Congenital Disorders Of Glycosylation Type Research Articles

Congenital Disorders of Glycosylation

Congenital disorders of glycosylation (CDG) are a group of disorders involving a defect in the synthesis of oligosaccharides. Oligosaccharides are fundamental for protein stability and cellular communication and are present in almost every cell in the human body. A defect in the synthesis of oligosaccharides can result in multisystemic effects. Congenital disorders of glycosylation are classified into type I and type II disorders, each with subgroup classifications. All CDGs are autosomal recessive disorders, with CDG type I being the most common. This article will explore both types of CDG, their clinical presentation, diagnosis, and management.

Congenital Disorder of Glycosylation

Congenital disorders of glycosylation (CDG) are a group of rare genetically inherited disorders that involve the malfunction of attaching sugar molecules to lipids, proteins, or other organic molecules through an enzymatic process. The resulting defect in glycoprotein and glycolipid synthesis often has a heterogeneous range of multisystemic effects ranging from mild dysmorphism to profound organ failure and subsequent death. There are 2 types of CDG, type I and type II, with multiple subtypes within each. This column is a case presentation about an infant who presented with CDG type Ik.

DDOST Mutations Identified by Whole-Exome Sequencing Are Implicated in Congenital Disorders of Glycosylation

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.

SRD5A3-CDG: A patient with a novel mutation

Congenital disorders of glycosylation (CDG) are genetic diseases with an extremely broad spectrum of clinical presentations due to defective glycosylation of glycoproteins and glycolipids. Some 45 CDG types have been reported since the first clinical description in 1980. Protein glycosylation disorders are defects in protein N- and/or O-glycosylation. Dolichol phosphate is the carrier of the N-glycan during their assembly first at the outside and subsequently at the inside of the endoplasmic reticulum (ER) membrane, and hence is a key molecule in protein glycosylation. Recently, defects have been identified in the last three steps of the dolichol phosphate biosynthesis: dolicholkinase deficiency (DK1-CDG), steroid 5alpha-reductase type 3 deficiency (SRD5A3-CDG), and dehydrodolichyl diphosphate synthase deficiency (DHDDS-CDG). We report on a patient with SRD5A3-CDG carrying a novel (homozygous) mutation. The diagnostic features of this novel inborn error of glycosylation are psychomotor retardation, nystagmus, visual impairment due to variable eye malformations, cerebellar abnormalities/ataxia, and often ichthyosiform skin lesions.

Insights into complexity of congenital disorders of glycosylation

Biochemical and biological properties of glycoconjugates are strongly determined by the specific structure of its glycan parts. Glycosylation, the covalent attachment of sugars to proteins and lipids, is very complex and highly-coordinated process involving > 250 gene products. Deficiency of glycosylation enzymes or transporters results in impaired glycosylation, and consequently pathological modulation of many physiological processes. Inborn defects of glycosylation enzymes, caused by the specific mutations, lead to the development of rare, but severe diseases - congenital disorders of glycosylation (CDGs). Up today, there are more than 45 known CDGs. Their clinical manifestations range from very mild to extremely severe (even lethal) and unfortunately, only three of them can be effectively treated nowadays. CDG symptoms highly vary, though some are common for several CDG types but also for other unrelated diseases, especially neurological ones, leaving the possibility that many CDGs cases are under- or misdiagnosed. Glycan analysis of serum transferrin (by isoelectric focusing or more sophisticated methods, such as HPLC (high-performance liquid chromatography) or MALDI (matrix-assisted laser desorption/ionization)) or serum N-glycans (by MS), enzyme activity assays and DNA sequence analysis are the most frequently used methods for CDG screening and identification, since no specific tests are available yet. In this review we summarize the current knowledge on the clinical, biochemical and genetic characteristic of distinct CDGs, as well as existing diagnostic and therapeutic procedures, aiming to contribute to the awareness on the existence of these rare diseases and encourage the efforts to elucidate its genetic background, improve diagnostics and develop new strategies for their treatment.

Congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) have grown enormously since the discovery of the first protein glycosylation defect in 1980, presenting with a broad clinical spectrum. Expansion in number and complexity of the CDG group has even necessitated a new nomenclature. By 2011, the CDG group includes lipid glycosylation disorders and other related processes and almost 50 distinct disorders. Current research has not only expanded the spectrum of CDG types, but has also given novel insight into those previously described. The discovery of genetic defects in the conserved oligomeric Golgi complex, affecting protein glycosylation and processing through the secretory pathway, raised the concept of 'secondary' glycosylation disorders. The number of lipid glycosylation disorders, linking lipid synthesis to CDG, that were previously regarded as rare, is also increasing rapidly. In other areas of research, the bridge between muscular dystrophies and metabolic disorders is being further reinforced with the discovery of additional defects in the DPM-CDG subgroup, a CDG characterized by significant muscle involvement. It is of great importance that clinicians stay up-to-date on the field of CDG and consider it in their differential diagnosis of unknown syndromal presentations. Nevertheless, many advances have yet to be made, including information on the natural course of CDG. The lack of treatment for nearly all CDG types is striking, and the field must continue to push for innovative therapies. Clinicians and researchers must work together to describe the natural course and, most importantly, collaborate to find new therapies.

Oligosaccharyltransferase: the central enzyme of N‐linked protein glycosylation

N-linked glycosylation is one of the most abundant modifications of proteins in eukaryotic organisms. In the central reaction of the pathway, oligosaccharyltransferase (OST), a multimeric complex located at the membrane of the endoplasmic reticulum, transfers a preassembled oligosaccharide to selected asparagine residues within the consensus sequence asparagine-X-serine/threonine. Due to the high substrate specificity of OST, alterations in the biosynthesis of the oligosaccharide substrate result in the hypoglycosylation of many different proteins and a multitude of symptoms observed in the family of congenital disorders of glycosylation (CDG) type I. This review covers our knowledge of human OST and describes enzyme composition. The Stt3 subunit of OST harbors the catalytic center of the enzyme, but the function of the other, highly conserved, subunits are less well defined. Some components seem to be involved in the recognition and utilization of glycosylation sites in specific glycoproteins. Indeed, mutations in the subunit paralogs N33/Tusc3 and IAP do not yield the pleiotropic phenotypes typical for CDG type I but specifically result in nonsyndromic mental retardation, suggesting that the oxidoreductase activity of these subunits is required for glycosylation of a subset of proteins essential for brain development.

Plasma N-Glycan Profiling by Mass Spectrometry for Congenital Disorders of Glycosylation Type II

Determination of the genetic defect in patients with a congenital disorder of glycosylation (CDG) is challenging because of the wide clinical presentation, the large number of gene products involved, and the occurrence of secondary causes of underglycosylation. Transferrin isoelectric focusing has been the method of choice for CDG screening; however, improved methods are required for the molecular diagnosis of patients with CDG type II. Plasma samples with a typical transferrin isofocusing profile were analyzed. N-glycans were released from these samples by PNGase F [peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase] digestion, permethylated and purified, and measured on a MALDI linear ion trap mass spectrometer. A set of 38 glycans was used for quantitative comparison and to establish reference intervals for such glycan features as the number of antennae, the level of truncation, and fucosylation. Plasma N-glycans from control individuals, patients with known CDG type II defects, and patients with a secondary cause of underglycosylation were analyzed. CDGs due to mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT2), β-1,4-galactosyltransferase 1 (B4GALT1), and SLC35C1 (a GDP-fucose transporter) defects could be diagnosed directly from the N-glycan profile. CDGs due to defects in proteins involved in Golgi trafficking, such as subunit 7 of the conserved oligomeric Golgi complex (COG7) and subunit V0 a2 of the lysosomal H(+)-transporting ATPase (ATP6V0A2) caused a loss of triantennary N-glycans and an increase of truncated structures. Secondary causes with liver involvement were characterized by increased fucosylation, whereas the presence of plasma sialidase produced isolated undersialylation. MALDI ion trap analysis of plasma N-glycans documents features that discriminate between primary and secondary causes of underglycosylation and should be applied as the first step in the diagnostic track of all patients with an unsolved CDG type II.

