Congenital Disorders Of Glycosylation Type Research Articles

81 Liver Transplant as Definitive Therapy for Immune Defects Associated with Congenital Disorder of Glycosylation Type 1 B

81 Liver Transplant as Definitive Therapy for Immune Defects Associated with Congenital Disorder of Glycosylation Type 1 B

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17–34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8–12%), with hair and dental abnormalities present in only 4–7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Genetic counseling for congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDGs) are a genetically and clinically diverse group of disorders that arise as a result of defects within glycosylation synthetic pathways. CDGs are caused by pathogenic variants in many different genes in the glycosylation network. With over 160 different CDG types currently identified and a vast range of severity and presentations existing within and across those types, the road to a CDG diagnosis is often lengthy and complicated. The perils of this arduous CDG diagnostic odyssey are fraught with various genetic counseling uncertainties: (1) confusion about family planning, (2) queries about inheritance, (3) managing treatment, and (4) dealing with the uncertainty of rare diseases. Thus, the role of the genetic counselor is paramount in helping affected individuals and their families navigate these genetic counseling complexities. Case examples of common genetic counseling difficulties for CDGs are outlined, providing clinical applications of what CDG presentations, diagnostic processes, and common difficulties look like. Information on the nomenclature, incidence, prevalence, diagnostic testing, treatment, and management of CDGs are also discussed to provide a comprehensive summary of CDGs for genetic counselors, and subsequently to affected individuals and their families.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Clinical and biochemical footprints of inherited metabolic diseases. XII. Immunological defects.

Immunological problems are increasingly acknowledged manifestations in many inherited metabolic diseases (IMDs), ranging from exaggerated inflammation, autoimmunity and abnormal cell counts to recurrent microbial infections. A subgroup of IMDs, the congenital disorders of glycosylation (CDG), includes CDG types that are even classified as primary immunodeficiencies. Here, we reviewed the list of metabolic disorders reported to be associated with various immunological defects and identified 171 IMDs accompanied by immunological manifestations. Most IMDs are accompanied by immune dysfunctions of which immunodeficiency and infections, innate immune defects, and autoimmunity are the most common abnormalities reported in 144/171 (84%), 44/171 (26%) and 33/171 (19%) of IMDs with immune system involvement, respectively, followed by autoinflammation 17/171 (10%). This article belongs to a series aiming at creating and maintaining a comprehensive list of clinical and metabolic differential diagnoses according to organ system involvement.

Epidemiology and Prevalence of Patients with Congenital Disorders of Glycosylation in Malaysia

Congenital disorders of glycosylation (CDG) are rarely, inherited metabolic diseases in which the glycosylation process is severely impaired. The prevalence has not been well determined and the aims of this study were to describe the epidemiology and calculate the birth prevalence of CDG in the Malaysian population. From 2018 to 2022, 548 patients were suspected with CDG. Sample were collected and analysed using isoelectric focusing (IEF) and capillary zone electrophoresis (CZE) methods. Qualitative measurement of sialo pattern was determined to categorised CDG Type I, II and abnormal transferrin pattern. Transferrin level was quantitated using automated BN ProSpec® System as well as demographic and clinical features were documented. The distribution of CDG was 0.4% and 2.9% was diagnosed as abnormal transferrin types. The prevalence of CDG among Malaysian was 0.22 per 100,000 live births and the combined prevalence of abnormal pattern was 0.85 per 100,000 live births. Positive cases were found in infants of Malay ethnicity. Overall, the prevalence of CDG in Malaysia was low and may be underestimated yet consistent with other reported in other countries.

N-Glycoprofiling of SLC35A2-CDG: Patient with a Novel Hemizygous Variant

Congenital disorders of glycosylation (CDG) are a group of rare inherited metabolic disorders caused by a defect in the process of protein glycosylation. In this work, we present a comprehensive glycoprofile analysis of a male patient with a novel missense variant in the SLC35A2 gene, coding a galactose transporter that translocates UDP-galactose from the cytosol to the lumen of the endoplasmic reticulum and Golgi apparatus. Isoelectric focusing of serum transferrin, which resulted in a CDG type II pattern, was followed by structural analysis of transferrin and serum N-glycans, as well as the analysis of apolipoprotein CIII O-glycans by mass spectrometry. An abnormal serum N-glycoprofile with significantly increased levels of agalactosylated (Hex3HexNAc4-5 and Hex3HexNAc5Fuc1) and monogalactosylated (Hex4HexNAc4 ± NeuAc1) N-glycans was observed. Additionally, whole exome sequencing and Sanger sequencing revealed de novo hemizygous c.461T > C (p.Leu154Pro) mutation in the SLC35A2 gene. Based on the combination of biochemical, analytical, and genomic approaches, the set of distinctive N-glycan biomarkers was characterized. Potentially, the set of identified aberrant N-glycans can be specific for other variants causing SLC35A2-CDG and can distinguish this disorder from the other CDGs or other defects in the galactose metabolism.

Neurological Consequences of Congenital Disorders of Glycosylation.

