18 research outputs found
Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik
Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 β1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-I
High Residual Activity of PMM2 in Patients’ Fibroblasts: Possible Pitfall in the Diagnosis of CDG-Ia (Phosphomannomutase Deficiency)
Congenital disorders of glycosylation (CDGs) are a rapidly enlarging group of inherited diseases with abnormal N-glycosylation of glycoconjugates. Most patients have CDG-Ia, which is due to a phosphomannomutase (PMM) deficiency. In this article, we report that a significant portion (9 of 54) of patients with CDG-Ia had a rather high residual PMM activity in fibroblasts included in the normal range (means of the controls ± 2 SD) and amounting to 35%–70% of the mean control value. The clinical diagnosis of CDG-Ia was made difficult by the fact that most (6 of 9) of these patients belong to a subgroup characterized by a phenotype that is milder than classical CDG-Ia. These patients lack some of the symptoms that are suggestive for the diagnosis, such as inverted nipples and abnormal fat deposition, and, as a mean, had higher residual PMM activities in fibroblasts (2.05±0.61 mU/mg protein, n=9; vs. controls 5.34±1.74 mU/mg protein, n=22), compared with patients with moderate (1.32±0.86 mU/mg protein, n=18) or severe (0.63±0.56 mU/mg protein, n=27, P<.001) cases. Yet they all showed mild mental retardation, hypotonia, cerebellar hypoplasia, and strabismus. All of them had an abnormal serum transferrin pattern and a significantly reduced PMM activity in leukocytes. Six of the nine patients with mild presentations were compound heterozygotes for the C241S mutation, which is known to reduce PMM activity by only ∼2-fold. Our results indicate that intermediate PMM values in fibroblasts may mask the diagnosis of CDG-Ia, which is better accomplished by measurement of PMM activity in leukocytes and mutation search in the PMM2 gene. They also indicate that there is some degree of correlation between the residual activity in fibroblasts and the clinical phenotype
Lack of Hardy-Weinberg equilibrium for the most prevalent PMM2 mutation in CDG-Ia (congenital disorders of glycosylation type Ia)
Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome)
Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken syndrome) is the prototype of a class of genetic multisystem disorders characterized by defective glycosylation of glycoconjugates(1-4). It is mostly a severe disorder which presents neonatally. There is a severe encephalopathy with axial hypotonia, abnormal eye movements and pronounced psychomotor retardation, as well as a peripheral neuropathy, cerebellar hypoplasia and retinitis pigmentosa. The patients show a peculiar distribution of subcutaneous fat, nipple retraction and hypogonadism. There is a 20% lethality in the first years of life due to severe infections, liver insufficiency or cardiomyopathy(2,3,5). CDG1 shows an autosomal recessive mode of inheritance and has been mapped to chromosome 16p(6,7). Most patients show a deficiency of phosphomannomutase (PMM)(8), an enzyme necessary for the synthesis of GDP-mannose. We have cloned the PMM1 gene, which is on chromosome 22q13 (ref, 9), We now report the identification of a second human PMM gene, PMM2, which is located on 16p13 and which encodes a protein with 66% identity to PMM1. We found eleven different missense mutations in PMM2 in 16 CDG1 patients from different geographical origins and with a documented phosphomannomutase deficiency. Our results give conclusive support to the biochemical finding that the phosphomannomutase deficiency is the basis for CDG1
Prenatal diagnosis in CDG1 families: beware of heterogeneity
Carbohydrate-deficient glycoprotein syndrome type I (CDG1) is an autosomal recessive, metabolic disorder with severe psychomotor retardation and a high mortality rate in early childhood. Most patients have a deficiency of phosphomannomutase, due to mutations in PMM2, a gene located on chromosome 16p13, Over a period of 18 months we offered prenatal diagnosis to eight families. In six cases and prior to the identification of the gene, the diagnosis was based on linkage analysis and phosphomannomutase measurements. Subsequently direct mutation analysis has been used in two families. It is shown here that phosphomannomutase activities are strongly reduced in cultured amniocytes and trophoblasts of affected foetuses, We refrained from offering prenatal testing in two other families, because either the disease did not link to chromosome 16 and/or normal phosphomannomutase activities were measured in fibroblasts from the proband, This confirms earlier suggestions of heterogeneity for CDG1
Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2.
Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified. These mutants had a Vmax of 0.2-50% of the wild enzyme and were unstable. The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype. We conclude that total lack of phosphomannomutase 2 is incompatible with life. Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2
PMM (PMM1), the human homologue of SEC53 or yeast phosphomannomutase, is localized on chromosome 22q13
We have cloned the human homologue of SEC53 or yeast phosphomannomutase (HGMW-approved symbol PMM1) from a liver cDNA library. This cDNA encodes a protein of 262 amino acids with a predicted molecular mass of 29 kDa and 54% identity with yeast phosphomannomutase. Expression of the human cDNA in Escherichia coli yielded an active phosphomannomutase, which was purified to homogeneity. Northern blot analysis of human tissues showed strong expression in liver, heart, brain, and pancreas and a lower expression in skeletal muscle. The gene was assigned to chromosome 22q13.1 by the use of hybrid cell lines and by fluorescence in situ hybridization. Most patients presenting with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken disease) have a greatly reduced phosphomannomutase activity; the gene encoding this enzyme is a likely candidate for CDG1. Since the CDG1 locus maps else where in the genome (16p13), mutations in the phosphomannomutase gene encoded by chromosome 22 are not a major cause of CDG1