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PURPOSE: Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence of Tf protein variants within the general population. Although reports describe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic. EXPERIMENTAL DESIGN: Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot, N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing of PMM2 was performed. RESULTS: In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies. PMM2 causative variants were identified in both patients. CONCLUSION AND CLINICAL RELEVANCE: We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.
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Trastornos Congénitos de Glicosilación , Fosfotransferasas (Fosfomutasas)/deficiencia , Humanos , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/complicaciones , Transferrina/genética , Transferrina/metabolismo , Neuraminidasa/metabolismo , GlicosilaciónRESUMEN
TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.
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Proteínas de Transporte de Catión , Trastornos Congénitos de Glicosilación , Humanos , Manganeso/metabolismo , Galactosa , Antiportadores/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Proteínas de Transporte de Catión/metabolismo , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismoRESUMEN
BACKGROUND: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia (XMEN) disease is a primary immunodeficiency due to loss-of-function mutations in the gene encoding for magnesium transporter 1 (MAGT1). Furthermore, as MAGT1 is involved in the N-glycosylation process, XMEN disease is classified as a congenital disorder of glycosylation. Although XMEN-associated immunodeficiency is well described, the mechanisms underlying platelet dysfunction and those responsible for life-threatening bleeding events have never been investigated. OBJECTIVES: To assess platelet functions in patients with XMEN disease. METHODS: Two unrelated young boys, including one before and after hematopoietic stem cell transplantation, were investigated for their platelet functions, glycoprotein expression, and serum and platelet-derived N-glycans. RESULTS: Platelet analysis highlighted abnormal elongated cells and unusual barbell-shaped proplatelets. Platelet aggregation, integrin αIIbß3 activation, calcium mobilization, and protein kinase C activity were impaired between both patients. Strikingly, platelet responses to protease-activated receptor 1 activating peptide were absent at both low and high concentrations. These defects were also associated with decreased molecular weights of glycoprotein Ibα, glycoprotein VI, and integrin αIIb due to partial impairment of N-glycosylation. All these defects were corrected after hematopoietic stem cell transplantation. CONCLUSION: Our results highlight prominent platelet dysfunction related to MAGT1 deficiency and defective N-glycosylation in several platelet proteins that could explain the hemorrhages reported in patients with XMEN disease.
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Infecciones por Virus de Epstein-Barr , Magnesio , Masculino , Humanos , Magnesio/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Glicosilación , Herpesvirus Humano 4/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismoRESUMEN
The microbiome and immune system of infants are shaped by various bioactive components of human breastmilk, notably human milk oligosaccharides (HMOs). HMOs represent the third component of breastmilk and exhibit extremely high structural diversity with many isomers. Here, we propose a high throughput and high resolution approach to characterize main oligosaccharides present in breastmilk with high identification level thanks to ion mobility spectrometry. Four pairs of standard HMO isomers, that are (LNT/LNnT), (LNFP I/LNFP V), (3'-SL/6'-SL) and (2'-FL/3-FL), were first investigated under both positive and negative ionization mode using direct introduction-trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOF). By examining all the ionic species formed (i.e. protonated and deprotonated ions as well as adduct species), every isomer pair could be distinguished through the separation of at least one species, even with a small difference in collision cross section values (as small as 1.5%) thanks to the flexible resolution capacity of the TIMS instrument. Although multiple mobility peaks resulting from different glycan anomeric conformers, open-ring and/or different ionic isomer structures (i.e. various charge site locations), could be observed for some HMO species. The reduction at the reducing-end of HMOs did not significantly facilitate the isomer distinction. Finally, the unambiguous identification of the studied HMOs in a breastmilk sample showed the potential of the approach combining ion mobility separation and MS/MS experiments for high throughput distinction of HMO isomers in complex breastmilk samples without laborious sample preparation.
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Espectrometría de Movilidad Iónica , Leche Humana , Humanos , Isomerismo , Oligosacáridos , Espectrometría de Masas en TándemRESUMEN
SLC37A4-CDG is an emerging congenital disorder of glycosylation which is characterized by a dominant inheritance and a major coagulopathy originating from the liver. Recent studies took interest in the biochemical alterations found in this CDG and showed that they consisted of multiple glycosylation abnormalities, which result from mislocalization of the endoplasmic reticulum glucose-6-phosphate transporter and associated Golgi homeostasis defects. In this work, we highlight in six affected individuals abnormal patterns for various serum N-glycoproteins and bikunin proteoglycan isoforms, together with specific alterations of the mass spectra of endoglycosidase H-released serum N-glycans. Collectively, these data complement previous findings, help to better delineate SLC37A4-CDG and could present interest in diagnosing this disease.
