Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Interest of large-scale metabolomic analysis for the characterization of immune deficits in humans

La métabolomique est la discipline qui vise à analyser l'ensemble des petites molécules d'un liquide biologique ou d'un tissu. Elle utilise des outils aujourd'hui à fort potentiel et suscite un vif intérêt dans la communauté scientifique et particulièrement médicale. Le profilage à large spectre de ces petites molécules ou métabolites dans les liquides biologiques et les tissus chez l'humain est un enjeu majeur en analyse métabolomique, mais des analyses plus ciblées sont aussi pertinentes. De par la grande diversité physico-chimique des petites molécules, il est nécessaire d'utiliser des méthodes complémentaires afin d'assurer une couverture optimale. Cela implique généralement l'utilisation combinée de techniques de chromatographies liquides différentes couplées à de la spectrométrie de masse. La polyvalence des couplages LC-HRMS permet dans la grande majorité des cas de surmonter ce problème et il est complété par des outils bio-informatiques dédiés assurant différentes fonctions, à savoir la détection et l'alignement des variables, les analyses statistiques, la fusion de données et l'identification des métabolites. Dans ce contexte, notre travail vise à développer des approches de LC-MS pour l'analyse métabolomique d'échantillons humain pour l'étude de différents déficits immunitaires. La déficience en CTPS1 a été associée à une capacité altérée de prolifération des lymphocytes activés conduisant donc à une maladie d'immunodéficience combinée. Dans un premier temps, nous avons développé une méthode ciblée de mesure de l'activité de CTPS, par LC-MS, dans les lymphocytes humains et ce afin de pouvoir documenter son rôle dans l'immuno-modulation. Cette technique a été développée, validée et utilisée sur des échantillons humains lymphocytaires, afin de mettre en évidence ce déficit enzymatique en CTPS1 chez des patients porteurs de mutations nouvellement identifiées. CTPS1 étant une enzyme clé dans la synthèse des pyrimidines, nous avons poursuivi la caractérisation métabolique de ce déficit en développant une analyse métabolomique non ciblée par spectrométrie de masse (dite aussi à large spectre). Nous avons utilisé 2 méthodes LC-HRMS dans l'annotation du métabolome de cellules mononuclées du sang périphérique (PBMC) humains. Le développement de cette méthode a été réalisé sur des PBMC issus de donneurs sains, mis en culture, stimulés de manière artificielle et traités par un inhibiteur spécifique de la CTPS, la 3 Déazauridine (3DAU). Des milliers de signaux ont été détectés à partir des extraits cellulaires, toutefois l'identification d'une grande partie de ces signaux demeurait inconnue. Dans ce contexte, une base de données a été élaborée à l'aide de composés chimiques de références. La combinaison de méthodes chromatographiques différentes (RP et HILIC) a permis l'identification de près de 400 métabolites dans les PBMC étudiés. Outre les voies métaboliques perturbées lors de la stimulation par le couplage PMA-Ionomycine, nos analyses préliminaires ont montré, comme attendu, l'impact délétère de l'inhibiteur 3DAU sur le métabolisme des pyrimidines, accompagné d'une augmentation de marqueurs de l'apoptose tels que les acides oléique et linoléique. Par la suite, nous sommes passés à l'analyse d'échantillons érythrocytaires de patients, collectés au sein du laboratoire pour le diagnostic d'autres déficits immunitaires combinés sévères (DICS) comme les déficiences en Adénosine Désaminase et Purine nucléoside phosphorylase. Ces études sont parmi les premières à combiner des analyses LC/ESI-HRMS métabolomiques sur des échantillons érythrocytaires rares issus de patients atteints de DICS. Elles constituent la première étape de construction d'une base de données relationnelle incluant données spectrales et biologiques (variations des conditions physiologiques et physio-pathologiques observées) qui pourrait être utilisée pour la caractérisation de biomarqueurs dans le cas d'études cliniques et diagnostiques.Metabolomics is the discipline that aims to analyze all small molecules of a biological fluid or tissue. It uses tools with high potential today and arouses a keen interest in the scientific and especially medical community. The large-scale profiling of these small molecules or metabolites in body fluids and tissues in humans is a major goal in metabolomic analysis, but more targeted analyzes are also relevant. Due to the large physico-chemical diversity of small molecules, it is necessary to use complementary methods to ensure optimal coverage. This usually involves the combined use of different liquid chromatography techniques coupled with mass spectrometry (LC-MS or LC-HRMS for high resolution devices). The versatility of LC-HRMS couplings in the vast majority of cases overcomes this problem and is complemented by dedicated bioinformatics tools providing different functions, namely the detection and alignment of variables, statistical analysis, fusion data and the identification of metabolites. In this context, our work aims to develop LC-MS approaches for the metabolomic analysis of human samples for the study of different immune deficits. The deficiency of CTPS1 has been associated with an impaired capacity for proliferation of activated lymphocytes thus leading to a combined immunodeficiency disease. Firstly, we developed a targeted method for measuring CTPS activity, by LC-MS, in human lymphocytes in order to document its role in immunomodulation. This technique has been developed, validated and used on human lymphocyte samples, in order to highlight this enzymatic deficiency in CTPS1 in patients with newly identified mutations. Since CTPS1 is a key enzyme in the synthesis of pyrimidines, we continued the metabolic characterization of this deficiency by developing a non-targeted metabolomic analysis by mass spectrometry (also called large scale). We used 2 LC-HRMS methods in the annotation of the metabolome of human peripheral blood mononuclear cells (PBMCs). The development of this method was performed on PBMCs from healthy donors, cultured, artificially stimulated and treated with a specific CTPS inhibitor, the 3DAU. Thousands of signals were detected from cell extracts, however identification of many of these signals remained unknown. In this context, a database has been developed using reference chemical compounds. The combination of different chromatographic methods (RP and HILIC) allowed the identification of nearly 400 metabolites in the PBMCs studied. In addition to the disturbed metabolic pathways during stimulation by PMA-Ionomycin coupling (glycolysis, beta-oxidation), our preliminary analyzes showed, as expected, the deleterious impact of the 3DAU inhibitor on the pyrimidine metabolism, accompanied by an increase in markers of apoptosis such as oleic and linoleic acids. Subsequently, we proceeded to the analysis of erythrocyte samples of patients, collected in the laboratory for the diagnosis of other severe combined immunodeficiencies (SCID) such as Adenosine Deaminase (ADA) and Purine nucleoside phosphorylase (PNP). These studies are among the first to combine metabolomic LC / ESI-HRMS analyzes on rare erythrocyte samples from SCID patients. They constitute the first step in the construction of a relational database including spectral data (MS spectra, MS / MS spectra) and biological data (variations of physiological and physio-pathological conditions observed) that could be used for the characterization of biomarkers in the field case of clinical and diagnostic studies

