Cloning, expression and characterization of the murine orthologue of SBF2, the gene mutated in Charcot-Marie-Tooth disease type 4B2

Autosomal recessive hereditary motor and sensory neuropathy (HMSN) or Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. The clinical picture includes progressive distal weakness and atrophy, foot deformities, and distal sensory loss. For autosomal recessive CMT type 4B2 one locus was mapped to chromosome 11p15. Recently, mutations in SET binding factor 2 (SBF2), were identified as cause of CMT4B2. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and all disease-associated mutations known to date lead to shortened or truncated proteins, also implicating loss-of-function. Here, we describe the molecular cloning and the expression pattern of Sbf2. The mRNA spans around 8 kb, and the protein shares high amino acid identity compared to the human protein suggesting a conserved function. Sbf2 is encoded by 40 exons on murine chromosome 7. In situ hybridization, Northern blots and RT-analysis revealed a very broad pattern of Sbf2 expression. Overexpressed epitope tagged Sbf2 showed cytoplasmic distribution. Taken together, this study provides information about the mRNA expression and subcellular localization of Sbf2 and as such helps in further understanding its function in development and disease.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

The CMT4B disease‐causing proteins MTMR2 and MTMR13/SBF2 regulate AKT signalling

Charcot‐Marie‐Tooth disease type 4B is caused by mutations in the genes encoding either the lipid phosphatase myotubularin‐related protein‐2 (MTMR2) or its regulatory binding partner MTMR13/SBF2. Mtmr2 dephosphorylates PI‐3‐P and PI‐3,5‐P2 to form phosphatidylinositol and PI‐5‐P, respectively, while Mtmr13/Sbf2 is an enzymatically inactive member of the myotubularin protein family. We have found altered levels of the critical signalling protein AKT in mouse mutants for Mtmr2 and Mtmr13/Sbf2. Thus, we analysed the influence of Mtmr2 and Mtmr13/Sbf2 on signalling processes. We found that overexpression of Mtmr2 prevents the degradation of the epidermal growth factor receptor (EGFR) and leads to sustained Akt activation whereas Erk activation is not affected. Mtmr13/Sbf2 counteracts the blockage of EGFR degradation without affecting prolonged Akt activation. Our data indicate that Mtmr2 and Mtmr13/Sbf2 play critical roles in the sorting and modulation of cellular signalling which are likely to be disturbed in CMT4B.     

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.     

Molecular characterization and expression analysis of Mtmr2, mouse homologue of MTMR2, the Myotubularin-related 2 gene, mutated in CMT4B

Charcot-Marie-Tooth type 4B (CMT4B) is caused by mutations in the myotubularin-related 2 gene, MTMR2, on chromosome 11q22. To date, six loss of function mutations and one missense mutation have been demonstrated in CMT4B patients. It remains to be determined how dysfunction of a ubiquitously expressed phosphatase causes a demyelinating neuropathy. An animal model for CMT4B would provide insights into the pathogenesis of this disorder. We have therefore characterized the mouse homologue of MTMR2 by reconstructing the full-length Mtmr2 cDNA as well as the genomic structure. The 1932 nucleotide open reading frame corresponds to 15 coding exons, spanning a genomic region of approximately 55 kilobases, on mouse chromosome 9 as demonstrated by fluorescence in situ hybridization analysis. A comparison between the mouse and human genes revealed a similar genomic structure, except for the number of alternatively spliced exons in the 5'-untranslated region, two in mouse and three in man. In situ hybridization analysis of mouse embryos showed that Mtmr2 was ubiquitously expressed during organogenesis at E9.5, with some areas of enriched expression. At E14.5, Mtmr2 mRNA was more abundant in the peripheral nervous system, including in dorsal root ganglia and spinal roots.     

Small heat-shock protein 22 mutated in autosomal dominant Charcot-Marie-Tooth disease type 2L

Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. We have previously described a large Chinese CMT family and assigned the locus underlying the disease (CMT2L; OMIM 608673) to chromosome 12q24. Here, we report a novel c.423GT (Lys141Asn) missense mutation of small heat-shock protein 22-kDa protein 8 (encoded by HSPB8), which is also responsible for distal hereditary motor neuropathy type (dHMN) II. No disease-causing mutations have been identified in another 114 CMT families.     

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 (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 p_o gene localized on chromosome Iq21-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.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.     

