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A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency.

ABSTRACT: Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.

SUBMITTER: Cline A

PROVIDER: S-EPMC3484097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPhosphomannomutase deficiency (PMM2-CDG) is the most frequent congenital disorder of glycosylation. The cerebellum is nearly always affected in PMM2-CDG patients, a cerebellar atrophy progression is observed, and cerebellar dysfunction is their main daily functional limitation. Different therapeutic agents are under development, and clinical evaluation of drug candidates will require a standardized score of cerebellar dysfunction. We aim to assess the validity of the International Cooperative Ataxia Rating Scale (ICARS) in children and adolescents with genetically confirmed PMM2-CDG deficiency. We compare ICARS results with the Nijmegen Pediatric CDG Rating Scale (NPCRS), neuroimaging, intelligence quotient (IQ) and molecular data.MethodsOur observational study included 13 PMM2-CDG patients and 21 control subjects. Ethical permissions and informed consents were obtained. Three independent child neurologists rated PMM2-CDG patients and control subjects using the ICARS. A single clinician administered the NPCRS. All patients underwent brain MRI, and the relative diameter of the midsagittal vermis was measured. Psychometric evaluations were available in six patients. The Mann-Whitney U test was used to compare ICARS between patients and controls. To evaluate inter-observer agreement in patients' ICARS ratings, intraclass correlation coefficients (ICC) were calculated. ICARS internal consistency was evaluated using Cronbach's alpha. Spearman's rank correlation coefficient test was used to correlate ICARS with NPCRS, midsagittal vermis relative diameter and IQ.ResultsICARS and ICARS subscores differed between patients and controls (p < 0.001). Interobserver agreement of ICARS was "almost perfect" (ICC = 0.99), with a "good" internal reliability (Cronbach's alpha = 0.72). ICARS was significantly correlated with the total NPCRS score (rs 0.90, p < 0.001). However, there was no agreement regarding categories of severity. Regarding neuroimaging, inverse correlations between ICARS and midsagittal vermis relative diameter (rs -0.85, p = 0.003) and IQ (rs -0.94, p = 0.005) were found. Patients bearing p.E93A, p.C241S or p.R162W mutations presented a milder phenotype.ConclusionsICARS is a reliable instrument for assessment of PMM2-CDG patients, without significant inter-rater variability. Despite our limited sample size, the results show a good correlation between functional cerebellar assessment, IQ and neuroimaging. For the first a correlation between ICARS, neuroimaging and IQ in PMM2-CDG patients has been demonstrated.

Project description:BackgroundPMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder.ObjectiveTo evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors.MethodsWe studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay.ResultsWe found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules.ConclusionsOur results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present work improves our understanding of the immunological functions in PMM2-CDG and possibly in other CDG-I types.

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A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency.

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