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de Almeida R.A.,University of Manchester | Fogli A.,French Institute of Health and Medical Research | Fogli A.,University Of Clermont | Fogli A.,Estaing University Hospital Center | And 7 more authors.
PLoS ONE | Year: 2013

Recessive inherited mutations in any of five subunits of the general protein synthesis factor eIF2B are responsible for a white mater neurodegenerative disease with a large clinical spectrum. The classical form is called Childhood Ataxia with CNS hypomyelination (CACH) or Vanishing White Matter Leukoencephalopathy (VWM). eIF2B-related disorders affect glial cells, despite the fact that eIF2B is a ubiquitous protein that functions as a guanine-nucleotide exchange factor (GEF) for its partner protein eIF2 in the translation initiation process in all eukaryotic cells. Decreased eIF2B activity measured by a GEF assay in patients' immortalised lymphocytic cells provides a biochemical diagnostic assay but is limited by the availability of eIF2 protein, which is classically purified from a mammalian cell source by column chromatography. Here we describe the generation of a recombinant expression system to produce purified human eIF2 from yeast cells. We demonstrate that human eIF2 can function in yeast cells in place of the equivalent yeast factor. We purify human eIF2 and the C-terminal domain of human eIF2Bε using affinity chromatography from engineered yeast cells and find that both function in a GEF assay: the first demonstration that this human eIF2Bε domain has GEF function. We show that CACH/VWM mutations within this domain reduce its activity. Finally we demonstrate that the recombinant eIF2 functions similarly to eIF2 purified from rat liver in GEF assays with CACH/VWM eIF2B-mutated patient derived lymphocytic cells. © 2013 de Almeida et al.

Ferreira M.-C.,Molecular Biology Laboratory | Ferreira M.-C.,French Institute of Health and Medical Research | Dorboz I.,Molecular Biology Laboratory | Dorboz I.,French Institute of Health and Medical Research | And 3 more authors.
Clinical Biochemistry | Year: 2015

Objectives: The aim of this study was to develop a reliable, rapid and cost-effective molecular diagnostic assay allowing widespread routine investigation of eIF2B-related disorders (CACH/VWM syndrome). This heterogeneous disease is caused by autosomal recessive mutations in the genes encoding the five subunits of the translation-initiation factor eIF2B. Such a diagnostic method would be particularly adapted to the apparently acute presentation of the disease. Design and methods: We developed a multiplex PCR amplification method for 7 genomic regions of the eIF2B genes in a single run. This method targeted the 8 most frequent mutations representing 61.4% of all mutations identified to date in our laboratory. These mutations affected eIF2B2 exon 5, eIF2B3 exon 2, eIF2B4 exons 8 and 11 and eIF2B5 exons 5, 7 and 8. PCR products were then pooled and subjected to a primer-extension assay validated using previously genotyped samples. Results: The results were compared to screening and/or direct sequencing methods: 100% agreement between methods confirmed equivalent sensitivity and specificity. The new assay was highly superior in terms of cost, time to results and robustness despite sample heterogeneity. Conclusions: This genotyping strategy allows the detection of all eIF2B mutations targeted. A second multiplex primer-extension assay is in development to detect the 11 next-most frequent mutations, thus raising the global detection rate to 76.8%. Our approach is widely applicable as it involves standard techniques and equipment. Moreover, it can easily be further adapted to the clinical and genetic heterogeneity of eIF2B-related disorders by including or excluding mutations. © 2015 The Canadian Society of Clinical Chemists.

Ferreira M.-C.,Molecular Biology Laboratory | Ferreira M.-C.,French Institute of Health and Medical Research | Ferreira M.-C.,Laboratoire Gen Bio | Dorboz I.,University Paris Diderot | And 4 more authors.
European Journal of Medical Genetics | Year: 2015

