Liesbeth Keldermans

The Normal Phenotype of Pmm1-Deficient Mice Suggests that Pmm1 Is Not Essential for Normal Mouse Development

ABSTRACT Phosphomannomutases (PMMs) are crucial for the glycosylation of glycoproteins. In humans, two highly conserved PMMs exist: PMM1 and PMM2. In vitro both enzymes are able to convert mannose-6-phosphate (mannose-6-P) into mannose-1-P, the key starting compound for glycan biosynthesis. However, only mutations causing a deficiency in PMM2 cause hypoglycosylation, leading to the most frequent type of the congenital disorders of glycosylation (CDG): CDG-Ia. PMM1 is as yet not associated with any disease, and its physiological role has remained unclear. We generated a mouse deficient in Pmm1 activity and documented the expression pattern of murine Pmm1 to unravel its biological role. The expression pattern suggested an involvement of Pmm1 in (neural) development and endocrine regulation. Surprisingly, Pmm1 knockout mice were viable, developed normally, and did not reveal any obvious phenotypic alteration up to adulthood. The macroscopic and microscopic anatomy of all major organs, as well as animal behavior, appeared to be normal. Likewise, lectin histochemistry did not demonstrate an altered glycosylation pattern in tissues. It is especially striking that Pmm1, despite an almost complete overlap of its expression with Pmm2, e.g., in the developing brain, is apparently unable to compensate for deficient Pmm2 activity in CDG-Ia patients. Together, these data point to a (developmental) function independent of mannose-1-P synthesis, whereby the normal knockout phenotype, despite the stringent conservation in phylogeny, could be explained by a critical function under as-yet-unidentified challenge conditions.

COG5-CDG: expanding the clinical spectrum

BackgroundThe Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350–6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia.MethodsCDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis.Results and conclusionWe identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.

Quality control of glycoproteins bearing truncated glycans in an ALG9-defective (CDG-IL) patient

We describe an ALG9-defective (congenital disorders of glycosylation type IL) patient who is homozygous for the p.Y286C (c.860A>G) mutation. This patient presented with psychomotor retardation, axial hypotonia, epilepsy, failure to thrive, inverted nipples, hepatomegaly, and pericardial effusion. Due to the ALG9 deficiency, the cells of this patient accumulated the lipid-linked oligosaccharides Man(6)GlcNAc(2)-PP-dolichol and Man(8)GlcNAc(2)-PP-dolichol. It is known that the oligosaccharide structure has a profound effect on protein glycosylation. Therefore, we investigated the influence of these truncated oligosaccharide structures on the protein transfer efficiency, the quality control of newly synthesized glycoproteins, and the eventual degradation of the truncated glycoproteins formed in this patient. We demonstrated that lipid-linked Man(6)GlcNAc(2) and Man(8)GlcNAc(2) are transferred onto proteins with the same efficiency. In addition, glycoproteins bearing these Man(6)GlcNAc(2) and Man(8)GlcNAc(2) structures efficiently entered in the glucosylation/deglucosylation cycle of the quality control system to assist in protein folding. We also showed that in comparison with control cells, patients cells degraded misfolded glycoproteins at an increasing rate. The Man(8)GlcNAc(2) isomer C on the patients glycoproteins was found to promote the degradation of misfolded glycoproteins.

Increased recurrence risk in congenital disorders of glycosylation type Ia (CDG-Ia) due to a transmission ratio distortion

Congenital disorders of glycosylation type Ia or CDG-Ia (MIM 212065) is the most common type of a group of recessive disorders characterised by deficient glycosylation.1 The disease is caused by mutations in the PMM2 gene, coding for a phosphomannomutase (PMM). PMM converts mannose-6-phosphate to mannose-1-phosphate, a precursor of the mannosyl donor in N-and O-glycosylation and the synthesis of GPI anchors. PMM deficiency leads to underglycosylation and altered processing of the N-glycans in serum proteins of CDG-Ia patients.2,3 A plethora of different mutations, mostly missense mutations, results in a clinical spectrum ranging from mild to very severe with neonatal death.4,5 A founder effect for the mutation F119L (c.357C→A) in the Scandinavian population results in a more homogenous group of patients in these countries.6 Around 37% of CDG-Ia patients are heterozygous for the missense mutation R141H (c.422G→A), which has never been observed in the homozygous state.7,8 The very low residual (<1%) activity of the mutant R141H protein is probably not sufficient for viability.9 In spite of this genetic lethality, the carrier frequency for R141H is rather high, being 1/72 in the Dutch and Danish populations.8 Without a mechanism counteracting the constant loss of recessive disease alleles, this R141H mutation would have vanished. The most obvious explanations for its persistence are a high mutation rate, genetic drift, a heterozygous advantage, or a transmission distortion. The first possibility has been discounted by the observation that, in most patients and carriers, the mutation is associated with a specific haplotype, and thus represents a single, ancestral event.6,8 Based on linkage disequilibrium with marker D16S3020 the most recent common ancestor was calculated to have lived at least 250 generations ago (R Colombo and E Schollen, unpublished data). Genetic drift could be an important factor in …