腓骨肌萎缩症4型遗传学研究进展

腓骨肌萎缩症也称夏科-马利-杜斯氏病(Charcot-Marie-Tooth disease, CMT),是人类最常见的遗传性周围神经病之一,其遗传方式以常染色体显性遗传为主,也有部分呈常染色体隐性遗传或X连锁显性或隐性遗传。根据临床表型将CMT分为脱髓鞘型(CMT1)、轴突型(CMT2)和中间型（DI-CMT）。常染色体隐性遗传的CMT1(AR-CMT1,也称CMT4型)临床表现除了CMT常见的四肢远端进行性肌无力和萎缩,以及高足弓和爪形手外,常起病早,进展迅速,并有不同程度的感觉障碍和脊柱畸形(以脊柱侧凸为主)。近年来的研究显示,CMT4有11种亚型,其中有些亚型的致病机制较明确,有些亚型存在建立者突变,有些亚型还局限在临床描述和突变检出上。文章综述了CMT4的最新研究进展,包括各亚型的临床表现、致病机制和小鼠模型等。     

Identification of a New Locus for Autosomal Recessive Charcot–Marie–Tooth Disease with Focally Folded Myelin on Chromosome 11p15

Autosomal recessive Charcot–Marie–Tooth disease type 4B (CMT4B) is a demyelinating hereditary motor and sensory neuropathy characterized by abnormal folding of myelin sheaths. A locus for CMT4B has previously been mapped to chromosome 11q23 in a southern Italian pedigree. We initially excluded linkage in two Tunisian families with CMT4B to chromosome 11q23, demonstrating genetic heterogeneity within the CMT4B phenotype. Subsequently, using homozygosity mapping and linkage analysis in the largest Tunisian pedigree, we mapped a new locus to chromosome 11p15. A maximum two-point lod score of 6.05 was obtained with the marker D11S1329. Recombination events refined the CMT4B locus region to a 5.6-cM interval between markers D11S1331 and D11S4194. The second Tunisian CMT4B family was excluded from linkage to the new locus, demonstrating the existence of at least a third locus for the CMT4B phenotype.     

Axonal Transport Defects in a Mitofusin 2 Loss of Function Model of Charcot-Marie-Tooth Disease in Zebrafish

Charcot-Marie-Tooth disease (CMT) represents a group of neurodegenerative disorders typically characterised by demyelination (CMT1) or distal axon degeneration (CMT2) of motor and sensory neurons. The majority of CMT2 cases are caused by mutations in mitofusin 2 (MFN2); an essential gene encoding a protein responsible for fusion of the mitochondrial outer membrane. The mechanism of action of MFN2 mutations is still not fully resolved. To investigate a role for loss of Mfn2 function in disease we investigated an ENU-induced nonsense mutation in zebrafish MFN2 and characterised the phenotype of these fish at the whole organism, pathological, and subcellular level. We show that unlike mice, loss of MFN2 function in zebrafish leads to an adult onset, progressive phenotype with predominant symptoms of motor dysfunction similar to CMT2. Mutant zebrafish show progressive loss of swimming associated with alterations at the neuro-muscular junction. At the cellular level, we provide direct evidence that mitochondrial transport along axons is perturbed in Mfn2 mutant zebrafish, suggesting that this is a key mechanism of disease in CMT. The progressive phenotype and pathology suggest that zebrafish will be useful for further investigating the disease mechanism and potential treatment of axonal forms of CMT. Our findings support the idea that MFN2 mutation status should be investigated in patients presenting with early-onset recessively inherited axonal CMT.     

Truncating and Missense Mutations in IGHMBP2 Cause Charcot-Marie Tooth Disease Type 2

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-μ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5′ region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.     

Mutations in FGD4 encoding the Rho GDP/GTP exchange factor FRABIN cause autosomal recessive Charcot-Marie-Tooth type 4H

Charcot-Marie-Tooth (CMT) disorders are a clinically and genetically heterogeneous group of hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities, and electrophysiological changes. The CMT4H subtype is an autosomal recessive demyelinating form of CMT that was recently mapped to a 15.8-Mb region at chromosome 12p11.21-q13.11, in two consanguineous families of Mediterranean origin, by homozygosity mapping. We report here the identification of mutations in FGD4, encoding FGD4 or FRABIN (FGD1-related F-actin binding protein), in both families. FRABIN is a GDP/GTP nucleotide exchange factor (GEF), specific to Cdc42, a member of the Rho family of small guanosine triphosphate (GTP)-binding proteins (Rho GTPases). Rho GTPases play a key role in regulating signal-transduction pathways in eukaryotes. In particular, they have a pivotal role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division. Consistent with these reported functions, expression of truncated FRABIN mutants in rat primary motoneurons and rat Schwann cells induced significantly fewer microspikes than expression of wild-type FRABIN. To our knowledge, this is the first report of mutations in a Rho GEF protein being involved in CMT.     

