Alexander disease: putative mechanisms of an astrocytic encephalopathy.

Alexander disease (AXD) is the first primary astrocytic disorder. This encephalopathy is caused by dominant mutations in the glial fibrillary acidic protein (GFAP) gene, encoding the main intermediate filament of astrocyte. Pathologically, this neurodegenerative disease is characterised by dystrophic astrocytes containing intermediate filament aggregates associated with myelin abnormalities. More than 20 GFAP mutations have been reported. Many of them cluster in highly conserved regions between several intermediate filaments. Contrary to other intermediate filament-related diseases, AXD seems to be the consequence of a toxic gain of function induced by aggregates. This is supported by the phenotype of mice overexpressing human GFAP. Nevertheless, GFAP null mice display myelin abnormalities and blood-brain barrier dysfunction that are present in AXD. Given the pivotal role of astrocytes in brain physiology, there are many possibilities for astrocytes to dysfunction and to impair the functions of other cells. Physiopathological hypotheses are discussed in the frame of AXD.

Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation.

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism.

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein.

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.

Linkage analysis of candidate myelin genes in familial multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. A complex genetic etiology is thought to underlie susceptibility to this disease. The present study was designed to analyze whether differences in genes that encode myelin proteins influence susceptibility to MS. We performed linkage analysis of MS to markers in chromosomal regions that include the genes encoding myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMGP), and myelin oligodendrocyte glycoprotein (MOG) in a well-characterized population of 65 multiplex MS families consisting of 399 total individuals, 169 affected with MS and 102 affected sibpairs. Physical mapping data permitted placement of MAG and PLP genes on the Genethon genetic map; all other genes were mapped on the Genethon genetic map by linkage analysis. For each gene, at least one marker within the gene and/or two tightly linked flanking markers were analyzed. Marker data analysis employed a combination of genetic trait model-dependent (parametric) and model-independent linkage methods. Results indicate that MAG, MBP, OMGP, and PLP genes do not have a significant genetic effect on susceptibility to MS in this population. As MOG resides within the MHC, a potential role of the MOG gene could not be excluded.

Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases.

We report neuropathological, biochemical and molecular studies on two patients with childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome, a leukodystrophy recently defined according to clinical and radiological criteria. Both had severe cavitating orthochromatic leukodystrophy without atrophy, predominating in hemispheric white matter, whereas U-fibers, internal capsule, corpus callosum, anterior commissure and cerebellar white matter were relatively spared. The severity of white matter lesions contrasted with the rarity of myelin breakdown products and astroglial and microglial reactions. In the white matter, there was an increase in a homogeneous cell population with the morphological features of oligodendrocytes, in many instances presenting an abundant cytoplasm like myelination glia. These cells were negative for glial fibrillary acidic protein and antibodies PGM1 and MIB1. Some were positive for myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, but the majority were positive for human 2'-3' cyclic nucleotide 3' phosphodiesterase and all were positive for carbonic anhydrase II, confirming that they are oligodendrocytes. Myelin protein and lipid content were reduced. The PLP gene, analyzed in one case, was not mutated or duplicated. The increased number of oligodendrocytes without mitotic activity suggests an intrinsic oligodendroglial defect or an abnormal interaction with axons or other glial cells. This neuropathological study supports the notion that CACH syndrome constitutes a specific entity.

Connexin32 mutations associated with X-linked Charcot-Marie-Tooth disease show two distinct behaviors: loss of function and altered gating properties.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in the gene encoding connexin32 (Cx32), which is expressed in Schwann cells. We have compared the functional properties of 11 Cx32 mutations with those of the wild-type protein by testing their ability to form intercellular channels in the paired oocyte expression system. Although seven mutations were functionally incompetent, four others were able to generate intercellular currents of the same order of magnitude as those induced by wild-type Cx32 (Cx32wt). In homotypic oocyte pairs, CMTX mutations retaining functional activity induced the development of junctional currents that exhibited changes in the sensitivity and kinetics of voltage dependence with respect to that of Cx32wt. The four mutations were also capable of interacting in heterotypic configuration with the wild-type protein, and in one case the result was a marked rectification of junctional currents in response to voltage steps of opposite polarity. In addition, the functional CMTX mutations displayed the same selective pattern of compatibility as Cx32wt, interacting with Cx26, Cx46, and Cx50 but failing to do so with Cx40. Although the functional mutations exhibited sensitivity to cytoplasmic acidification, which induced a >/=80% decrease in junctional currents, both the rate and extent of channel closure were enhanced markedly for two of them. Together, these results indicate that the functional consequences of CMTX mutations of Cx32 are of two drastically distinct kinds. The presence of a functional group of mutations suggests that a selective deficit of Cx32 channels may be sufficient to impair the homeostasis of Schwann cells and lead to the development of CMTX.

Membrane topology of the myelin/oligodendrocyte glycoprotein.

