Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation (original) (raw)
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The Emerging Field of Diagnostics in Glycosylation Disorders
Pediatrics & Therapeutics, 2013
Protein glycosylation is an important post-translational modification. It enhances the functional diversity of proteins, half-life and influences their biological activity. Defective glycosylation often leads to multisystem disease and adds itself to the expanding group of 'Congenital disorders or glycosylation' which are predominantly disorders of N-linked glycosylation. Another rapidly growing group of disorders are defects in O-linked glycosylation, including a subset of dystroglycanopathies. Current diagnostic strategies for glycosylation disorders are compounded by the multivariate clinical phenotype of many of the diseases. Biochemical tests such as the isoelectric focusing of transferrin and apolipoprotein CIII are used to assess a patient's glycoform profile before in depth enzyme and genetic analysis is initiated. Whilst the glycoform profiling has been instrumental in screening for many glycosylation disorders, there is a need for a more sensitive and informative test. This short review gives an overview of the recent methods used in glycobiology research that could be used to devise such a test, which alongside currently used diagnostic tests should further facilitate the delineation of CDG subtypes. It provides a view to a potential strategy using marker glycopeptides to develop a mass spectrometry based assay that could be implemented into clinical diagnostic laboratories.
Clinical glycomics for the diagnosis of congenital disorders of glycosylation
Journal of inherited metabolic disease, 2018
Clinical glycomics comprises a spectrum of different analytical methodologies to analyze glycan structures, which provides insights into the mechanisms of glycosylation. Within clinical diagnostics, glycomics serves as a functional readout of genetic variants, and can form a basis for therapy development, as was described for PGM1-CDG. Integration of glycomics with genomics has resulted in the elucidation of previously unknown disorders of glycosylation, namely CCDC115-CDG, TMEM199-CDG, ATP6AP1-CDG, MAN1B1-CDG, and PGM1-CDG. This review provides an introduction into protein glycosylation and presents the different glycomics methodologies ranging from gel electrophoresis to mass spectrometry (MS) and from free glycans to intact glycoproteins. The role of glycomics in the diagnosis of congenital disorders of glycosylation (CDG) is presented, including a diagnostic flow chart and an overview of glycomics data of known CDG subtypes. The review ends with some future perspectives, showing...
Electrophoresis, 2018
Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and non-specific, a combination of electrophoretic, mass spectrometric and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N-and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG and SLC35A3-CDG, are described as illustrative examples.
International Journal of Molecular Sciences
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...
Orphanet Journal of Rare Diseases, 2021
Background Congenital disorders of glycosylation (CDG) result from defects in the synthesis of glycans and the attachment of glycans to proteins and lipids. Our study aimed to describe the clinical, biochemical, and molecular findings of CDG patients, and to present the long-term follow-up. Material and methods A single-center study (1995–2019 years) of patients with congenital disorders of N-glycosylation and combined N- and O-hypoglycosylation was performed. Results Among 32 patients included into the study, there were 12 PMM2-CDG, 3 ALG13-CDG, 3 ALG1-CDG, 1 ALG3-CDG, 3 MPI-CDG, 1 PGM1-CDG, 4 SRD5A3-CDG, 1 DPAGT1-CDG, 3 ATP6AP1-CDG, 1 ATP6V0A2-CDG. The phenotypic and genotypic spectrum during long-term (in some cases over 20 years) observation was characterised and several measurements of serum Tf isoforms taken. Statistical analysis revealed strong negative correlation between asialo-Tf and tetrasialo-Tf, as well as between disialo-Tf and tetrasialo-Tf. Within CDG type I, no diff...
Update and perspectives on congenital disorders of glycosylation
Glycobiology, 2001
Defects in nine genes of the N-linked glycosylation pathway cause congenital disorders of glycosylation (CDGs) and serious medical consequences. Although glycobiology is seldom featured in a general medical education, an increasing number of physicians are becoming acquainted with the field because it directly impacts patient diagnosis and care. Medical practice and attitudes will change in the postgenomic era, and glycobiology has an opportunity to be a cornerstone of part of that new perspective. This review of recent developments in the CDG field describes the biochemical and molecular basis of these disorders, describes successful experimental approaches, and points out a few perspectives on current problems. The broad, multisystemic presentations of these patients emphasize that glycobiology is very much a general medical science, cutting across many traditional medical specialties. The glycobiology community is well poised to provide novel perspectives for the dedicated clinicians treating both wellknown and emerging human diseases.
Dystroglycanopathies: About Numerous Genes Involved in Glycosylation of One Single Glycoprotein
Journal of neuromuscular diseases, 2015
Dystroglycanopathies are neuromuscular disorders due to abnormal glycosylation of dystroglycan which is a cellsurface glycoprotein that acts as a receptor for extracellular matrix proteins containing laminin-G domains. The reduced ability of abnormally glycosylated ␣−DG to bind laminin is associated with abnormal neuronal migration and muscular dystrophy. Clinical manifestations are extremely variable, and include a wide spectrum of phenotypic severity: some mutations are associated with adult-onset Limb-girdle muscular dystrophy and other mutations with a congenital onset, determining the more complex disorder Congenital Muscular Dystrophy which includes severe structural brain and eye anomalies such as Muscle-Eye-Brain Disease, Walker-Warburg Syndrome, and Fukuyama Congenital Muscular Dystrophy. So far, mutations in eighteen different genes have been identified in patients with dystroglycanopathies, all of them demonstrating autosomal recessive inheritance. Most genes code for glycosyltransferases (POMT1, POMT2, POMGNT1, LARGE, GTDC2, B4GAT1, B3GALNT2) although a minority does not (DPM1, DPM2, DPM3, DOLK, POMK, GMPPB). Others genes code for proteins of unknown function in the ␣−dystroglycan glycosylation (FKTN, FKRP, ISPD, and TMEM5) or ␣−dystroglycan itself, DAG1. The biochemical picture becomes a little bit more complete, but also more complex, with each new identified gene. In the majority of cases the identity of the defective gene cannot be predicted from the clinical phenotype. Considering the number of causative genes in dystroglycanopathies, targeted sequencing comprising genes of all glycosylation, whatever the type, would appear at present to be the best way of tackling molecular diagnosis.
Plasma N-Glycan Profiling by Mass Spectrometry for Congenital Disorders of Glycosylation Type II
Clinical Chemistry, 2011
BACKGROUND: 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.
Glycoforms of six serum glycoproteins in a patient with congenital disorder of glycosylation type I
Archivum immunologiae et therapiae experimentalis, 2002
In this paper the occurrence and relative content of defectively glycosylated serum glycoforms in transferrin (Tf), alpha1-acid glycoprotein (AGP), haptoglobin (Hp), alpha1-antitrypsin (alpha1-AT), alpha2-macroglobulin (alpha2-MG) and ceruloplasmin (Cpl) in the serum of a patient with congenital disorder of glycosylation type I are reported. Blood samples were taken when the patient was 14 years old and then after a one-year interval. The patterns of glycoforms in both samples were compared. In 4 out of 6 examined glycoproteins, glycoforms lacking one and two oligosaccharide chains occurred. "Underglycosylated" glycoforms of alpha2-MG and Cpl were not clearly detectable. Tf was shown to be affected with this defect to a higher extent than other glycoproteins, containing only 30% properly glycosylated molecules and also as much as 30% of the molecules lacking two glycan units. In Hp and alpha1-AT the proportions of properly and defectively glycosylated forms were similar. T...
Congenital Disorders of Glycosylation: What Clinicians Need to Know?
Frontiers in Pediatrics, 2021
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...