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Trastornos Congénitos de Glicosilación , Antiportadores/metabolismo , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi , Humanos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , PolisacáridosRESUMEN
Congenital disorders of glycosylation (CDG) constitute an ever-growing group of genetic diseases affecting the glycosylation of proteins. CDG individuals usually present with severe multisystem disorders. MAN1B1-CDG is a CDG with nonspecific clinical symptoms such as intellectual deficiency and developmental delay. Although up to 40 affected individuals were described so far, its final diagnosis is not straightforward using common biochemical methods due to the trace-level accumulation of defective glycan structures. In this study, we present three unreported MAN1B1-CDG individuals and propose a decision tree to reach diagnosis using a panel of techniques ranging from exome sequencing to gel electrophoresis and mass spectrometry. The occurrence of MAN1B1-CDG in patients showing unexplained intellectual disability and development delay, as well as a particular transferrin glycosylation profile, can be ascertained notably using matrix assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry analysis of endo-ß-acetylglucosaminidase H-released serum N-glycans. In addition to reporting new pathogenic variants and additional clinical signs such as hypersialorrhea, we highlight particular biochemical features of MAN1B1-CDG with potential glycoprotein-specific glycosylation defects.
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We identified three cases of congenital disorders of glycosylation (CDG) with Golgi homeostasis disruption, one ATP6V0A2-CDG and two COG4-CDG, with normal transferrin screening analyses. Patient 1 (P1) presented at birth with cutis laxa. Patient 2 (P2) and patient 3 (P3) are adult siblings and presented with severe symptoms evocative of inborn errors of metabolism. Targeted gene sequencing in P1 revealed pathogenic ATP6V0A2 variants, shared by her affected older brother. In P2 and P3, whole exome sequencing revealed a homozygous COG4 variant of unknown significance. In all affected individuals, transferrin analysis was normal. Mass-spectrometry based serum N-glycome analysis and two-dimensional electrophoresis (2-DE) of haptoglobin and of mucin core 1 O-glycosylated apolipoprotein C-III (apoC-III) were performed. All results of second-line N-glycosylation analyses were initially normal. However, apoC-III 2-DE revealed characteristic "apoC-III1" pattern in P1 and specific "apoC-III0" patterns in P2 and P3. In P2 and P3, this allowed reclassifying the variant as likely pathogenic according to ACMG guidelines. These cases highlight the existence of normal transferrin patterns in CDG with Golgi homeostasis disruption, putting the clinicians at risk of misdiagnosing patients. Furthermore, they show the potential of apoC-III 2-DE in diagnosing this type of CDG, with highly specific patterns in COG-CDG.
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Trastornos Congénitos de Glicosilación , Transferrina , Apolipoproteína C-III/genética , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Femenino , Glicosilación , Homeostasis , Humanos , Recién Nacido , Masculino , Transferrina/metabolismoRESUMEN
SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.
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Antiportadores/genética , Trastornos Congénitos de Glicosilación/etiología , Retículo Endoplásmico/patología , Hepatopatías/complicaciones , Proteínas de Transporte de Monosacáridos/genética , Mutación , Adulto , Niño , Preescolar , Trastornos Congénitos de Glicosilación/patología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Genes Dominantes , Glicosilación , Humanos , Lactante , Recién Nacido , Masculino , LinajeRESUMEN
Recombinant human erythropoietin (rEPO) biosimilars are copies of epoetin drugs developed after the first patents ended. However differences in the process of production can result in small structural differences when compared to the reference product. Differences in N-glycosylation profiles are of particular importance for rEPOs, because they can drastically impact the half-life in circulation and activity. Changes of structure can also impact electrophoretic profiles that are used to reveal the presence of a rEPO in a doping control sample. In this study three not well characterized biosimilars were evaluated (Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria). As these products could be used for doping, first their EPO profiles were determined using the antidoping methods (electrophoretic separation by the charge (isolectric focusing, IEF-PAGE) or the molecular weight (SDS-PAGE) and specific EPO immunodetection). Compared to the original epoetin alfa Eprex®, it revealed more basic isoforms for Epotin™ and Jimaixin™ after IEF-PAGE and a slightly lower molecular weight after SDS-PAGE in particular for Hemax®. To better understand the reason for these differences, EPO specific N-glycans were evaluated using two complementary approaches: MALDI-TOF mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. All three biosimilars presented a significant decrease in the major glycan forms of Eprex® along with an increase in less complex forms. Jimaixin™ and Epotin™ presented also a lower amount of fully sialylated forms. HILIC method also showed that O-acetylation level of sialic acid residues might vary from one rEPO to the other.