Alexander disease (AxD), a fatal degenerative leukoencephalopathy, is caused by de novo heterozygous missense mutations in the Glial Fibrillary Acidic Protein (GFAP) gene. The pathological hallmark of the disease is the presence of Rosenthal fibers, cytoplasmic inclusions in astrocytes, composed mainly of GFAP, αB-crystallin and HSP27.To date, several patients with a typical presentation of the disease or displaying characteristic Rosenthal fibers in brain material have been reported with no GFAP mutation. Recently, several studies have demonstrated a correlation between Rosenthal fiber formation and wild-type GFAP overexpression, despite the absence of mutations.We tested the hypothesis that a GFAP gene rearrangement could modulate AxD severity or promote GFAP overexpression and aggregation, resulting in leukoencephalopathy. A QMPSF assay was validated for 11 exonic fragments: 3 in control genes (. CFTR, DSCR1, F9) and 8 corresponding to GFAP exons. A total of 97 patients suspected of AxD were analyzed: 28 with a GFAP mutation; 69 with clinical and magnetic resonance imaging criteria compatible with the disease. Neither duplications nor deletions of GFAP were found, suggesting that GFAP coding-region rearrangements may not be involved in AxD or Alexander-related leukoencephalopathies.In addition, 80 patients with undetermined leukodystrophies, and negative for PLP1, GJA12, Sox10 and MCT8 mutations and PLP1 and Lamin B1 rearrangements, were tested. These patients were also negative for GFAP rearrangements.Other hypotheses should be investigated for a molecular diagnosis in patients with undetermined leukoencephalopathy: mutations in GFAP isoforms, splicing sites or regulatory regions, or defaults in genes encoding molecular partners of GFAP. © 2015 Elsevier Masson SAS.

Svenstrup K.,Copenhagen University | Giraud G.,French Institute of Health and Medical Research | Boespflug-Tanguy O.,French Institute of Health and Medical Research | Boespflug-Tanguy O.,Reference Center for Leukodystrophies | And 9 more authors.
Journal of Neurology, Neurosurgery and Psychiatry | Year: 2010

Background: Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterised by progressive spasticity and weakness in the lower limbs. Mutations in PLP1 on the X chromosome cause spastic paraplegia type 2 (SPG2) or the allelic Pelizaeus-Merzbacher Disease (PMD). The PLP1 protein is a major myelin protein involved in stabilisation and maintenance of the myelin sheath. The function of the protein has been studied in the rumpshaker mouse, which is a model of SPG2/PMD. Objective: To characterise the phenotype of patients with the 'rumpshaker mutation.' Patients: A family with HSP caused by the 'rumpshaker mutation.' Results: The patients showed nystagmus during infancy and had early onset of HSP. They had normal cognition, and cerebral MRI showed relatively unspecific white matter abnormalities on T2 sequences without clear progression. Urinary urgency was reported among the female carriers. MRS of both patients showed increased myo-inositol in the white matter, while decreased N-acetylaspartate was found exclusively in the oldest patient. All evoked potential examinations were compatible with severe central demyelination, while no signs of peripheral demyelination or axonal degeneration were found. 18F-FDG-PET scans were normal. Conclusion: The phenotypes of the patients reported here are the mildest described to be caused by the rumpshaker mutation and represent the mildest form among the spectrum of PLP1 related disorders. No definite symptoms in the female carriers could be ascribed to the mutation. These data suggest the pathology to be an underlying dysmyelinating disorder in combination with a central axonal degeneration.

Tonduti D.,Neurological Institute C Besta Foundation Irccs | Tonduti D.,University Paris Diderot | Aiello C.,Bambino Gesu Childrens Research Hospital | Renaldo F.,University Paris Diderot | And 18 more authors.
European Journal of Paediatric Neurology | Year: 2016

Background Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) was first described in 2002. After the recent identification of TUBB4A mutation as the genetic basis of the disease, the clinical and neuroimaging phenotype related to TUBB4A mutations expanded, ranging from primary dystonia type 4 with normal MRI to severe H-ABC cases. Patients and methods The study included patients referred to us for an unclassified hypomyelinating leukodystrophy. We selected patients with deleterious heterozygous TUBB4A mutations. Molecular analysis of TUBB4A was performed on genomic DNA extracted from peripheral blood. Results The series included 12 patients (5 females and 7 males). Five patients carried the common mutation c.745G > A (p.Asp249Asn), while the remaining harbored different mutations. Three new mutations were found in 5 patients. Clinical and neuroimaging observations are described. A clear correlation between the clinical presentation and the genotype seems to be absent in our group of 12 patients. Conclusions TUBB4A-mutated patients manifest a comparable clinical and neuroimaging picture but they can differ from each other in terms of rate of disease progression. Extrapyramidal signs can be absent in the first stages of the disease, and a careful evaluation of MRI is fundamental to obtain the final diagnosis. From a therapeutic perspective a trial with l-dopa should be considered in all patients presenting extrapyramidal symptoms. © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

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