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the mammalian central nervous system, is located on the surface of the oligodendrocyte plasma membrane and the outermost lamellae of mature myelin; it is expressed during the latter steps of myelinogenesis. It has been shown that MOG may play a pathological role in autoimmune demyelinating diseases of the central nervous system, although its physiological function remains unknown. MOG is an integral membrane glycoprotein with an extracellular immunoglobulin-like domain and two hydrophobic segments which were predicted to be membrane-spanning on the basis of hydropathy analysis. As a first step in elucidation of MOG function, we have investigated its membrane topology, combining immunofluorescence studies on cultured oligodendrocytes and MOG-transfected Chinese hamster ovary cells with biochemical analyses, including in vitro translation, membrane insertion and protease-digestion assays. Our results indicate that the C-terminal tail of MOG is located into the cytoplasm, and that only the first hydrophobic region of MOG spans the membrane whereas the second hydrophobic region appears to be semi-embedded in the lipid bilayer, lying partially buried in the membrane with its N-terminal and C-terminal boundaries facing the cytoplasm.

Functional analysis of the mouse myelin/oligodendrocyte glycoprotein gene promoter in the oligodendroglial CG4 cell line.

Myelin/oligodendrocyte glycoprotein (MOG) is a late phylogenetic acquisition among vertebrates that is found only in mammals. MOG is a minor component of myelin protein, representing approximately 0.01-0.05% of the total. Regulatory elements in the MOG gene were identified by transfecting the oligodendroglial CG4 cell line with chimeric MOG-luciferase genes. Only a few hundred base pairs upstream of the coding sequence were necessary for high-level activity of the mouse MOG promoter. More distal recognition sites may exist, because silencing activity, indicative of negative regulatory elements, was detected upstream of base pair 657. Transcriptional activity of chimeric MOG- and myelin basic protein-luciferase genes was greater in CG4 cells than in 3T3 fibroblasts or C6 glioblastoma, demonstrating their superiority for functional analysis of myelin gene regulatory elements.

New mutations in the X-linked form of Charcot-Marie-Tooth disease.

Mutations in the gene for connexin 32 are associated with a chromosome X-linked form of Charcot-Marie-Tooth disease. The prevalence of this form is probably underestimated. We screened 12 candidate families and found 7 missense mutations of which 4 are new. These mutations are located in intra- and extramembraneous parts of the protein. Some mutations are probably present with a higher frequency. This study further confirms variation of connexin 32 mutations with scarcity in the second transmembrane domain and, so far, absence in the fourth transmembrane domain and in the carboxy-terminal region.

Identification of a Val 145 Ile substitution in the human myelin oligodendrocyte glycoprotein: lack of association with multiple sclerosis. The Réseau de Recherche Clinique INSERM sur la Susceptibilité Génétique à la Sclérose en Plaques.

Myelin/oligodendrocyte glycoprotein (MOG) is a major target antigen in experimental autoimmune encephalomyelitis and it has been suggested that it may as well play a key role in the demyelination process in multiple sclerosis (MS). As MOG variants could be pathogenic in autoimmune demyelinating diseases of the central nervous system, we analysed the coding sequence of MOG in MS patients and described a G-->A transition occurring in exon 3 of the human MOG gene. The mutation predicts that isoleucine substitutes for a valine at codon 145 (Val 145 Ile) in the transmembrane region of the protein. This is the first aminoacid substitution reported in human MOG. The polymorphism can be detected by restriction enzyme digestion of genomic DNA or reverse-transcribed PCR amplified products, making it a simple tool to detect a potential implication of MOG alleles in susceptibility to MS by association study. The analysis of 83 unrelated MS patients and 82 unrelated healthy controls showed that the polymorphism is found in similar proportions in MS patients (18%) and controls (14.6%). It is therefore unlikely that the MOG Val 145 Ile variant is responsible for genetic susceptibility to MS.

X-linked dominant Charcot-Marie-Tooth neuropathy (CMTX): new mutations in the connexin32 gene.

X-linked dominant Charcot-Marie-Tooth (CMTX) neuropathy has been mapped to the Xq13 region. Subsequently, several mutations that could account for CMTX have been detected in the coding part of the connexin32 (Cx32) gene, which is located within this region. In order to develop more specific diagnostic tools, we have begun a systematic screening of families with dominant CMTX for mutations in the coding region of the Cx32 gene. This report describes a study of ten families and different mutations segregating with the disease were detected in five of them. In addition to the previously reported Arg22stop and Arg215Trp substitutions, three novel mutations are described, including two different missense mutations at codon Arg22 (Arg22Pro and Arg22Gly), and a nonsense mutation at codon Trp133. The identification of new CMTX-causing mutations is a critical step for carrier detection and presymptomatic diagnosis, and should provide essential information on the structure-function relationship of Cx32 in vitro as well as in vivo.

Charcot-Marie-Tooth type 1B neuropathy: third mutation of serine 63 codon in the major peripheral myelin glycoprotein PO gene.

