Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric Golgi complex leading to a new type of congenital disorders of glycosylation

Deficiency of subunit 6 of the conserved oligomeric Golgi (COG6) complex causes a new combined N- and O-glycosylation deficiency of the congenital disorders of glycosylation, designated as CDG-IIL (COG6-CDG). The index patient presented with a severe neurologic disease characterized by vitamin K deficiency, vomiting, intractable focal seizures, intracranial bleedings and fatal outcome in early infancy. Analysis of oligosaccharides from serum transferrin by HPLC and mass spectrometry revealed the loss of galactose and sialic acid residues, whereas import and transfer of these sugar residues into Golgi-enriched vesicles or onto proteins, respectively, were normal to slightly reduced. Western blot examinations combined with gel filtration chromatography studies in patient-derived skin fibroblasts showed a severely reduced expression of the mentioned subunit and the occurrence of COG complex fragments at the expense of the integral COG complex. Sequencing of COG6-cDNA and COG6 gene resulted in a homozygous mutation (c.G1646T), leading to amino acid exchange p.G549V in the COG6 protein. Retroviral complementation of the patients' fibroblasts with the wild-type COG6-cDNA led to normalization of the COG complex-depending retrograde protein transport after Brefeldin A treatment, demonstrated by immunofluorescence analysis

Congenital Disorders of Glycosylation IIj (CDG-IIj): identification of a defect in COG6 subunit of conserved oligomeric Golgi complex

`Congenital Disorders of Glycosylation` (CDG) ist eine schnell wachsende Gruppe mit bislang 23 autosomal rezessiven, multisystemischen Erkrankungen, welche mit einem Defizit in der Glykoproteinbiosynthese einhergeht. Die Übertragung der Oligosaccharidketten auf neu synthetisierte Proteine beeinflusst ihre Faltung, ihren Transport durch Zellkompartimente ebenso wie ihre biologische Aktivität und Stabilität.Ziel dieser Arbeit ist die Identifizierung einer neuen Erkrankung hervorgerufen durch eine Defizienz der Untereinheit 6 des Conserved Oligomeric Golgi (COG6) Komplexes. Die Patientin zeigt schwere neurologische Erkrankungen, Erbrechen, hartnäckige fokale Krampfanfälle, intrakraniale Blutungen und den tödlichen Ausgang im Säuglingsalter. Die grundlegenden Untersuchungen auf CDG mittels isoelektrischer Fokussierung (IEF) des Serumtransferrins und Apolipoprotein CIII ergab einen kombinierten N- und O-Glykosylierungsdefekt. Die nachfolgende Analyse der Transferrin-verknüpften Oligosaccharide mittels HPLC und Massenspektrometrie zeigt das Fehlen von Galaktose- und Sialinsäureresten. Western-Blot Analysen kombiniert mit gelfiltrationschromatographischen Studien in Hautfibroblasten der Patientin zeigt eine drastisch reduzierte Expression der COG6 Untereinheit und gleichzeitig die Fragmentierung des COG-Komplexes. Die Sequenzierung der COG6-cDNA und des COG6-Gens ergibt die homozygote Punktmutation (c.G1646T), was zum Aminosäureaustausch p.G549V im COG6 Protein führt. Die retrovirale Komplementation der Patientenfibroblasten mit Wildtyp COG6-cDNA führt zur Normalisierung des COG-Komplex abhängigen Proteintransportes nach Brefeldin A Behandlung, bewiesen durch Immunfluoreszenzanalysen

Deficiency of GDP-Man:GlcNAc(2)-PP-Dolichol Mannosyltransferase Causes Congenital Disorder of Glycosylation Type Ik

The molecular nature of a severe multisystemic disorder with a recurrent nonimmune hydrops fetalis was identified as deficiency of GDP-Man:GlcNAc(2)-PP-dolichol mannosyltransferase, the human orthologue of the yeast ALG1 gene (MIM 605907). The disease belongs to the group of congenital disorders of glycosylation (CDG) and is designated as subtype CDG-Ik. In patient-derived serum, the total amount of the glycoprotein transferrin was reduced. Moreover, a partial loss of N-glycan chains was observed, a characteristic feature of CDG type I forms. Metabolic labeling with [6-(3)H]glucosamine revealed an accumulation of GlcNAc(2)-PP-dolichol and GlcNAc(1)-PP-dolichol in skin fibroblasts of the patient. Incubation of fibroblast extracts with [(14)C]GlcNAc(2)-PP-dolichol and GDP-mannose indicated a severely reduced activity of the β1,4-mannosyltransferase, elongating GlcNAc(2)-PP-dolichol to Man(1)GlcNAc(2)-PP-dolichol at the cytosolic side of the endoplasmic reticulum. Genetic analysis of the patient’s hALG1 gene identified a homozygous mutation leading to the exchange of a serine residue to leucine at position 258 in the hALG1 protein. The disease-causing nature of the hALG1 mutation for the glycosylation defect was verified by a retroviral complementation approach in patient-derived primary fibroblasts and was confirmed by the expression of wild-type and mutant hALG1 in the Saccharomyces cerevisiae alg1-1 strain