The chapter is devoted to neurological aspects of congenital disorders of glycosylation (CDG). At the beginning, the various types of CDG with neurological presentation of symptoms are summarized. Then, the occurrence of various neurological constellation of abnormalities (for example: epilepsy, brain anomalies on neuroimaging, ataxia, stroke-like episodes, autistic features) in different CDG types are discussed followed by data on possible biomarkers and limited treatment options.

Nutrition interventions in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a group of more than 160 inborn errors of metabolism affecting multiple pathways of protein and lipid glycosylation. Patients present with a wide range of symptoms and therapies are only available for very few subtypes. Specific nutritional treatment options for certain CDG types include oral supplementation of monosaccharide sugars, manganese, uridine, or pyridoxine. Additional management includes specific diets (i.e., complex carbohydrate or ketogenic diet), iron supplementation, and albumin infusions. We review the dietary management in CDG with a focus on two subgroups: N-linked glycosylation defects and GPI-anchor disorders.

Evaluation of Cell Models to Study Monocyte Functions in PMM2 Congenital Disorders of Glycosylation.

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.

Electrospray Ionization Mass Spectrometry of Transferrin: Use of Quadrupole Mass Analyzers for Congenital Disorders of Glycosylation.

Electrospray ionization (ESI) mass spectrometry of transferrin can be used to diagnose congenital disorders of glycosylation (CDG) by detecting abnormal N-glycosylation due to reduced site occupancy or processing failure. Time-of-flight mass spectrometers are widely used to separate 25–45 charged ions in the m/z 1,700–3,000 range, and a summed zero-charge mass distribution is generated despite the risk of improper deconvolution. In this study, the low m/z region of the multiply-charged ion mass spectrum enabled a robust analysis of CDG. A triple quadrupole mass spectrometer, the standard instrument for newborn screening for inborn errors of metabolism, permitted the identification of the key ions characteristic of different types of CDG affecting PMM2, ALG14, SLC35A1, SLC35A2, MAN1B1 and PGM1 in the m/z 1,970–2,000 region. Charge deconvolution was used as a complementary tool for validating the findings. It was necessary to set a cutoff level for the evaluation, since small peaks indicating glycosylation failure or reduced sialylation were observed, even in control subjects. This method and workflow facilitates the implementation of MS-based analyses and the screening of CDG in clinical laboratories.

Synergistic use of glycomics and single-molecule molecular inversion probes for identification of congenital disorders of glycosylation type-1.

Congenital disorders of glycosylation type 1 (CDG‐I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG‐I is a partial absence of complete N‐glycans on transferrin. However, recent findings of a diagnostic N‐tetrasaccharide for ALG1‐CDG and increased high‐mannose N‐glycans for a few other CDG suggested the potential of glycan structural analysis for CDG‐I gene discovery. We analyzed the relative abundance of total plasma N‐glycans by high resolution quadrupole time‐of‐flight mass spectrometry in a large cohort of 111 CDG‐I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single‐molecule molecular inversion probes (smMIPs) for sequencing of CDG‐I candidate genes on the basis of specific N‐glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N‐tetrasaccharide in ALG2‐CDG patients and a novel fucosylated N‐pentasaccharide as specific glycomarker for ALG1‐CDG. Moreover, group‐specific high‐mannose N‐glycan signatures were found in ALG3‐, ALG9‐, ALG11‐, ALG12‐, RFT1‐, SRD5A3‐, DOLK‐, DPM1‐, DPM3‐, MPDU1‐, ALG13‐CDG, and hereditary fructose intolerance. Further differential analysis revealed high‐mannose profiles, characteristic for ALG12‐ and ALG9‐CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG‐I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG‐I patient cohorts.

First person – Giovanna Lucrecia Gallo and Ayelen Valko

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Giovanna Lucrecia Gallo and Ayelen Valko are co-first authors on ‘ A novel fission yeast platform to model N-glycosylation and the bases of congenital disorders of glycosylation type I’, published in JCS. Giovanna conducted the research described in this article while a PhD student in Cecilia D'Alessio's lab at Fundación Instituto Leloir, Buenos Aires, Argentina. She is now a postdoc in the lab of Nora Lopez at Centro de Virologia Humana y Animal, Buenos Aires, Argentina, investigating the biological mechanisms underlying virus–host cell interactions, intracellular viral strategies to survive host immune responses and the control mechanisms displayed by the host. Ayelen conducted the research described in this article while a postdoc in Cecilia D'Alessio's lab. She is now a postdoc in the lab of Sebastian Schuck at Heidelberg University Biochemistry Center, Germany, investigating the underlying mechanisms of micro-ER-phagy, an autophagic process that is essential for elimination of the ER during ER stress.

A novel fission yeast platform to model N-glycosylation and the bases of congenital disorders of glycosylation type I.