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Biosimilares Farmacéuticos , Eritropoyetina , Preparaciones Farmacéuticas , Argelia , China , Epoetina alfa , Humanos , Polisacáridos , Proteínas RecombinantesRESUMEN
BACKGROUND: Glycosylation is one of the most complex post-translational modifications of proteins and lipids, notably requiring many glycosyltransferases, glycosidases and sugar transporters encoded by about 1-2% of all human genes. Deleterious variants in any of them may result in improper protein or lipid glycosylation, thus yielding the so-called 'congenital disorders of glycosylation' or CDG. SCOPE OF REVIEW: We first review the current state of knowledge on the common blood and cellular glycoproteins used in the biochemical screening of CDG, as well as the emerging ones for an improved diagnosis. We then provide an overview of the current state-of-the-art methodologies ranging from gel electrophoresis to mass spectrometry to measure improper glycosylation. Finally, we discuss how additional tools such as metabolomics and microfluidics can be added to the current toolbox to better diagnose and delineate CDG. MAJOR CONCLUSIONS: Combining several biochemical indicators and related methods is often required to cope with the large clinical heterogeneity of CDG and establish a definitive diagnosis. GENERAL SIGNIFICANCE: This review aims to critically present current available CDG biochemical biomarkers and dedicated methods in the context of highly diverse glycosylation pathways and related inherited diseases.
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Trastornos Congénitos de Glicosilación/diagnóstico , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Trastornos Congénitos de Glicosilación/metabolismo , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Glicosilación , Glicosilfosfatidilinositoles/análisis , Glicosilfosfatidilinositoles/metabolismo , Humanos , Metabolómica/métodos , Técnicas Analíticas Microfluídicas/métodos , Procesamiento Proteico-PostraduccionalRESUMEN
The authors wish to make a correction to Section 2 [...].
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BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. Therefore, we aimed to analyze the blood metabolome in patients with cirrhosis, with and without ACLF. METHODS: We performed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 650 patients with AD (acute decompensation of cirrhosis, without ACLF), 181 with ACLF, 43 with compensated cirrhosis, and 29 healthy individuals. RESULTS: Of the 137 annotated metabolites identified, 100 were increased in patients with ACLF of any grade, relative to those with AD, and 38 comprised a distinctive blood metabolite fingerprint for ACLF. Among patients with ACLF, the intensity of the fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that the fingerprint reflected not only decreased kidney excretion but also altered cell metabolism. The higher the ACLF-associated fingerprint intensity, the higher the plasma levels of inflammatory markers, tumor necrosis factor α, soluble CD206, and soluble CD163. ACLF was characterized by intense proteolysis and lipolysis; amino acid catabolism; extra-mitochondrial glucose metabolism through glycolysis, pentose phosphate, and D-glucuronate pathways; depressed mitochondrial ATP-producing fatty acid ß-oxidation; and extra-mitochondrial amino acid metabolism giving rise to metabotoxins. CONCLUSIONS: In ACLF, intense systemic inflammation is associated with blood metabolite accumulation and profound alterations in major metabolic pathways, in particular inhibition of mitochondrial energy production, which may contribute to the development of organ failures. LAY SUMMARY: Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. We identified a 38-metabolite blood fingerprint specific for ACLF that revealed mitochondrial dysfunction in peripheral organs. This may contribute to organ failures.