We report studies on two patients (a mother and her daughter) presenting with a Charcot-Marie-Tooth type 1 (CMT1) phenotype: low nerve conduction velocities of 13-15 m/s and an early onset at the age of walking. DNA analysis of the gene coding for the major peripheral myelin protein PO showed a new point mutation in exon 2, which resulted in substitution of a phenylalanine for serine at amino acid position 63 of PO. This is the third mutation reported at this codon, the two previously described leading to CMT1B (serine 63 deletion), or to Dejerine-Sottas disease (cysteine for serine 63 substitution), suggesting that different phenotypes can result from alteration of a single amino acid, depending on the type of the change involved.

Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms.

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the central nervous system localized on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major. MOG isoform, the human MOG gene also contains, in the 3' region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement of the MOG gene in hereditary neurological disorders.

Mutations in the myelin protein zero gene associated with Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth type 1 (CMT1) disease is an autosomal dominant neuropathy of the peripheral nerve. The majority of CMT 1 cases are due to a duplication of an 1.5-Mb DNA fragment on chromosome 17p11.2 (CMT 1a). Micromutations were found in the gene for peripheral myelin protein 22 (PMP22) located in the duplicated region of CMT 1a, and in the peripheral myelin protein zero (PO) located on chromosome 1q21-q23 (CMT 1b). We have characterized two new mutations in the PO gene in two french families presenting CMT disease. Both mutations occur in the extracellular domain of the PO protein. One mutation is a de novo mutation and is from paternal origin.

Physical mapping of the human and mouse MOG gene at the distal end of the MHC class Ib region.

Myelin/oligodendrocyte glycoprotein (MOG) is expressed specifically in the central nervous system (CNS) by myelinating glial cells, the oligodendrocytes. The external location of MOG on myelin sheaths and its late expression during myelinogenesis argue for a role of MOG in the completion of myelin and maintenance of its integrity. MOG is a target autoantigen in demyelinating diseases, such as experimental autoimmune encephalomyelitis (EAE) in animals and multiple sclerosis (MS) in humans. We previously located the gene encoding MOG to the major histocompatibility complex (MHC), both in human, by cytogenetics, and in mouse, by analysis of recombinants. To refine the position, we have now selected yeast artificial chromosome clones (YAC) which contain the MOG gene. Physical mapping of the human MOG and the mouse Mog genes by characterization of these YAC clones indicated that the gene is located at the distal end of the major histocompatibility complex (MHC) class Ib region in both species. The human MOG gene lies 60 kilobases (kb) telomeric to HLA-F in a head-to-head orientation; the mouse Mog gene lies 25 (kb) telomeric to H2-M5 in a tail-to-head orientation. These orthologous genes provide markers for comparative analysis of the evolution of the MHC in the two species. The physical mapping of MOG should facilitate analysis of its role in hereditary neurological diseases, and the YAC clones identified here will permit the identification of new genes in the region.

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene.

Charcot-Marie-Tooth type 1 (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein P0 gene localized on chromosome 1q21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P0 genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P0 gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.

Characterization and expression of the cDNA coding for the human myelin/oligodendrocyte glycoprotein.

We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.

Genetic homogeneity of Pelizaeus-Merzbacher disease: tight linkage to the proteolipoprotein locus in 16 affected families. PMD Clinical Group.

Among the numerous leukodystrophies that have an early onset and no biochemical markers, Pelizaeus-Merzbacher disease (PMD) is one that can be identified using strict clinical criteria and demonstrating an abnormal formation of myelin that is restricted to the CNS in electrophysiological studies and brain magnetic resonance imaging (MRI). In PMD, 12 different base substitutions and one total deletion of the genomic region containing the PLP gene have been reported, but, despite extensive analysis, PLP exon mutations have been found in only 10%-25% of the families analyzed. To test the genetic homogeneity of this disease, we have carried out linkage analysis with polymorphic markers of the PLP genomic region in 16 families selected on strict diagnostic criteria of PMD. We observed a tight linkage of the PMD locus with markers of the PLP gene (cDNA PLP, exon IV polymorphism) and of the Xq22 region (DXS17, DXS94, and DXS287), whereas the markers located more proximally (DXYS1X and DXS3) or distally (DXS11) were not linked to the PMD locus. Multipoint analysis gave a maximal location score for the PMD locus (13.98) and the PLP gene (8.32) in the same interval between DXS94 and DXS287, suggesting that in all families PMD is linked to the PLP locus. Mutations of the extraexonic PLP gene sequences or of another unknown close gene could be involved in PMD. In an attempt to identify molecular defects of this genomic region that are responsible for PMD, these results meant that RFLP analysis could be used to improve genetic counseling for the numerous affected families in which a PLP exon mutation could not be demonstrated.

Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene.

We have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5' flanking region reveals that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerase chain reaction method.