Omics approach to characterize milk-derived extracellular vesicles/exosomes isolated from goats expressing or not alphas1-CN

International audienceBackgroundGoats homozygous for a null allele (O/O) at the CSN1S1 locus encoding αs1-casein display a chronic endoplasmic reticulum (ER) stress (Unfolded Protein Response) due to an accumulation of the other caseins in this compartment, thus triggering a general MEC dysfunction with a strong impact on milk composition. Milk-derived extracellular vesicles (MEVs) contain molecular information, which are thus recognized as mediators of intercellular communication. We made the assumption that the absence of CSN1S1 expression may influence MEVs cargos including miRNA, proteins, lipids and metabolites. The objective of this study was to assess the impact of αs1-casein deficit on MEVs cargos and to compare the biological material they convey.Material & MethodsWe have developed an improved method based on a density gradient ultracentrifugation to isolate MEVs. The quality of MEVs was analyzed morphologically by transmission electron microscopy (TEM) with negative staining (uranyl acetate), the specific “exosome” protein markers were detected by Western blot and ELISA and the size distribution and particle concentration were measured by NTA. The MEV’s proteome was acquired by LC-MS/MS and nucleic acid content (mRNA and miRNA) by NGS and qPCR. EV lipid content was extracted with the MTBE method. Data acquisition was performed by an Orbitrap-MS and analyzed using Lipid Data Analyzer. MEV metabolites were extracted using MPLEx protocol, data acquired by LC coupled with HRMS and analyzed using Metaboanalyst online workbenches. Differential statistical and bioinformatic analyses were performed using appropriate softwares.ResultsThe novel purification method gives MEV populations, free of contamination by other EVs and milk components, at sufficient concentrations to perform subsequent analyses. Nearly 280 proteins involved in the biogenesis of exosomes and MVB formation, their adhesion and internalization as well as proteins associated with membrane transport and enzymes involved in cellular metabolism were identified, among which 41 exosomal proteins differed between CSN1S1 O/O (null) and A/A (wildtype) genotypes. Ongoing profiling of RNA from MEVs has already identified over 230 miRNA and confirmed MEC origin due to the presence of mRNA encoding specific major milk proteins. The comparison of exosomal miRNomes of goat homozygous for A and O alleles at the CSN1S1 locus pointed out 15 miRNAs differentially abundant, potentially related to the MEC phenotype. Sphingomyelin and phosphatidylcholin were the major phospholipids observed in MEV populations. We have totally identified ca. 4,000 compounds using pHILIC and RPLC, 79 of which were significantly up or down-regulated in studied genotypes.ConclusionSeveral differences distinguishing goats according to the genotype at the CSN1S1 locus were found at each level of the omic analysis of MEVs. Differentially abundant miRNAs and transcriptome analyses are in agreement with UPR phenotype and confirmed their involvement in post-transcriptional regulatory mechanisms

Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation

International audienceCytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1 T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1 T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1 T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1 T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma

The equilibrative nucleoside transporter ENT1 is critical for nucleotide homeostasis and optimal erythropoiesis

This is a related article to: Nucleoside ENTry modulates erythropoiesis (cf ci-dessous)International audienceAbstract The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1−/− mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis

Defects in t6A tRNA modification due to GON7 and YRDC mutations lead to Galloway-Mowat syndrome

N6-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t6A) is a universal modification essential for translational accuracy and efficiency. The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex