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Introduction: The human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease., Objectives: Here, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics., Methods: Comparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data., Results: Glycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors., Conclusion: Our work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Contributing authors Pierre-Olivier Schmit, Stuart Pengelley, and Kristina Marx are employees of Bruker Daltonics which is the manufacturer of some of the hardware and software that were used in this work., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-β-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond., (© 2024. The Author(s).)

Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein ( ISPD ), fukutin-related protein ( FKRP ), and fukutin ( FKTN ) genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous FKRP mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation ( p  < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Although hereditary fructose intolerance (HFI) is an inborn error of fructose metabolism that classically presents at infancy, the diagnosis is often missed or delayed. In this study, we aimed to develop tools to facilitate the diagnosis of HFI. The intake of fructose-containing food products, that is, fruit, fruit juice and sugar-sweetened beverages, was assessed by a 3-day food diary in adult HFI patients ( n  = 15) and age, sex, and BMI-matched controls ( n  = 15). Furthermore, glycosylation of transferrin was examined using high-resolution mass spectrometry and abnormally glycosylated transferrin was expressed as ratio of normal glycosylated transferrin. We found that the sensitivity and specificity of the 3-day food diary for the intake of at least one fructose-containing food product were both 100%. Both mono-glyco:diglyco transferrin and a-glyco+mono-glyco:di-glyco transferrin were greater in HFI patients and had a high-discriminatory power (area under the receiver operating characteristic curve: 0.97 and 0.94, respectively). In this well-characterized cohort of adult HFI patients, the 3-day food questionnaire and the glycosylation pattern of transferrin are valuable tools to facilitate the recognition and diagnosis of HFI in adult patients., Competing Interests: Bianca Panis, Lise E.F. Janssen, Dirk J. Lefeber, Nynke Simons and M. Estela Rubio‐Gozalbo declare that they have no conflict of interest. Martijn C.G.J. Brouwers received consultancy fees from Arrowhead and Editas Medicine., (© 2023 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By 13 C 6 -glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose.

Deleterious variants in N- acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: Npl R63C , carrying the human p.Arg63Cys variant, and Npl del116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N- acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in Npl R63C mice, suggesting a potential treatment for human patients.

Heterozygous variants in GBA1, encoding glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease (PD). Moreover, sporadic PD patients also have a substantial reduction of GCase activity. Genetic variants of SMPD1 are also overrepresented in PD cohorts, whereas a reduction of its encoded enzyme (acid sphingomyelinase or ASM) activity is linked to an earlier age of PD onset. Despite both converging on the ceramide pathway, how the combined deficiencies of both enzymes might interact to modulate PD has yet to be explored. Therefore, we created a double-knockout (DKO) zebrafish line for both gba1 (or gba) and smpd1 to test for an interaction in vivo, hypothesising an exacerbation of phenotypes in the DKO line compared to those for single mutants. Unexpectedly, DKO zebrafish maintained conventional swimming behaviour and had normalised neuronal gene expression signatures compared to those of single mutants. We further identified rescue of mitochondrial Complexes I and IV in DKO zebrafish. Despite having an unexpected rescue effect, our results confirm ASM as a modifier of GBA1 deficiency in vivo. Our study highlights the need for validating how genetic variants and enzymatic deficiencies may interact in vivo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Heart failure (HF) is a progressive chronic disease that remains a primary cause of death worldwide, affecting over 64 million patients. HF can be caused by cardiomyopathies and congenital cardiac defects with monogenic etiology. The number of genes and monogenic disorders linked to development of cardiac defects is constantly growing and includes inherited metabolic disorders (IMDs). Several IMDs affecting various metabolic pathways have been reported presenting cardiomyopathies and cardiac defects. Considering the pivotal role of sugar metabolism in cardiac tissue, including energy production, nucleic acid synthesis and glycosylation, it is not surprising that an increasing number of IMDs linked to carbohydrate metabolism are described with cardiac manifestations. In this systematic review, we offer a comprehensive overview of IMDs linked to carbohydrate metabolism presenting that present with cardiomyopathies, arrhythmogenic disorders and/or structural cardiac defects. We identified 58 IMDs presenting with cardiac complications: 3 defects of sugar/sugar-linked transporters (GLUT3, GLUT10, THTR1); 2 disorders of the pentose phosphate pathway (G6PDH, TALDO); 9 diseases of glycogen metabolism (GAA, GBE1, GDE, GYG1, GYS1, LAMP2, RBCK1, PRKAG2, G6PT1); 29 congenital disorders of glycosylation (ALG3, ALG6, ALG9, ALG12, ATP6V1A, ATP6V1E1, B3GALTL, B3GAT3, COG1, COG7, DOLK, DPM3, FKRP, FKTN, GMPPB, MPDU1, NPL, PGM1, PIGA, PIGL, PIGN, PIGO, PIGT, PIGV, PMM2, POMT1, POMT2, SRD5A3, XYLT2); 15 carbohydrate-linked lysosomal storage diseases (CTSA, GBA1, GLA, GLB1, HEXB, IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, ARSB, GUSB, ARSK). With this systematic review we aim to raise awareness about the cardiac presentations in carbohydrate-linked IMDs and draw attention to carbohydrate-linked pathogenic mechanisms that may underlie cardiac complications.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1 , according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13 C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila , CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance., Competing Interests: HS, BN, BU, BA, IM, PM, MK, SB, KA, RV, DL, MT, HB, VP No competing interests declared, (© 2023, Scott, Novikov et al.)

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood-CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes.

Purpose: A protein quantitative trait locus (pQTL) analysis recently revealed a strong association between hemopexin (HPX) levels and genetic variants at the complement factor H ( CFH ) locus. In this study, we aimed to determine HPX plasma levels in patients with age-related macular degeneration (AMD) and to compare them with those in controls. We also investigated whether genetic variants at the CFH locus are associated with HPX plasma levels., Methods: HPX levels were quantified in 200 advanced AMD cases and 200 controls using an enzyme-linked immunosorbent assay and compared between the two groups. Furthermore, HPX levels were analyzed per genotype group of three HPX-associated variants (rs61818956, rs10494745, and rs10801582) and four AMD-associated variants (rs794362 [proxy for rs187328863], rs570618, rs10922109, and rs61818924 [proxy for rs61818925]) at the CFH locus., Results: HPX levels were similar in the control group compared with the AMD group. The three variants at the CFH locus, which were previously associated with the HPX levels, showed no association with the HPX levels in our data set. No significant differences in HPX levels were detected between the different genotype groups of AMD-associated variants at the CFH locus., Conclusions: In this study, HPX levels were not associated with AMD or AMD-associated variants at the CFH locus. The finding of a previous pQTL study that variants at the CFH locus were associated with HPX levels was also not confirmed in this study., (Copyright © 2022 Molecular Vision.)

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Haas, de Jonge, Koenen, Joosten, Janssen, de Boer, Hendriks, Lefeber and Cambi.)

Ribitol-phosphate modification is crucial for the functional maturation of α-dystroglycan. Its dysfunction is associated with muscular dystrophy, cardiomyopathy, and central nervous system abnormalities; however, no effective treatments are currently available for diseases caused by ribitol-phosphate defects. In this study, we demonstrate that prodrug treatments can ameliorate muscular dystrophy caused by defects in isoprenoid synthase domain containing (ISPD), which encodes an enzyme that synthesizes CDP-ribitol, a donor substrate for ribitol-phosphate modification. We generated skeletal muscle-selective Ispd conditional knockout mice, leading to a pathogenic reduction in CDP-ribitol levels, abnormal glycosylation of α-dystroglycan, and severe muscular dystrophy. Adeno-associated virus-mediated gene replacement experiments suggested that the recovery of CDP-ribitol levels rescues the ISPD-deficient pathology. As a prodrug treatment strategy, we developed a series of membrane-permeable CDP-ribitol derivatives, among which tetraacetylated CDP-ribitol ameliorated the dystrophic pathology. In addition, the prodrug successfully rescued abnormal α-dystroglycan glycosylation in patient fibroblasts. Consequently, our findings provide proof-of-concept for supplementation therapy with CDP-ribitol and could accelerate the development of therapeutic agents for muscular dystrophy and other diseases caused by glycosylation defects., (© 2022. The Author(s).)

