Screening Using Serum Percentage of Carbohydrate-Deficient Transferrin for Congenital Disorders of Glycosylation in Children with Suspected Metabolic Disease

Abstract Background: Diagnoses of congenital disorders of glycosylation (CDG) are based on clinical suspicion and analysis of transferrin (Tf) isoforms. Here we present our experience of CDG screening in children with a suspected metabolic disease by determination of serum percentage of carbohydrate-deficient transferrin (%CDT) in tandem with isoelectric focusing (IEF) analysis of Tf and α1-antitrypsin (α1-AT). Methods: We performed approximately 8000 serum %CDT determinations using %CDT turbidimetric immunoassay (TIA). In selected samples, IEF analysis of Tf and α1-AT was carried out on an agarose gel (pH 4–8) using an electrophoresis unit. The isoforms were detected by Western blotting and visualized by color development. We performed neuraminidase digestion of serum to detect polymorphic variants of Tf. Results: We established a cutoff value for serum %CDT of 2.5% in our pediatric population. Sixty-five patients showed consistently high values of serum %CDT. In accordance with Tf and α1-AT IEF profiles, enzyme assays, and mutation analysis, we made the following diagnoses: 23 CDG-Ia, 1 CDG-Ib, and 1 conserved oligomeric Golgi 1 (COG-1) deficiency. In addition, we identified 13 CDG-Ix non Ia, non-Ib; 3 CDG-Ix; and 9 CDG-IIx cases, albeit requiring further characterization; 9 patients with a secondary cause of hypoglycosylation and 6 with a polymorphic Tf variant were also detected. Conclusion: The combined use of CDT immunoassay with IEF of Tf and α1-AT is a useful 1st-line screening tool for identifying CDG patients with an N-glycosylation defect. Additional molecular investigations must of course be carried out to determine the specific genetic disease.

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Quantification of total serum transferrin and transferrin sialoforms in human serum; an alternative method for the determination of carbohydrate-deficient transferrin in clinical samples

Analytical Methods ◽

10.1039/c4ay00159a ◽

2014 ◽

Vol 6 (12) ◽

pp. 3967-3974 ◽

Cited By ~ 9

Author(s):

Yoana Nuevo Ordonez ◽

Raymond F. Anton ◽

W. Clay Davis

Keyword(s):

Biochemical Marker ◽

Clinical Samples ◽

Total Serum ◽

Congenital Disorders ◽

Serum Transferrin ◽

Congenital Disorders Of Glycosylation ◽

Chronic Alcohol ◽

Chronic Alcohol Consumption ◽

Carbohydrate Deficient Transferrin

Carbohydrate deficient transferrin (CDT) is a biochemical marker for congenital disorders of glycosylation (CDG), chronic alcohol consumption, and forensic medicine diagnosis.

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Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection–Electrospray Ionization–Quadrupole Time-of-Flight Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2018.296780 ◽

2019 ◽

Vol 65 (5) ◽

pp. 653-663 ◽

Cited By ~ 8

Author(s):

Jie Chen ◽

Xueli Li ◽

Andrew Edmondson ◽

Gail Ditewig Meyers ◽

Kosuke Izumi ◽

...

Keyword(s):

Mass Spectrometry ◽

Electrospray Ionization ◽

Flow Injection ◽

Time Of Flight ◽

Congenital Disorders ◽

Congenital Disorders Of Glycosylation ◽

Detection Range ◽

Flight Mass Spectrometry ◽

Carbohydrate Deficient Transferrin ◽

Glycan Profiling

Abstract BACKGROUND Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection–electrospray ionization–quadrupole time-of-flight mass spectrometry. METHODS After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

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Personal Experience with the Application of Carbohydrate-Deficient Transferrin (CDT) Assays to the Detection of Congenital Disorders of Glycosylation

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2000.143 ◽

2000 ◽

Vol 38 (10) ◽

Cited By ~ 9

Author(s):

Catrina Colomé ◽

Imma Ferrer ◽

Rafael Artuch ◽

M. Antònia Vilaseca ◽

Mercè Pineda ◽

...