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Insuficiencia Hepática Crónica Agudizada/sangre , Insuficiencia Hepática Crónica Agudizada/complicaciones , Glucólisis , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Metaboloma , Metabolómica/métodos , Mitocondrias/metabolismo , Adulto , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la EnfermedadRESUMEN
We report the first pretargeting in vivo study using the Strain-Promoted Sydnone-Alkyne Cycloaadition (SPSAC) reaction. The injection of a fluorine-18 labeled cyclooctyne three days after cetuximab bearing chlorosydnone moieties allowed a significant detection of the tumor by PET imaging suggesting an efficient click reaction inside the tumoral site. With a kinetic constant superior to 300 M-1 s-1, the SPSAC reaction might be an interesting tool, in addition to tetrazine-cyclooctene ligation, for in vivo chemistry.
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Alquinos/química , Química Clic/métodos , Radioisótopos de Flúor/química , Tomografía de Emisión de Positrones/métodos , Animales , Ciclización , Xenoinjertos , Humanos , RatonesRESUMEN
Early nutrition impacts preterm infant early growth rate and brain development but can have long lasting effects as well. Although human milk is the gold standard for feeding new born full-term and preterm infants, little is known about the effects of its bioactive compounds on breastfed preterm infants' growth outcomes. This study aims to determine whether breast milk metabolome, glycome, lipidome, and free-amino acids profiles analyzed by liquid chromatography-mass spectrometry had any impact on the early growth pattern of preterm infants. The study population consisted of the top tercile-Z score change in their weight between birth and hospital discharge ("faster grow", n = 11) and lowest tercile ("slower grow", n = 15) from a cohort of 138 premature infants (27â»34 weeks gestation). This holistic approach combined with stringent clustering or classification statistical methods aims to discriminate groups of milks phenotype and identify specific metabolites associated with early growth of preterm infants. Their predictive reliability as biomarkers of infant growth was assessed using multiple linear regression and taking into account confounding clinical factors. Breast-milk associated with fast growth contained more branched-chain and insulino-trophic amino acid, lacto-N-fucopentaose, choline, and hydroxybutyrate, pointing to the critical role of energy utilization, protein synthesis, oxidative status, and gut epithelial cell maturity in prematurity.
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Desarrollo Infantil , Recien Nacido Prematuro/crecimiento & desarrollo , Leche Humana/química , Leche Humana/metabolismo , Adulto , Metabolismo de los Hidratos de Carbono , Estudios de Cohortes , Femenino , Humanos , Lactante , Fenómenos Fisiológicos Nutricionales del Lactante , Metabolismo de los Lípidos , Masculino , MetabolómicaRESUMEN
A general approach for the efficient hydrogen-isotope exchange of nucleobase derivatives is described. Catalyzed by ruthenium nanoparticles, using mild reaction conditions, and involving either D2 or T2 as isotopic sources, this reaction possesses a wide substrate scope and a high solvent tolerability. This novel method facilitates the access to essential diagnostic tools in drug discovery and development: tritiated pharmaceuticals with high specific activities and deuterated oligonucleotides suitable for use as internal standards during LC-MS quantification.
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Medición de Intercambio de Deuterio , Deuterio/química , Hidrógeno/química , Oligonucleótidos/química , Preparaciones Farmacéuticas/química , Cromatografía Liquida , Espectrometría de MasasRESUMEN
Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N- and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG, and SLC35A3-CDG, are described as illustrative examples.
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Trastornos Congénitos de Glicosilación , Electroforesis/métodos , Espectrometría de Masas/métodos , Análisis de Secuencia de ADN/métodos , Adolescente , Niño , Preescolar , Trastornos Congénitos de Glicosilación/sangre , Trastornos Congénitos de Glicosilación/diagnóstico , Femenino , Glicómica , Glicoproteínas/sangre , Glicoproteínas/química , Humanos , Lactante , Masculino , Polisacáridos/análisis , Polisacáridos/químicaRESUMEN
Congenital disorders of glycosylation (CDG) linked to defects in Golgi apparatus homeostasis constitute an increasing part of these rare inherited diseases. Among them, COG-CDG, ATP6V0A2-CDG, TMEM199-CDG and CCDC115-CDG have been shown to disturb Golgi vesicular trafficking and/or lumen pH acidification. Here, we report 3 new unrelated cases of CCDC115-CDG with emphasis on diagnosis difficulties related to strong phenotypic similarities with mitochondriopathies, Niemann-Pick disease C and Wilson Disease. Indeed, while two individuals clinically presented with early and severe liver fibrosis and cirrhosis associated with neurological symptoms, the other one "only" showed isolated and late severe liver involvement. Biological results were similar to previously described patients, including hypercholesterolemia, elevated alkaline phosphatases and defects in copper metabolism. CDG screening and glycosylation study finally led to the molecular diagnosis of CCDC115-CDG. Besides pointing to the importance of CDG screening in patients with unexplained and severe liver disease, these reports expand the clinical and molecular phenotypes of CCDC115-CDG. The hepatic involvement is particularly addressed. Furthermore, hypothesis concerning the pathogenesis of the liver disease and of major biological abnormalities are proposed.
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Trastornos Congénitos de Glicosilación/complicaciones , Aparato de Golgi/genética , Hepatopatías/etiología , Mutación , Proteínas del Tejido Nervioso/genética , Adulto , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/patología , Femenino , Glicosilación , Aparato de Golgi/metabolismo , Aparato de Golgi/patología , Humanos , Recién Nacido , Hepatopatías/patología , Masculino , Pronóstico , Adulto JovenRESUMEN
Human milk oligosaccharides (HMOs) represent the third most abundant components of milk after lactose and lipids. HMOs are indigestible by the suckling infant but can act as prebiotics and have significant biological functions regarding the organism defense against pathogens (such as bacteria or viruses) by preventing interactions with their receptors. Although constituted of only five distinct monosaccharide building blocks, HMOs are highly structurally diverse compounds with many co-existing structural isomers. Here we report the development and comparison of two distinct glycomic platforms based on liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for analyzing HMOs. We have implemented and thoroughly compared the LC-MS of permethylated and native HMOs on reversed phase (RP) and porous graphitic carbon (PGC) columns for their ability to resolve the natural heterogeneity of milk oligosaccharides at the highest sensitivity. Our data essentially underlines the usefulness of analyzing HMOs as permethylated derivatives especially for getting more precise structural information at high sensitivity. For instance, permethylation annihilates gas-phase fucose migration during MS/MS experiments, thus facilitating spectra interpretation and giving access to relevant information regarding oligosaccharide branching and isomer distinction. At the opposite, LC-MS profiling of native HMOs (using PGC) in milk performed best in terms of detected species, while also being much faster in terms of sample preparation. Although less efficient than PGC chromatography, RPLC proved successful for separating pairs of permethylated isomeric HMOs. A key advantage of RP over PGC liquid chromatography is that retention times can be correlated to molecular weights, which can greatly facilitate further HMO identification using retention time prediction. Altogether these data lead us to think that LC-MS analysis of native HMOs (using PGC) can be used as first-line profiling approach while permethylation can be performed afterwards for facilitating structural characterization.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Leche Humana/química , Oligosacáridos , Humanos , Isomerismo , Oligosacáridos/análisis , Oligosacáridos/química , Oligosacáridos/aislamiento & purificaciónRESUMEN
RATIONALE: Glycosylation is one of the most complex types of post-translational modifications of proteins. The alteration of glycans bound to proteins from cerebrospinal fluid (CSF) in relation to disorders of the central nervous system is a highly relevant subject, but only few studies have focused on the glycosylation of CSF proteins. METHODS: Reproducible profiles of CSF N-glycans were first obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after permethylation. Tryptic glycopeptides from CSF proteins were also enriched by hydrophilic interaction, and the resulting extracts divided into two equal aliquots. A first aliquot was enzymatically deglycosylated and analyzed by nano-liquid chromatography/tandem mass spectrometry while the second one, containing intact enriched glycopeptides, was directly analyzed. Site-specific data were obtained by combining the data from these three experiments. RESULTS: We describe the development of a versatile approach for obtaining site-specific information on the N-glycosylation of CSF glycoproteins. Under these conditions, 124 N-glycopeptides representing 55 N-glycosites from 36 glycoproteins were tentatively identified. Special emphasis was placed on the analysis of glycoproteins/glycopeptides bearing 'brain-type' N-glycans, representing potential biologically relevant structures in the field of neurodegenerative disorders. Using our workflow, only a few proteins were shown to carry such particular glycan motifs. CONCLUSIONS: We developed an approach combining N-glycomics and N-glycoproteomics and underline its usefulness to study the site-specific glycosylation of major human CSF proteins. The final rather long-term objective is to combine these data with those from other omics approaches to delve deeper into the understanding of particular neurological disorders.