Synthetic sugar analogs are widely applied in metabolic oligosaccharide engineering (MOE) and as novel drugs to interfere with glycoconjugate biosynthesis. However, mechanistic insights on their exact cellular metabolism over time are mostly lacking. We combined ion-pair ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry mass spectrometry using tributyl- and triethylamine buffers for sensitive analysis of sugar metabolites in cells and organisms and identified low abundant nucleotide sugars, such as UDP-arabinose in human cell lines and CMP-sialic acid (CMP-NeuNAc) in Drosophila. Furthermore, MOE revealed that propargyloxycarbonyl (Poc)-labeled ManNPoc was metabolized to both CMP-NeuNPoc and UDP-GlcNPoc. Finally, time-course analysis of the effect of antitumor compound 3Fax-NeuNAc by incubation of B16-F10 melanoma cells with N-acetyl-D-[UL-13C6]glucosamine revealed full depletion of endogenous ManNAc 6-phosphate and CMP-NeuNAc within 24 h. Thus, dynamic tracing of sugar metabolic pathways provides a general approach to reveal time-dependent insights into the metabolism of synthetic sugars, which is important for the rational design of analogs with optimized effects., (© The Author(s) 2021. Published by Oxford University Press.)

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background & Aims: Recently, novel inborn errors of metabolism were identified because of mutations in V-ATPase assembly factors TMEM199 and CCDC115. Patients are characterized by generalized protein glycosylation defects, hypercholesterolemia, and fatty liver disease. Here, we set out to characterize the lipid and fatty liver phenotype in human plasma, cell models, and a mouse model., Methods and Results: Patients with TMEM199 and CCDC115 mutations displayed hyperlipidemia, characterized by increased levels of lipoproteins in the very low density lipoprotein range. HepG2 hepatoma cells, in which the expression of TMEM199 and CCDC115 was silenced, and induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells from patients with TMEM199 mutations showed markedly increased secretion of apolipoprotein B (apoB) compared with controls. A mouse model for TMEM199 deficiency with a CRISPR/Cas9-mediated knock-in of the human A7E mutation had marked hepatic steatosis on chow diet. Plasma N-glycans were hypogalactosylated, consistent with the patient phenotype, but no clear plasma lipid abnormalities were observed in the mouse model. In the siTMEM199 and siCCDC115 HepG2 hepatocyte models, increased numbers and size of lipid droplets were observed, including abnormally large lipid droplets, which colocalized with lysosomes. Excessive de novo lipogenesis, failing oxidative capacity, and elevated lipid uptake were not observed. Further investigation of lysosomal function revealed impaired acidification combined with impaired autophagic capacity., Conclusions: Our data suggest that the hypercholesterolemia in TMEM199 and CCDC115 deficiency is due to increased secretion of apoB-containing particles. This may in turn be secondary to the hepatic steatosis observed in these patients as well as in the mouse model. Mechanistically, we observed impaired lysosomal function characterized by reduced acidification, autophagy, and increased lysosomal lipid accumulation. These findings could explain the hepatic steatosis seen in patients and highlight the importance of lipophagy in fatty liver disease. Because this pathway remains understudied and its regulation is largely untargeted, further exploration of this pathway may offer novel strategies for therapeutic interventions to reduce lipotoxicity in fatty liver disease., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The sugar fucose is expressed on mammalian cell membranes as part of glycoconjugates and mediates essential physiological processes. The aberrant expression of fucosylated glycans has been linked to pathologies such as cancer, inflammation, infection, and genetic disorders. Tools to modulate fucose expression on living cells are needed to elucidate the biological role of fucose sugars and the development of potential therapeutics. Herein, we report a class of fucosylation inhibitors directly targeting de novo GDP-fucose biosynthesis via competitive GMDS inhibition. We demonstrate that cell permeable fluorinated rhamnose 1-phosphate derivatives (Fucotrim I & II) are metabolic prodrugs that are metabolized to their respective GDP-mannose derivatives and efficiently inhibit cellular fucosylation., (© 2021. The Author(s).)

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae serotype 19A prevalence has increased after the implementation of the PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period from 2004 to 2016. The whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule ( cps ) loci compared to examine their diversity and determine the impact on the production of capsular polysaccharide (CPS) sugar precursors and CPS shedding. We discovered 79 types with a unique cps locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway and in the wzg gene, which is of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single-nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, significant differences in multiple nucleotide sugar levels were observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB , rmlD , and wzg in serotype 19A in the Netherlands, for which we have not discovered an associated phenotype.

EDEM3 encodes a protein that converts Man 8 GlcNAc 2 isomer B to Man 7-5 GlcNAc 2 . It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man 8 GlcNAc 2 isomer B to Man 7 GlcNAc 2 , consistent with loss of EDEM3 enzymatic activity. In human cells, Man 5 GlcNAc 2 to Man 4 GlcNAc 2 conversion is also diminished with an increase of Glc 1 Man 5 GlcNAc 2 . Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG., (Copyright © 2021 American Society of Human Genetics. All rights reserved.)

Background: In NANS deficiency, biallelic mutations in the N -acetylneuraminic acid synthase ( NANS ) gene impair the endogenous synthesis of sialic acid ( N -acetylneuraminic acid) leading to accumulation of the precursor, N -acetyl mannosamine (ManNAc), and to a multisystemic disorder with intellectual disability. The aim of this study was to determine whether sialic acid supplementation might be a therapeutic avenue for NANS-deficient patients., Methods: Four adults and two children with NANS deficiency and four adult controls received oral NeuNAc acid (150 mg/kg/d) over three days. Total NeuNAc, free NeuNAc and ManNAc were analyzed in plasma and urine at different time points., Results: Upon NeuNAc administration, plasma free NeuNAc increased within hours ( P  < 0.001) in control and in NANS-deficient individuals. Total and free NeuNAc concentrations also increased in the urine as soon as 6 h after beginning of oral administration in both groups. NeuNAc did not affect plasma and urinary ManNAc, that remained higher in NANS deficient subjects than in controls (day 1-3; all P  < 0.01). Oral NeuNAc was well tolerated with no significant side effects., Discussion: Orally administered free NeuNAc was rapidly absorbed but also rapidly excreted in the urine. It did not change ManNAc levels in either patients or controls, indicating that it may not achieve enough feedback inhibition to reduce ManNAc accumulation in NANS-deficient subjects. Within the limitations of this study these results do not support a potential for oral free NeuNAc in the treatment of NANS deficiency but they provide a basis for further therapeutic approaches in this condition., Competing Interests: The authors declare no potential conflict of interest., (© 2021 The Authors.)