Transferrin mutations at the glycosylation site complicate diagnosis of congenital disorders of glycosylation type I

Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. Isoelectric focusing (IEF) of serum transferrin is the most common screening method to detect abnormalities of protein N-glycosylation. On the basis of the IEF profile, patients can be grouped into CDG type I or CDG type II. Several protein variants of transferrin are known that result in a shift in isoelectric point (pI). In some cases, these protein variants co-migrate with transferrin glycoforms, which complicates interpretation. In two patients with abnormal serum transferrin IEF profiles, neuraminidase digestion and subsequent IEF showed profiles suggestive of the diagnosis of CDG type I. Mass spectrometry of tryptic peptides of immunopurified transferrin, however, revealed a novel mutation at the N-glycan attachment site. In case 1, a peptide with mutation p.Asn630Thr in the 2nd glycosylation site was identified, resulting in an additional band at disialotransferrin position on IEF. After neuraminidase digestion, a single band was found at the asialotransferrin position, indistinguishable from CDG type I patients. In case 2, a peptide with mutation p.Asn432His was found. These results show the use of mass spectrometry of transferrin peptides in the diagnostic track of CDG type I.

The impact of mass spectrometry in the diagnosis of congenital disorders of glycosylation

Contribution of mass spectrometry (MS) in the diagnosis and characterization of congenital disorders of glycosylation (CDG) has long been known. CDG type I diseases are characterized by the under-occupancy of protein N-glycosylation sites. Electrospray (ESI) MS and matrix assisted laser desorption ionization (MALDI) MS are effective for underglycosylation analyses of intact serum Transferrin (Tf) in CDG-I patients by mass determination of individual component glycoforms. Thus, high-throughput methods developed to speed-up analytical times found increasing application in clinical testing for CDG detection. ESI MS recognizable glycoform profiles of serum Tf have been reported in CDG-I different from PMM2-CDG and in individual CDG-II defects. MALDI MS analysis of acidic and neutral N-linked glycans released from total plasma or targeted glycoproteins, is the mainstream tool to explore abnormal oligosaccharide structure and changes in the relative amount of individual oligosaccharides in CDG-II patients. Here we briefly review state-of-the-art and updates of MS-based applications for the diagnosis of CDG with special emphasis to detectable glycosylation profiles reported in different CDG types.

Mouse models for congenital disorders of glycosylation

Glycoprotein biosynthesis describes the process of co- and posttranslational attachment of sugar chains to proteins, a process that has been found in nearly all known organisms. Human deficiencies evoked by mutations in the glycosylation pathway of glycoproteins lead to congenital disorders of glycosylation (CDG), a rapidly expanding group of autosomal recessive inherited metabolic diseases with multisystemic phenotypes that are mostly combined with severe neurological impairment. Although investigations on new types of CDG have proceeded rapidly in recent years, the correlation between inaccurate protein glycosylation and pathological loss of functionality of distinct organ systems remains widely unknown, and therapeutics for the patients are mostly not available. Therefore, mouse models provide an outstanding helpful tool for investigations on different aspects of glycosylation deficiencies that cannot be performed in patients or cell culture. This review focuses on existing mouse models generated for the types of CDG that affect the N-glycosylation pathway.

Anesthetic Management of a Boy with Congenital Disorder of Glycosylation (CDG) I-x

Congenital disorders of glycosylation (CDGs) are group group of genetic defects in the assembly and processing pathway of protein glycosylation, which cause a wide range of multi system dysfunction. This paper describes the anesthetic management of 6 year old boy with CDG type I-x for upper airway surgery. We used a sevoflurane-nitrous oxide-remifentanil regime with no complications and good results. As for now, the literature lacks reports of anesthetic management of children with CDG I-x, and this report may provide clinicians an option for safe anesthetic management.

Re-evaluation of the dysequilibrium syndrome

To re-evaluate middle-aged Swedish patients diagnosed with dysequilibrium syndrome (DES) in childhood and to compare their clinical and neuroimaging features to DES with VLDLR gene mutations (DES-VLDR). Six patients from five families underwent neurological examination and magnetic resonance imaging (MRI) of the brain. Blood samples from the patients were screened for serum carbohydrate-deficient transferrin (s-CDT; disialotransferrin). The very-low-density lipoprotein receptor (VLDLR) gene was sequenced. Five patients had non-progressive cerebellar ataxia (NPCA), dysarthria and short stature. Mental retardation and strabismus, characteristic for DES-VLDLR, were inconsistent among our patients. None of our patients had VLDLR mutations or MRI findings characteristic of DES-VLDLR. MRI findings were variable from a normal cerebellum to marked cerebellar hypoplasia or atrophy and signal intensity changes. One patient was diagnosed with congenital disorder of glycosylation type 1a (CDG-1a). DES was originally coined on mainly clinical grounds before MRI and specific genetic tests were available, both of which should be used to arrive at an appropriate diagnosis.

