segregation analysis in first degree relatives.

Conclusions

High throughput sequencing using the Laminopathies/ Premature Aging disorders panel allowed molecular diagnosis of rare disorders associated with premature aging features and genetic counseling for families, representing an interesting first-level analysis before whole genome sequencing may be proposed, as a future second step, by the National high throughput sequencing platforms (“Medicine France Genomics 2025” Plan), in families without molecular diagnosis.

Distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of diseases, resembling Charcot–Marie–Tooth syndromes, but characterized by an exclusive involvement of the motor part of the peripheral nervous system.

Here, we describe two new compound heterozygous mutations in VRK1, the vaccinia-related kinase 1 gene, in two siblings from a Lebanese family, affected with dHMN associated with upper motor neurons (MNs) signs. The mutations lead to severely reduced levels of VRK1 by impairing its stability, and to a shift of nuclear VRK1 to cytoplasm. Depletion of VRK1 from the nucleus alters the dynamics of coilin, a phosphorylation target of VRK1, by reducing its stability through increased proteasomal degradation. In human-induced pluripotent stem cell-derived MNs from patients, we demonstrate that this drop in VRK1 levels leads to Cajal bodies (CBs) disassembly and to defects in neurite outgrowth and branching. Mutations in VRK1 have been previously reported in several neurological diseases affecting lower or both upper and lower MNs. Here, we describe a new phenotype linked to VRK1 mutations, presenting as a classical slowly progressive motor neuropathy, beginning in the second decade of life, with associated upper MN signs. We provide, for the first time, evidence for a role of VRK1 in regulating CB assembly in MNs. The observed MN defects are consistent with a length dependent axonopathy affecting lower and upper MNs, and we propose that diseases due to mutations in VRK1 should be grouped under a unique entity named `VRK1-related motor neuron disease’.

Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19–21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.