Background: NANS-CDG is a recently described congenital disorder of glycosylation caused by biallelic genetic variants in NANS , encoding an essential enzyme in de novo sialic acid synthesis. Sialic acid at the end of glycoconjugates plays a key role in biological processes such as brain and skeletal development. Here, we present an observational cohort study to delineate the genetic, biochemical, and clinical phenotype and assess possible correlations. Methods: Medical and laboratory records were reviewed with retrospective extraction and analysis of genetic, biochemical, and clinical data (2016-2020). Results: Nine NANS-CDG patients (nine families, six countries) referred to the Radboudumc CDG Center of Expertise were included. Phenotyping confirmed the hallmark features including intellectual developmental disorder (IDD) ( n = 9/9; 100%), facial dysmorphisms ( n = 9/9; 100%), neurologic impairment ( n = 9/9; 100%), short stature ( n = 8/9; 89%), skeletal dysplasia ( n = 8/9; 89%), and short limbs ( n = 8/9; 89%). Newly identified features include ophthalmological abnormalities ( n = 6/9; 67%), an abnormal septum pellucidum ( n = 6/9; 67%), (progressive) cerebral atrophy and ventricular dilatation ( n = 5/9; 56%), gastrointestinal dysfunction ( n = 5/9; 56%), thrombocytopenia ( n = 5/9; 56%), and hypo-low-density lipoprotein cholesterol ( n = 4/9; 44%). Biochemically, elevated urinary excretion of N -acetylmannosamine (ManNAc) is pathognomonic, the concentrations of which show a significant correlation with clinical severity. Genotypically, eight novel NANS variants were identified. Three severely affected patients harbored identical compound heterozygous pathogenic variants, one of whom was initiated on experimental prenatal and postnatal treatment with oral sialic acid. This patient showed markedly better psychomotor development than the other two genotypically identical males. Conclusions: ManNAc screening should be considered in all patients with IDD, short stature with short limbs, facial dysmorphisms, neurologic impairment, and an abnormal septum pellucidum +/- congenital and neurodegenerative lesions on brain imaging, to establish a precise diagnosis and contribute to prognostication. Personalized management includes accurate genetic counseling and access to proper supports and tailored care for gastrointestinal symptoms, thrombocytopenia, and epilepsy, as well as rehabilitation services for cognitive and physical impairments. Motivated by the short-term positive effects of experimental treatment with oral sialic, we have initiated this intervention with protocolized follow-up of neurologic, systemic, and growth outcomes in four patients. Research is ongoing to unravel pathophysiology and identify novel therapeutic targets., Competing Interests: BJ and KS were employed by the company Decode Genetics/Amgen, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 den Hollander, Rasing, Post, Klein, Oud, Brands, de Boer, Engelke, van Essen, Fuchs, Haaxma, Jensson, Kluijtmans, Lengyel, Lichtenbelt, Østergaard, Peters, Salvarinova, Simon, Stefansson, Thorarensen, Ulmen, Coene, Willemsen, Lefeber and Karnebeek.)

Behçet's disease (BD) is an inflammatory disease mainly affecting men along the ancient Silk Route. In the present study we describe a Dutch family suffering from BD-like disease with extreme pathergic responses, but without systemic inflammation. Genetic assessment revealed a combination of the human leukocyte antigen (HLA)-B*51 risk-allele together with a rare heterozygous variant in the CSF2 gene (c.130A>C, p.N44H) encoding for granulocyte-macrophage colony-stimulating factor (GM-CSF) found by whole exome sequencing. We utilized an over-expression vector system in a human hepatocyte cell line to produce the aberrant variant of GM-CSF. Biological activity of the protein was measured by signal transducer and activator of transcription 5 (STAT-5) phosphorylation, a downstream molecule of the GM-CSF receptor, in wild-type peripheral mononuclear cells (PBMCs) using flow cytometry. Increased STAT-5 phosphorylation was observed in response to mutated GM-CSF when compared to the wild-type or recombinant protein. CSF2 p.N44H results in disruption of one of the protein's two N-glycosylation sites. Enzymatically deglycosylated wild-type GM-CSF also enhanced STAT-5 phosphorylation. The patient responded well to anti-tumor necrosis factor (TNF)-α treatment, which may be linked to the capacity of TNF-α to induce GM-CSF in phorbol 12-myristate 13-acetate (PMA)-treated PBMCs, while GM-CSF itself only induced dose-dependent interleukin (IL)-1Ra production. The identified CSF2 pathway could provide novel insights into the pathergic response of BD-like disease and offer new opportunities for personalized treatment., (© 2021 British Society for Immunology.)

Purpose: To evaluate the plasma levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitors of metalloproteinase 3 (TIMP3) in neovascular age-related macular degeneration (nAMD) patients compared to controls, and to explore the potential effect of AMD-associated genetic variants on MMP9 and TIMP3 protein levels., Methods: nAMD and control patients were selected from the European Genetic Database (EUGENDA) based on different genotypes of rs142450006 near MMP9 and rs5754227 near TIMP3. Plasma total MMP9, active MMP9 and TIMP3 levels were measured using the enzyme linked immunosorbent assay (ELISA) and compared between nAMD patients and controls, as well as between different genotype groups., Results: nAMD patients had significantly higher total MMP9 levels compared to controls (median 46.58 versus 26.90 ng/ml; p = 0.0004). In addition, the median MMP9 level in the homozygous genotype group for the AMD-risk allele (44.23 ng/ml) was significantly higher than the median for the heterozygous genotype group (26.90 ng/ml; p = 0.0082) and the median for the homozygous group for the non-risk allele (28.55 ng/ml; p = 0.0355). No differences were detected for the active MMP9. TIMP3 levels did not significantly differ between the AMD and control groups, nor between the different genotype groups for rs5754227., Conclusions: The results of our MMP9 analyses indicate that nAMD patients have on average higher systemic MMP9 levels than control individuals, and that this is partly driven by the rs142450006 variant near MMP9 . This finding might be an interesting starting point for further exploration of MMP9 as a therapeutic target in nAMD, particularly among individuals carrying the risk-conferring allele rs142450006., (Copyright © 2021 Molecular Vision.)

Protein N -glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N -glycan biosynthesis. Here, we present insights into the disrupted N -glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N -glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.

Variants in SLC35C1 underlie leucocyte adhesion deficiency (LADII) or congenital disorder of glycosylation type 2c (CDGIIc), an autosomal recessive disorder of fucosylation. This immunodeficiency syndrome is generally characterized by severe recurrent infections, Bombay blood group, reduced growth and intellectual disability (ID). Features are all caused by an inability to generate key fucosylated molecules due to a defective transport of GDP-fucose into the Golgi. Here we report the use of exome sequencing to identify biallelic variants in SLC35C1 (c.501&#95;503delCTT, p.(Phe168del) and c.891T > G, p.(Asn297Lys)) in an individual with short stature and ID. Retrospective clinical examination based on the genetic findings revealed increased otitis media as the only immunological feature present in this child. Biochemical analysis of patient serum identified a clear but mild decrease in protein fucosylation. Modelling all described missense mutations on a SLC35C1 protein model showed pathogenic substitutions localise to close to the dimer interface, providing insight into the possible pathophysiology of non-synonymous causative variants identified in patients. Our evidence confirms this is the second family presenting with only a subset of features and broadens the clinical presentation of this syndrome. Of note, both families segregated a common allele (p.Phe168del), suggesting there could be an associated genotype-phenotype relationship for specific variants. Based on two out of 14 reported families not presenting with the characteristic features of SLC35C1-CDG, we suggest there is clinical utility in considering this gene in patients with short stature and ID.

Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glycan structures. Of utmost importance for this process is the intraorganellar environment of the Golgi. Each Golgi compartment has a distinct pH, which is maintained by the vacuolar H + -ATPase (V-ATPase). Moreover, tethering factors such as Golgins and the conserved oligomeric Golgi (COG) complex, in concert with coatomer (COPI) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion, efficiently deliver glycosylation enzymes to the right Golgi compartment. Together, these factors maintain intra-Golgi trafficking of proteins involved in glycosylation and thereby enable proper glycosylation. However, pathogenic mutations in these factors can cause defective glycosylation and lead to diseases with a wide variety of symptoms such as liver dysfunction and skin and bone disorders. Collectively, this group of disorders is known as congenital disorders of glycosylation (CDG). Recent technological advances have enabled the robust identification of novel CDGs related to membrane trafficking components. In this review, we highlight differences and similarities between membrane trafficking-related CDGs., Competing Interests: The authors declare no conflict of interest.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with β-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.