Congenital disorders of glycosylation presenting as epileptic encephalopathy with migrating partial seizures in infancy

Epilepsy is commonly observed in congenital disorders of glycosylation (CDG), but no distinctive electroclinical pattern has been recognized. We aimed at identifying a characteristic clinical presentation that might help targeted diagnostic work‐up.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol‐lipid linked oligosaccharide intermediate required for proper N‐linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1‐CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1‐CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1‐deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein‐linked xeno‐tetrasaccharide biomarker, NeuAc‐Gal‐GlcNAc2, was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Tissue distribution of the murine phosphomannomutases Pmm1 and Pmm2 during brain development.

The most common type of the congenital disorders of glycosylation, CDG‐Ia, is caused by mutations in the human PMM2 gene, reducing phosphomannomutase (PMM) activity. The PMM2 mutations mainly lead to neurological symptoms, while other tissues are only variably affected. Another phosphomannomutase, PMM1, is present at high levels in the brain. This raises the question why PMM1 does not compensate for the reduced PMM2 activity during CDG‐Ia pathogenesis. We compared the expression profile of the murine Pmm1 and Pmm2 mRNA and protein in prenatal and postnatal mouse brain at the histological level. We observed a considerable expression of both Pmms in different regions of the embryonic and adult mouse brain. Surprisingly, the expression patterns were largely overlapping. This data indicates that expression differences on the cellular and tissue level are an unlikely explanation for the absence of functional compensation. These results suggest that Pmm1 in vivo does not exert the phosphomannomutase‐like activity seen in biochemical assays, but either acts on as yet unidentified specific substrates or fulfils entirely different functions.

ALG11-CDG: Three novel mutations and further characterization of the phenotype

We report on two novel patients with ALG11-CDG. The phenotype was characterized by severe psychomotor disability, progressive microcephaly, sensorineural hearing loss, therapy-resistant epilepsy with burst suppression EEG, cerebral atrophy with, in one of them, neuronal heterotopia, and early lethality. Analysis of ALG11 revealed compound heterozygosity involving three novel mutations: the splice site mutation c.45-2A > T, the c.36dupG duplication, and the missense mutation c.479G > T (p.G160V) that was present in both.

Erratum: COG5-CDG: Expanding the clinical spectrum (Orphanet Journal of Rare Diseases (2013) 8 (120))

Author details Centre for Human Genetics, University of Leuven, Leuven, Belgium. Centre for Metabolic Diseases, University Hospital Gasthuisberg, Herestraat 49, BE-3000, Leuven, Belgium. University Children’s Hospital Queen Fabiola, Brussels, Belgium. Division of Metabolism, Bambino Gesu Hospital, Rome, Italy. Duchess of Kent Children’s Hospital, University of Hong Kong, Pokfulam, Hong Kong. Institute of Chemistry and Technology of Polymers, Catania, Sicily, Italy. Structural and Functional Glycobiology Unit, University of Lille 1, Lille 1, France.

Correction: COG5-CDG: expanding the clinical spectrum

Author details Centre for Human Genetics, University of Leuven, Leuven, Belgium. Centre for Metabolic Diseases, University Hospital Gasthuisberg, Herestraat 49, BE-3000, Leuven, Belgium. University Children’s Hospital Queen Fabiola, Brussels, Belgium. Division of Metabolism, Bambino Gesu Hospital, Rome, Italy. Duchess of Kent Children’s Hospital, University of Hong Kong, Pokfulam, Hong Kong. Institute of Chemistry and Technology of Polymers, Catania, Sicily, Italy. Structural and Functional Glycobiology Unit, University of Lille 1, Lille 1, France.