Keyword(s):

Personal Experience ◽

Congenital Disorders ◽

Congenital Disorders Of Glycosylation ◽

Carbohydrate Deficient Transferrin

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Diagnosis of Congenital Disorders of Glycosylation by Capillary Zone Electrophoresis of Serum Transferrin

Clinical Chemistry ◽

10.1373/clinchem.2003.021568 ◽

2004 ◽

Vol 50 (1) ◽

pp. 101-111 ◽

Cited By ~ 72

Author(s):

Hubert A Carchon ◽

Roland Chevigné ◽

Jean-Bernard Falmagne ◽

Jaak Jaeken

Keyword(s):

Capillary Zone Electrophoresis ◽

Reference Group ◽

Congenital Disorders ◽

Serum Transferrin ◽

Congenital Disorders Of Glycosylation ◽

Zone Electrophoresis ◽

Carbohydrate Deficient Transferrin ◽

Group A ◽

Capillary Zone ◽

Relative Migration

Abstract Background: Congenital disorders of glycosylation (CDG) are usually diagnosed by isoelectric focusing (IEF) of serum transferrin (Tf). The aim of this study was to evaluate capillary zone electrophoresis (CZE) as a diagnostic alternative to IEF. Methods: We performed 792 CZE analyses of Tf, using the CEofixTM-CDT (carbohydrate-deficient transferrin) assay. Peak identification was based on relative migration times (RMTs) to reduce migration variability. Results: Tf profiles comprised three main groups (A–C). Groups A and B were characterized by one or two dominant tetrasialo-Tf peaks, whereas group C showed a widely variable Tf isoform composition. Group A was composed of four subgroups: a major group with a typical Tf profile (considered as reference group), two minor groups with decreased or moderately increased trisialo-Tf isoform, and a group showing the presence of unknown compounds with RMTs similar to mono- and disialo-Tf. However, these compounds were absent on IEF. Group C contained all profiles from patients with confirmed as well as putative CDG. From the reference group, 99% confidence intervals were calculated for the RMTs of the Tf isoforms, and percentiles representing the Tf isoform distributions were defined. Conclusions: All patients with abnormal IEF results and confirmed CDG were identified by CZE; thus, this method can be used as a diagnostic alternative to IEF in a manner suitable for automation. Because whole serum is analyzed, it should be kept in mind that CZE profiles can show substances other than Tf.

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Our Experience with Diagnostics of Congenital Disorders of Glycosylation

Acta Medica (Hradec Kralove Czech Republic) ◽

10.14712/18059694.2018.103 ◽

2004 ◽

Vol 47 (4) ◽

pp. 267-272 ◽

Cited By ~ 2

Author(s):

Ziad Albahri ◽

Eliška Marklová ◽

Hubert Vaníček ◽

Lenka Minxová ◽

Petr Dědek ◽

...

Keyword(s):

Metabolic Disease ◽

Isoelectric Focusing ◽

Congenital Disorders ◽

Serum Transferrin ◽

Congenital Disorders Of Glycosylation ◽

Healthy Controls ◽

Protein Variants

The aim of this study is to report our 3years experience with the screening of congenital disorders of glycosylation. A common isoelectric focusing method with immunofixation was used for analysis of serum transferrin and α1-antitrypsin, apart from several other procedures. A group of about 1000 individuals, both healthy controls and patients, mostly with signs of a metabolic disease were examined. Here we present an overview of 1) hypoglycosylation findings, 2) distribution of protein variants, 3) misguiding rare Tf variants found in our set, and 4) association of some phenotypes with various diseases.

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Manganese-induced turnover of TMEM165

Biochemical Journal ◽

10.1042/bcj20160910 ◽

2017 ◽

Vol 474 (9) ◽

pp. 1481-1493 ◽

Cited By ~ 26

Author(s):

Sven Potelle ◽

Eudoxie Dulary ◽

Leslie Climer ◽

Sandrine Duvet ◽

Willy Morelle ◽

...

Keyword(s):

Glutamic Acid ◽

Mammalian Cells ◽

Congenital Disorders ◽

Congenital Disorders Of Glycosylation ◽

Crucial Importance ◽

Manganese Exposure ◽

Inherited Diseases ◽

Golgi Protein ◽

Glycosylation Defect

TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.

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