In 2016, 11 male patients were reported with immunodeficiency and hepatic, gastric and (in some) neurological disease due to X-linked ATP6AP1 deficiency (ATP6AP1-CDG). In 2018, three other patients were reported with additional features: connective tissue abnormalities, sensorineural hearing loss, hyperopia, glomerular and tubular dysfunction, exocrine pancreatic insufficiency and altered amino acid and lipid metabolism. We here present a follow-up of three reported siblings showing progression of deafness to total hearing loss, progressive loss of hair up to alopecia, chestnut skin and, at last follow-up, in some of them proteinuria. Three female carriers showed a normal serum transferrin isoelectrofocusing but in two of them there was a persistent proteinuria., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

NGLY1 encodes the enzyme N-glycanase that is involved in the degradation of glycoproteins as part of the endoplasmatic reticulum-associated degradation pathway. Variants in this gene have been described to cause a multisystem disease characterized by neuromotor impairment, neuropathy, intellectual disability, and dysmorphic features. Here, we describe four patients with pathogenic variants in NGLY1. As the clinical features and laboratory results of the patients suggested a multisystem mitochondrial disease, a muscle biopsy had been performed. Biochemical analysis in muscle showed a strongly reduced ATP production rate in all patients, while individual OXPHOS enzyme activities varied from normal to reduced. No causative variants in any mitochondrial disease genes were found using mtDNA analysis and whole exome sequencing. In all four patients, variants in NGLY1 were identified, including two unreported variants (c.849T>G (p.(Cys283Trp)) and c.1067A>G (p.(Glu356Gly)). Western blot analysis of N-glycanase in muscle and fibroblasts showed a complete absence of N-glycanase. One patient showed a decreased basal and maximal oxygen consumption rates in fibroblasts. Mitochondrial morphofunction fibroblast analysis showed patient specific differences when compared to control cell lines. In conclusion, variants in NGLY1 affect mitochondrial energy metabolism which in turn might contribute to the clinical disease course., (© 2020 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

N-glycosylation of membrane receptors is important for a wide variety of cellular processes. In the immune system, loss or alteration of receptor glycosylation can affect pathogen recognition, cell-cell interaction, and activation as well as migration. This is not only due to aberrant folding of the receptor, but also to altered lateral mobility or aggregation capacity. Despite increasing evidence of their biological relevance, glycosylation-dependent mechanisms of receptor regulation are hard to dissect at the molecular level. This is due to the intrinsic complexity of the glycosylation process and high diversity of glycan structures combined with the technical limitations of the current experimental tools. It is still challenging to precisely determine the localization and site-occupancy of glycosylation sites, glycan micro- and macro-heterogeneity at the individual receptor level as well as the biological function and specific interactome of receptor glycoforms. In addition, the tools available to manipulate N-glycans of a specific receptor are limited. Significant progress has however been made thanks to innovative approaches such as glycoproteomics, metabolic engineering, or chemoenzymatic labeling. By discussing examples of immune receptors involved in pathogen recognition, migration, antigen presentation, and cell signaling, this Mini Review will focus on the biological importance of N-glycosylation for receptor functions and highlight the technical challenges for examination and manipulation of receptor N-glycans., (Copyright © 2020 de Haas, Hendriks, Lefeber and Cambi.)

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients' primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.

Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in PGM3 in diagnostic, predictive and prenatal settings, and for risk assessment in relatives.