Cog2 Null Mutant CHO Cells Show Defective Sphingomyelin Synthesis

The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype with severe, early visual impairment and variable eye malformations, including optic nerve hypoplasia, retinal coloboma, congenital cataract and glaucoma. Some of the symptoms overlapped with the phenotype in other congenital disorders of glycosylation type I subtypes, such as vermis hypoplasia, anaemia, ichtyosiform dermatitis, liver dysfunction and coagulation abnormalities. We recently identified pathogenic mutations in the SRD5A3 gene, encoding steroid 5α-reductase type 3, in a group of patients who presented with this particular phenotype and a common metabolic pattern. Here, we report on the clinical, genetic and metabolic features of 12 patients from nine families with cerebellar ataxia and congenital eye malformations diagnosed with SRD5A3-congenital disorders of glycosylation due to steroid 5α-reductase type 3 defect. This enzyme is necessary for the reduction of polyprenol to dolichol, the lipid anchor for N-glycosylation in the endoplasmic reticulum. Dolichol synthesis is an essential metabolic step in protein glycosylation. The current defect leads to a severely abnormal glycosylation state already in the early phase of the N-glycan biosynthesis pathway in the endoplasmic reticulum. We detected high expression of SRD5A3 in foetal brain tissue, especially in the cerebellum, consistent with the finding of the congenital cerebellar malformations. Based on the overlapping clinical, biochemical and genetic data in this large group of patients with congenital disorders of glycosylation, we define a novel syndrome of cerebellar ataxia associated with congenital eye malformations due to a defect in dolichol metabolism.

Cog2 null mutant CHO cells show defective sphingomyelin synthesis

Abstract The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C6-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.

SRD5A3 Is Required for Converting Polyprenol to Dolichol and Is Mutated in a Congenital Glycosylation Disorder

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Should PMM2-deficiency (CDG Ia) be searched in every case of unexplained hydrops fetalis?

Hydrops fetalis (HF) is characterized by an accumulation of fluid in the extracellular compartments and in body cavities. Non-immune HF (NIHF) is caused by a wide variety of disorders and overall, 20–25% of NIHF remain unexplained. Inborn errors of metabolism, mostly lysosomal storage diseases have been estimated to account for 1–2% of cases, leading to HF by anemia or liver failure. Very few cases of NIHF and Congenital Disorder of Glycosylation (CDG) have been reported. We present here a case of recurrence of HF in a non-related couple in which the diagnosis of CDG type I was suspected at fetal pathological examination then confirmed at the enzymatic and molecular levels, as well as on a characteristic CDG I serum transferrin profile at 30weeks of gestation. We also provide a systematic review of reported cases with CDG type I and NIHF reported thus far. When NIHF remains unexplained despite exhaustive obstetrical screening, analysis of PMM activity in the parents' leucocytes is possible and might be performed easily during pregnancy. The accurate diagnosis is important in terms of counseling during pregnancy or later, in order to allow an early molecular prenatal diagnosis for the following pregnancies.

DFT investigation on the reaction mechanism catalyzed by α-phosphomannomutase1 in protonated/deprotonated states

Congenital disorder of glycosylation type 1a (CDG-1a) which is a congenital disease, is caused by mutations in α-Phosphomannomutase1. The reaction mechanism of the α-phosphomannomutase1 enzyme has been investigated by means of density functional theory using the hybrid functional B3LYP. The α-phosphomannomutase1 catalyzes the interconversion of the α-D-mannose 1-phosphate to D-mannose 6-phosphate via a mannose-1,6-(bis) phosphate intermediate. The quantum chemical models, which were chosen in protonated/deprotonated states models, were built on the basis of the docking result. The process of the phosphoryl group transferred from Asp19 to the mannose 6-phosphate is in different steps in the two states, but are both coupled with the protons transfer. Our computational results support the hypothesis that the Asp19 as a nucleophile plays an important role in the α-phosphomannomutase1 biology function, and indicate Gln62 could help to stabilize the phosphoryl group and the structure of the substrate. In addition, we can conjecture that the deprotonated state is more suitable for product release.