A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene

Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. The axonal form of the disease is designated as "CMT type 2" (CMT2). Although four loci known to be implicated in autosomal dominant CMT2 have been mapped thus far (on 1p35-p36, 3q13. 1, 3q13-q22, and 7p14), no one causative gene is yet known. A large Russian family with CMT2 was found in the Mordovian Republic (Russia). Affected members had the typical CMT2 phenotype. Additionally, several patients suffered from hyperkeratosis, although the association, if any, between the two disorders is not clear. Linkage with the CMT loci already known (CMT1A, CMT1B, CMT2A, CMT2B, CMT2D, and a number of other CMT-related loci) was excluded. Genomewide screening pinpointed the disease locus in this family to chromosome 8p21, within a 16-cM interval between markers D8S136 and D8S1769. A maximum two-point LOD score of 5.93 was yielded by a microsatellite from the 5' region of the neurofilament-light gene (NF-L). Neurofilament proteins play an important role in axonal structure and are implicated in several neuronal disorders. Screening of affected family members for mutations in the NF-L gene and in the tightly linked neurofilament-medium gene (NF-M) revealed the only DNA alteration linked with the disease: a A998C transversion in the first exon of NF-L, which converts a conserved Gln333 amino acid to proline. This alteration was not found in 180 normal chromosomes. Twenty unrelated CMT2 patients, as well as 26 others with an undetermined form of CMT, also were screened for mutations in NF-L, but no additional mutations were found. It is suggested that Gln333Pro represents a rare disease-causing mutation, which results in the CMT2 phenotype.     

Clinical and genetic heterogeneity of Charcot-Marie-Tooth disease.

The autosomal dominant forms of hereditary motor and sensory neuropathies include the hypertrophic form (CMT1) and the neuronal form of Charcot-Marie-Tooth disease (CMT2). While at least two distinct loci have been shown to be linked to the CMT1 phenotype (CMT1A and CMT1B, on chromosomes 17 and 1, respectively), whether the CMT2 phenotype results from mutations allelic to either of the CMT1 genes remains unknown. Studying one CMT1 and two CMT2 pedigrees, we were able to exclude the CMT2 disease locus from the region of chromosome 17 (Z = -2.80 at theta = 0.05 for D17S58) where the CMT1A gene maps (Z = +3.67 at theta = 0.00). Similarly, negative lod score values were obtained in CMT2 for the region of chromosome 1 where the CMT1B gene has been located (Z = -3.09 at theta = 0.05 for D1S61). The present study therefore provides evidence for genetic heterogeneity between the hypertrophic and the neuronal forms of Charcot-Marie-Tooth disease and demonstrates that the CMT2 gene is not allelic to either of the CMT1 genes mapped to date.     

Homozygous Defects in LMNA, Encoding Lamin A/C Nuclear-Envelope Proteins, Cause Autosomal Recessive Axonal Neuropathy in Human (Charcot-Marie-Tooth Disorder Type 2) and Mouse

The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.     

Identification of A Novel SBF2 Frameshift Mutation in Charcot–Marie–Tooth Disease Type 4B2 Using Whole-exome Sequencing

Abstract Charcot-Marie-Tooth disease type 4B2 with early-onset glaucoma （CMT4B2, OMIM 604563） is a genetically-heterogeneous childhood-onset neuromuscular disorder. Here, we report the case of a 15-year-old male adolescent with lower extremity weakness, gait abnormalities, foot deformities and early-onset glaucoma. Since clinical diagnosis alone was insufficient for providing pathogenetic evidence to indicate that the condition belonged to a consanguineous family, we applied whole-exome sequencing to samples from the patient, his parents and his younger brother, assuming that the patient＇s condition is transmitted in an autosomal recessive pattern. A frame-shift mutation, c.4571delG （P.Gly1524Glufs＊42）, was revealed in the CMT4B2-related gene SBF2 （also known as MTMR13, MIM 607697）, and this mutation was found to be homozygous in the proband and heterozygous in his parents and younger brother. Together with the results of clinical diagnosis, this case was diagnosed as CMT4B2. Our finding further demonstrates the use of whole-exome sequencing in the diagnosis and treatment of rare diseases.     

Characterization of the Rab7K157N mutant protein associated with Charcot-Marie-Tooth type 2B

Four missense mutations, that target highly conserved amino acid residues in the small GTPase Rab7, have been associated with the Charcot-Marie-Tooth (CMT) type 2B phenotype. CMT2B peripheral axonal neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Here, we have investigated the biochemical and functional properties of the Rab7 K157N mutated protein. Interestingly, Rab7 K157N showed altered nucleotide exchange rate and GTP hydrolysis compared to the wild type protein. Consistently, the majority of the expressed protein in HeLa cells was bound to GTP. In addition, Rab7 K157N was able to restore EGF degradation, previously inhibited by Rab7 silencing. Altogether these data indicate that Rab7 K157N, similarly to the other three mutated proteins causative of CMT2B, is predominantly in the GTP-bound form and behaves as an active mutant. Therefore, activated forms of Rab7 protein cause the CMT2B disease.