Background: Many muscular dystrophies currently remain untreatable. Recently, dietary ribitol has been suggested as a treatment for cytidine diphosphate (CDP)-l-ribitol pyrophosphorylase A (CRPPA, ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) myopathy, by raising CDP-ribitol concentrations. Thus, to facilitate fast diagnosis, treatment development, and treatment monitoring, sensitive detection of CDP-ribitol is required., Methods: An LC-MS method was optimized for CDP-ribitol in human and mice cells and tissues., Results: CDP-ribitol, the product of CRPPA, was detected in all major human and mouse tissues. Moreover, CDP-ribitol concentrations were reduced in fibroblasts and skeletal muscle biopsies from patients with CRPPA myopathy, showing that CDP-ribitol could serve as a diagnostic marker to identify patients with CRPPA with severe Walker-Warburg syndrome and mild limb-girdle muscular dystrophy (LGMD) phenotypes. A screen for potentially therapeutic monosaccharides revealed that ribose, in addition to ribitol, restored CDP-ribitol concentrations and the associated O-glycosylation defect of α-dystroglycan. As the effect occurred in a mutation-dependent manner, we established a CDP-ribitol blood test to facilitate diagnosis and predict individualized treatment response. Ex vivo incubation of blood cells with ribose or ribitol restored CDP-ribitol concentrations in a patient with CRPPA LGMD., Conclusions: Sensitive detection of CDP-ribitol with LC-MS allows fast diagnosis of patients with severe and mild CRPPA myopathy. Ribose offers a readily testable dietary therapy for CRPPA myopathy, with possible applicability for patients with FKRP and FKTN myopathy. Evaluation of CDP-ribitol in blood is a promising tool for the evaluation and monitoring of dietary therapies for CRPPA myopathy in a patient-specific manner., (© 2019 American Association for Clinical Chemistry.)

Congenital disorders of glycosylation type I (CDG-I) are inborn errors of metabolism, generally characterized by multisystem clinical manifestations, including developmental delay, hepatopathy, hypotonia, and skin, skeletal, and neurological abnormalities. Among others, dolichol-phosphate-mannose (DPM) is the mannose donor for N-glycosylation as well as O-mannosylation. DOLK-CDG, DPM1-CDG, DPM2-CDG, and DPM3-CDG are defects in the DPM synthesis showing both CDG-I abnormalities and reduced O-mannosylation of alpha-dystroglycan (αDG), which leads to muscular dystrophy-dystroglycanopathy. Mannose-phosphate-dolichol utilization defect 1 (MPDU1) plays a role in the utilization of DPM. Here, we report two MPDU1-CDG patients without skin involvement, but with massive dilatation of the biliary duct system and dystroglycanopathy characteristics including hypotonia, elevated creatine kinase, dilated cardiomyopathy, buphthalmos, and congenital glaucoma. Biochemical analyses revealed elevated disialotransferrin in serum, and analyses in fibroblasts showed shortened lipid linked oligosaccharides and DPM, and reduced O-mannosylation of αDG. Thus, MPDU1-CDG can be added to the list of disorders with overlapping biochemical and clinical abnormalities of CDG-I and dystroglycanopathy., Synopsis: Mannose-phosphate-dolichol utilization defect 1 patients can have overlapping biochemical and clinical abnormalities of congenital disorders of glycosylation type I and dystroglycanopathy., Competing Interests: The authors declare that they have no conflict of interest., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation., Methods: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied., Results: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator., Conclusions: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

The congenital disorders of glycosylation (CDG) are defects in glycoprotein and glycolipid glycan synthesis and attachment. They affect multiple organ/systems, but non-specific symptoms render the diagnosis of the different CDG very challenging. Phosphomannomutase 2 (PMM2)-CDG is the most common CDG, but advances in genetic analysis have shown others to occur more commonly than previously thought. The present work reports the clinical and mutational spectrum of 25 non-PMM2 CDG patients. The most common clinical symptoms were hypotonia (80%), motor or psychomotor disability (80%) and craniofacial dysmorphism (76%). Based on their serum transferrin isoform profile, 18 were classified as CDG-I and 7 as CDG-II. Pathogenic variations were found in 16 genes (ALG1, ALG6, ATP6V0A2, B4GALT1, CCDC115, COG7, DOLK, DPAGT1, DPM1, GFPT1, MPI, PGM1, RFT1, SLC35A2, SRD5A3, and SSR4). Overall, 27 variants were identified, 12 of which are novel. The results highlight the importance of combining genetic and biochemical analyses for the early diagnosis of this heterogeneous group of disorders., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)