Congenital disorders of glycosylation type I (CDG-I) are inherited human diseases caused by deficiencies in lipid-linked oligosaccharide (LLO) synthesis or the glycan transfer to proteins during N-glycosylation. We constructed a platform of 16 Schizosaccharomyces pombe strains that synthesize all possible theoretical combinations of LLOs containing three to zero glucose (Glc) residues and nine to five mannose (Man) residues. The occurrence of unexpected LLOs suggested the requirement of specific Man residues for glucosyltransferase activities. We then quantified protein hypoglycosylation in each strain and found that in S. pombe the presence of Glc in the LLO is more relevant to the transfer efficiency than the number of Man residues. Surprisingly, a decrease in the number of Man residues in glycans somehow improved the glycan transfer. The most severe hypoglycosylation was produced in cells that synthesized LLOs completely lacking Glc and having a high number of Man residues. This deficiency could be reverted by expressing a single-subunit oligosaccharyltransferase with a broad range of substrate specificity. Our work shows the usefulness of this new S. pombe set of mutants as a platform to model the molecular bases of human CDG-I diseases. This article has an associated First Person interview with the first authors of the paper.

TRAPPC9-CDG: A novel congenital disorder of glycosylation with dysmorphic features and intellectual disability

PurposeTRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities. MethodsExome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients’ blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics. ResultsAll 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites. ConclusionWe identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.

Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe.

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N -glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [ 14 C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms. Graphical abstract: Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe . (A) Radioactive pulse of S. pombe cells with [ 14 C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H 2 O, chloroform:methanol:H 2 O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H 2 O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.

Congenital Disorders of Glycosylation: What Clinicians Need to Know?

Congenital disorders of glycosylation (CDG) are a group of clinically heterogeneous disorders characterized by defects in the synthesis of glycans and their attachment to proteins and lipids. This manuscript aims to provide a classification of the clinical presentation, diagnostic methods, and treatment of CDG based on the literature review and our own experience (referral center in Poland). A diagnostic algorithm for CDG was also proposed. Isoelectric focusing (IEF) of serum transferrin (Tf) is still the method of choice for diagnosing N-glycosylation disorders associated with sialic acid deficiency. Nowadays, high-performance liquid chromatography, capillary zone electrophoresis, and mass spectrometry techniques are used, although they are not routinely available. Since next-generation sequencing became more widely available, an improvement in diagnostics has been observed, with more patients and novel CDG subtypes being reported. Early and accurate diagnosis of CDG is crucial for timely implementation of appropriate therapies and improving clinical outcomes. However, causative treatment is available only for few CDG types.

The Estimated Prevalence of N-Linked Congenital Disorders of Glycosylation Across Various Populations Based on Allele Frequencies in General Population Databases.

Congenital disorders of glycosylation (CDG) are a widely acknowledged group of metabolic diseases. PMM2-CDG is the most frequently diagnosed CDG with a prevalence as high as one in 20,000. In contrast, the prevalence of other CDG types remains unknown. This study aimed to analyze the estimated prevalence of different N-linked protein glycosylation disorders. We extracted allele frequencies for diverse populations from The Genome Aggregation Database (gnomAD), encompassing variant frequency information from 141,456 individuals. To identify pathogenic variants, we used the ClinVar database as a primary source. High confidence loss-of-function variants as defined by the LOFTEE algorithm were also classified as pathogenic. After summing up population frequencies for pathogenic alleles, estimated disease birth prevalence values with confidence intervals were calculated using the Bayesian method. We first validated our approach using two more common recessive disorders (cystic fibrosis and phenylketonuria) by showing that the estimated prevalences calculated from population allele frequencies were in accordance with previously published epidemiological studies. Among assessed 27 autosomal recessive N-glycosylation disorders, the only disease with estimated birth prevalence higher than one in 100,000 was PMM2-CDG (in both, all gnomAD individuals and those with European ancestry). The combined prevalence of 27 different N-glycosylation disorders was around one in 22,000 Europeans but varied considerably across populations. We will show estimated prevalence data from diverse populations and explain the possible pitfalls of this analysis. Still, we are confident that these data will guide CDG research and clinical care to identify CDG across populations.

Skeletal and Bone Mineral Density Features, Genetic Profile in Congenital Disorders of Glycosylation: Review.

Congenital disorders of glycosylation (CDGs) are a heterogeneous group of disorders with impaired glycosylation of proteins and lipids. These conditions have multisystemic clinical manifestations, resulting in gradually progressive complications including skeletal involvement and reduced bone mineral density. Contrary to PMM2-CDG, all remaining CDG, including ALG12-CDG, ALG3-CDG, ALG9-CDG, ALG6-CDG, PGM3-CDG, CSGALNACT1-CDG, SLC35D1-CDG and TMEM-165, are characterized by well-defined skeletal dysplasia. In some of them, prenatal-onset severe skeletal dysplasia is observed associated with early death. Osteoporosis or osteopenia are frequently observed in all CDG types and are more pronounced in adults. Hormonal dysfunction, limited mobility and inadequate diet are common risk factors for reduced bone mineral density. Skeletal involvement in CDGs is underestimated and, thus, should always be carefully investigated and managed to prevent fractures and chronic pain. With the advent of new therapeutic developments for CDGs, the severity of skeletal complications may be reduced. This review focuses on possible mechanisms of skeletal manifestations, risk factors for osteoporosis, and bone markers in reported paediatric and adult CDG patients.

Precision genetic cellular models identify therapies protective against ER stress

Rare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer’s disease and amyotrophic lateral sclerosis.