MPDU1 mutations underlie a novel human congenital disorder of glycosylation, designated type If.

Deficiencies in the pathway of N-glycan biosynthesis lead to severe multisystem diseases, known as congenital disorders of glycosylation (CDG). The clinical appearance of CDG is variable, and different types can be distinguished according to the gene that is altered. In this report, we describe the molecular basis of a novel type of the disease in three unrelated patients diagnosed with CDG-I. Serum transferrin was hypoglycosylated and patients' fibroblasts accumulated incomplete lipid-linked oligosaccharide precursors for N-linked protein glycosylation. Transfer of incomplete oligosaccharides to protein was detected. Sequence analysis of the Lec35/MPDU1 gene, known to be involved in the use of dolichylphosphomannose and dolichylphosphoglucose, revealed mutations in all three patients. Retroviral-based expression of the normal Lec35 cDNA in primary fibroblasts of patients restored normal lipid-linked oligosaccharide biosynthesis. We concluded that mutations in the Lec35/MPDU1 gene cause CDG. This novel type was termed CDG-If.

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.

Clinical and biochemical characteristics of congenital disorder of glycosylation type Ic, the first recognized endoplasmic reticulum defect in N-glycan synthesis.

We report on 8 patients with a recently described novel subtype of congenital disorder of glycosylation type Ic (CDG-Ic). Their clinical presentation was mainly neurological with developmental retardation, muscular hypotonia, and epilepsy. Several symptoms commonly seen in CDG-Ia such as inverted nipples, abnormal fat distribution, and cerebellar hypoplasia were not observed. The clinical course is milder overall, with a better neurological outcome, than in CDG-Ia. The isoelectric focusing pattern of serum transferrin in CDG-Ia and CDG-Ic is indistinguishable. Interestingly, beta-trace protein in cerebrospinal fluid derived from immunoblot analysis of the brain showed a less pronounced hypoglycosylation pattern in CDG-Ic patients than in CDG-Ia patients. Analysis of lipid-linked oligosaccharides revealed an accumulation of Man9GlcNAc2 intermediates due to dolichol pyrophosphate-Man9GlcNAc2 alpha-1,3 glucosyltransferase deficiency. All patients were homozygous for an A333V mutation.

Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.

alpha1,3Fucosyltransferase VI is expressed in HepG2 cells and codistributed with beta1,4galactosyltransferase I in the golgi apparatus and monensin-induced swollen vesicles.

The major alpha1,3fucosyltransferase activity in plasma, liver, and kidney is related to fucosyltransferase VI which is encoded by the FUT6 gene. Here we demonstrate the presence of alpha1, 3fucosyltransferase VI (alpha3-FucT VI) in the human HepG2 hepatoma cell line by specific activity assays, detection of transcripts, and the use of specific antibodies. First, FucT activity in HepG2 cell lysates was shown to prefer sialyl-N-acetyllactosamine as acceptor substrate indicating expression of alpha3-FucT VI. RT-PCR analysis further confirmed the exclusive presence of the alpha3-FucT VI transcripts among the five human alpha3-FucTs cloned to date. alpha3-FucT VI was colocalized with beta1,4galactosyltransferase I (beta4-GalT I) to the Golgi apparatus by dual confocal immunostaining. Pulse/chase analysis of metabolically labeled alpha3-FucT VI showed maturation of alpha3-FucT VI from the early 43 kDa form to the mature, endoglycosidase H-resistant form of 47 kDa which was detected after 2 h of chase. alpha3-FucT VI was released to the medium and accounted for 50% of overall cell-associated and released enzyme activity. Release occurred by proteolytical cleavage which produced a soluble form of 43 kDa. Monensin treatment segregated alpha3-FucT VI from the Golgi apparatus to swollen peripheral vesicles where it was colocalized with beta4-GalT I while alpha2,6(N)sialyltransferase remained associated with the Golgi apparatus. Both constitutive secretion of alpha3-FucT VI and its monensin-induced relocation to vesicles analogous to beta4-GalT I suggest a similar post-Golgi pathway of both alpha3-FucT VI and beta4-GalT I.

A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic.

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.