Nathalie Verpoorten

Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11–q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20–α-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.

Mutations in the Small GTP-ase Late Endosomal Protein RAB7 Cause Charcot-Marie-Tooth Type 2B Neuropathy

Charcot-Marie-Tooth type 2B (CMT2B) is clinically characterized by marked distal muscle weakness and wasting and a high frequency of foot ulcers, infections, and amputations of the toes because of recurrent infections. CMT2B maps to chromosome 3q13-q22. We refined the CMT2B locus to a 2.5-cM region and report two missense mutations (Leu129Phe and Val162Met) in the small GTP-ase late endosomal protein RAB7 which causes the CMT2B phenotype in three extended families and in three patients with a positive family history. The alignment of RAB7 orthologs shows that both missense mutations target highly conserved amino acid residues. RAB7 is ubiquitously expressed, and we found expression in sensory and motor neurons.

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein αA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

Heterozygous missense mutations in BSCL2 are associated with distal hereditary motor neuropathy and Silver syndrome

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12–q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.

Mutations in SEPT9 cause hereditary neuralgic amyotrophy

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy affecting the brachial plexus. HNA is triggered by environmental factors such as infection or parturition. We report three mutations in the gene septin 9 (SEPT9) in six families with HNA linked to chromosome 17q25. HNA is the first monogenetic disease caused by mutations in a gene of the septin family. Septins are implicated in formation of the cytoskeleton, cell division and tumorigenesis.

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity

Influx of Ca2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

Mutations in a Gene Encoding a Novel SH3/TPR Domain Protein Cause Autosomal Recessive Charcot-Marie-Tooth Type 4C Neuropathy

Charcot-Marie-Tooth disease type 4C (CMT4C) is a childhood-onset demyelinating form of hereditary motor and sensory neuropathy associated with an early-onset scoliosis and a distinct Schwann cell pathology. CMT4C is inherited as an autosomal recessive trait and has been mapped to a 13-cM linkage interval on chromosome 5q23-q33. By homozygosity mapping and allele-sharing analysis, we refined the CMT4C locus to a suggestive critical region of 1.7 Mb. We subsequently identified mutations in an uncharacterized transcript, KIAA1985, in 12 families with autosomal recessive neuropathy. We observed eight distinct protein-truncating mutations and three nonconservative missense mutations affecting amino acids conserved through evolution. In all families, we identified a mutation on each disease allele, either in the homozygous or in the compound heterozygous state. The CMT4C gene is strongly expressed in neural tissues, including peripheral nerve tissue. The translated protein defines a new protein family of unknown function with putative orthologues in vertebrates. Comparative sequence alignments indicate that members of this protein family contain multiple SH3 and TPR domains that are likely involved in the formation of protein complexes.

Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine-nucleotide exchange factor 10

Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.

Experimental Charcot-Marie-Tooth type 1A: A cDNA microarrays analysis

To reveal the spectrum of genes that are modulated in Charcot-Marie-Tooth neuropathy type 1A (CMT1A), which is due to overexpression of the gene coding for the peripheral myelin protein 22 (pmp22), we performed a cDNA microarray experiment with cDNA from sciatic nerves of a rat model of the disease. In homozygous pmp22 overexpressing animals, we found a significant down-regulation of 86 genes, while only 23 known genes were up-regulated, suggesting that the increased dosage of pmp22 induces a general down-regulation of gene expression in peripheral nerve tissue. Classification of the modulated genes into functional categories leads to the identification of some pathways altered by overexpression of pmp22. In particular, a selective down-regulation of the ciliary neurotrophic factor transcript and of genes coding for proteins involved in cell cycle regulation, for cytoskeletal components and for proteins of the extracellular matrix, was observed. Cntf expression was further studied by real-time PCR and ELISA technique in pmp22 transgenic sciatic nerves, human CMT1A sural nerve biopsies, and primary cultures of transgenic Schwann cells. According to the results of cDNA microarray analysis, a down-regulation of cntf, both at the mRNA and protein level, was found in all the conditions tested. These results are relevant to reveal the molecular function of PMP22 and the pathogenic mechanism of CMT1A. In particular, finding a specific reduction of cntf expression in CMT1A Schwann cells suggests that overexpression of pmp22 significantly affects the ability of Schwann cells to offer a trophic support to the axon, which could be a factor, among other, responsible for the development of axonal atrophy in human and experimental CMT1A.

Autosomal dominant axonal Charcot-Marie-Tooth disease type 2 (CMT2G) maps to chromosome 12q12–q13.3

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of disorders that involve the peripheral nervous system.1 It is characterised by progressive distal neurogenic muscular atrophy and weakness that initially affects the peroneal muscles and later the hands. Charcot-Marie-Tooth disease type 1 (CMT1), also called hereditary motor and sensory neuropathy type I (HMSN I), is a dominantly inherited demyelinating neuropathy characterised by reduced nerve conduction velocities (NCV) (motor median NCV <38 m/s). Charcot-Marie-Tooth disease type 2 (CMT) 2, or HMSN II, is a dominantly inherited axonal neuropathy characterised by normal or slightly reduced NCV. Both autosomal dominant CMT1 and autosomal dominant CMT2 are genetically heterogeneous, with five and six loci, respectively.2 Most patients with CMT1 have a 1.4 Mb tandem duplication on chromosome 17p11.2 (CMT1A (MIM 118220)).3,4 Other patients with CMT1 may have point mutations in the peripheral myelin protein 22 gene ( PMP22 in CMT1A (MIM 60197)),5 myelin protein zero gene ( MPZ in CMT1B (MIM 159440)),6 lipopolysaccharide induced tumour necrosis factor gene ( LITAF in CMT1C (MIM 603795)),7 early growth response 2 gene ( EGR2 in CMT1D (MIM 129010)),8 or the gap junction protein beta 1 gene ( GJB1 in CMT1X (MIM 304040)).9 Patients with CMT2 may have point mutations in the kinesin family member 1B gene ( KIF1B in CMT2A (MIM 605995)),10 RAB7, member RAS oncogene family ( RAB7 in CMT2B (MIM 602298)),11 glycyl tRNA synthetase ( GARS in CMT2D (MIM 600287)),12 or neurofilament light polypeptide ( NEFL in CMT2E (MIM 607684)).13 The genes for CMT2C and CMT2F have not been identified yet.14,15 Some patients with CMT2 have also been reported to have specific mutations in the MPZ gene.16,17 We previously described a large Spanish family diagnosed with autosomal dominant CMT2.18 For this study, we enlarged the …