LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症患者的突变分析

为了分析LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT)的突变特点, 文章分别应用PCR结合DNA序列分析方法和PCR-单链构象多态性(PCR-SSCP)结合DNA序列分析方法对6个常染色体显性遗传家系先证者和27个散发病例进行LITAF和RAB7基因突变分析; 应用PCR-SSCP结合DNA序列分析方法对14个常染色体遗传的CMT家系先证者和27个散发患者进行LMNA和MTMR2基因突变分析。结果发现: LITAF基因c.269G→A、c.274A→G序列变异和LMNA基因c.1243G→A、c.1910C→T序列变异, 未发现RAB7和MTMR2基因的序列变异。其中LITAF基因c.269G→A、LMNA基因c.1243G→A和c.1910C→T为新发现的单核苷酸多态; LITAF基因c.274A→G为已知多态。说明LITAF、RAB7、LMNA和MTMR2基因突变在中国人CMT患者中罕见。     

LITAF Mutations Associated with Charcot-Marie-Tooth Disease 1C Show Mislocalization from the Late Endosome/Lysosome to the Mitochondria

Charcot-Marie-Tooth (CMT) disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T). The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.     

DNA duplication associated with Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-tooth disease type 1A (CMT1A) was localized by genetic mapping to a 3 cM interval on human chromosome 17p. DNA markers within this interval revealed a duplication that is completely linked and associated with CMT1A. The duplication was demonstrated in affected individuals by the presence of three alleles at a highly polymorphic locus, by dosage differences at RFLP alleles, and by two-color fluorescence in situ hybridization. Pulsed-field gel electrophoresis of genomic DNA from patients of different ethnic origins showed a novel SacII fragment of 500 kb associated with CMT1A. A severely affected CMT1A offspring from a mating between two affected individuals was demonstrated to have this duplication present on each chromosome 17. We have demonstrated that failure to recognize the molecular duplication can lead to misinterpretation of marker genotypes for affected individuals, identification of false recombinants, and incorrect localization of the disease locus.     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Mosaicism for the Charcot-Marie-Tooth disease type 1A duplication suggests somatic reversion

A female patient with clinical signs and symptoms of a demyelinating neuropathy was shown to have a duplication of the 1.5-Mb region on chromosome 17p11.2, typical of the great majority of cases of Charcot-Marie-Tooth disease type 1A (CMT1A). However, analysis of DNA extracted from peripheral blood revealed a 2:2.4 instead of the usual 2:3 ratio between the 7.8- and 6.0-kb EcoRI fragments in the proximal and distal repetitive extragenic palindromic (REP) elements of CMT1A. Detection of a 3.2-kb EcoRI/SacI kb junction fragment with probe pLR7.8 confirmed the CMT1A duplication. The dosage of this junction fragment, compared with a 2.8-kb EcoRI/SacI fragment of the proximal REP elements of CMT1A, was 2:0.58 instead of the expected 2:1 dosage for heterozygous CMT1A duplications. We hypothesized that the lower dosages of these restriction fragments specific for the CMT1A duplication were due to mosaicism; this was confirmed by fluorescence in situ hybridization analysis with the D17S122-specific probe pVAW409R1. In peripheral blood lymphocytes the percentage of interphase nuclei with a duplication in 17p11.2 was 49%. In interphase nuclei extracted from buccal mucosa, hair-root cells or paraffin-embedded nervous tissue the duplication was detectable in 51%, 66% and 74%, respectively. This is the first report of mosaicism in a patient with a CMT1A duplication identified by three different and independent techniques. Received: 14 November 1995 / Revised: 13 February 1996     

A novel Gly137Asp MPZ mutation in a Charcot-Marie-Tooth disease type 1B family

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous peripheral neuropathy. Myelin protein zero (MPZ) is a major myelin protein of the peripheral nerve and mutations in its gene cause a wide phenotypic spectrum including CMT1B, CMT2I, CMT2J and Dejerine-Sottas syndrome. The objective of this study was to find the causative mutation in a Korean large autosomal dominant demyelinating CMT family (FC240). Clinical disabilities were measured by a functional disability scale (FDS), a CMT neuropathy score (CMTNS), and a 9-hole peg test (9-HPT). Mutational analysis of the FC240 family revealed a novel c.410G>A (Gly137Asp) mutation in the MPZ gene. The mutation was completely co-segregated with affected members in the family, and was not found in controls. The clinical features and electrophysiological findings were compatible with CMT1B. Clinical symptoms revealed phenotypic diversities among family members and generations within the same family. In addition, the present patients with Gly137Asp mutation showed earlier age at onset and slow progression than the reported patient with Gly137Ser. Therefore, it seems that there were wide phenotypic variations within the same family harboring Gly137Asp mutation, and between Gly137Asp and Gly137Ser mutations.     

Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bbeta, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.     

Localization of Charcot-Marie-Tooth disease type 1a (CMT1A) to chromosome 17p11.2.

Charcot-Marie-Tooth (CMT) disease type 1a has been previously localized to chromosome 17 using the markers D17S58 and D17S71. In that report we were unable to provide unequivocal localization of the CMT1A gene on either the proximal p or the q arm. Therefore, data from one additional CMT1A family and typing of other probes spanning the pericentromeric region of chromosome 17 (D17S73, D17S58, D17S122, D17S125, D17S124) were analyzed. Multipoint analysis demonstrates convincing evidence (log likelihood difference greater than 5) that the CMT1A gene lies within 17p11.2 and most likely between the flanking markers D17S122 and D17S124.     

Dominant intermediate Charcot-Marie-Tooth type C maps to chromosome 1p34-p35

Dominant intermediate Charcot-Marie-Tooth (DI-CMT) neuropathy is a genetic and phenotypic variant of classical CMT, characterized by intermediate nerve conduction velocities and histological evidence of both axonal and demyelinating features. We report two unrelated families with intermediate CMT linked to a novel locus on chromosome 1p34-p35 (DI-CMTC). The combined haplotype analysis in both families localized the DI-CMTC gene within a 6.3-cM linkage interval flanked by markers D1S2787 and D1S2830. The functional and positional candidate genes, Syndecan 3 (SDC3), and lysosomal-associated multispanning membrane protein 5 (LAPTM5) were excluded for pathogenic mutations.     

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6

Summary Essentially complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [¹⁵N]-and [¹⁵N, ¹³C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of ¹³C-chemical shift, amide hydrogen-bond exchange, and ¹⁵N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five -helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.     

Cell expression of GDAP1 in the nervous system and pathogenesis of Charcot-Marie-Tooth type 4A disease

Mutations in the mitochondrial protein GDAP1 are the cause of Charcot-Marie-Tooth type 4A disease (CMT4A), a severe form of peripheral neuropathy associated with either demyelinating, axonal or intermediate phenotypes. GDAP1 is located in the outer mitochondrial membrane and it seems that may be related with the mitochondrial network dynamics. We are interested to define cell expression in the nervous system and the effect of mutations in mitochondrial morphology and pathogenesis of the disease. We investigated GDAP1 expression in the nervous system and dorsal root ganglia (DRG) neuron cultures. GDAP1 is expressed in motor and sensory neurons of the spinal cord and other large neurons such as cerebellar Purkinje neurons, hippocampal pyramidal neurons, mitral neurons of the olfactory bulb and cortical pyramidal neurons. The lack of GDAP1 staining in the white matter and nerve roots suggested that glial cells do not express GDAP1. In DRG cultures satellite cells and Schwann cells were GDAP1-negative. Overexpression of GDAP1-induced fragmentation of mitochondria suggesting a role of GDAP1 in the fission pathway of the mitochondrial dynamics. Missense mutations showed two different patterns: most of them induced mitochondrial fragmentation but the T157P mutation showed an aggregation pattern. Whereas null mutations of GDAP1 should be associated with loss of function of the protein, missense mutations may act through different pathogenic mechanisms including a dominant-negative effect, suggesting that different molecular mechanisms may underlay the pathogenesis of CMT4A.     

The mutational spectrum in a cohort of Charcot-Marie-Tooth disease type 2 among the Han Chinese in Taiwan

Background

Charcot-Marie-Tooth disease type 2 (CMT2) is a clinically and genetically heterogeneous group of inherited axonal neuropathies. The aim of this study was to extensively investigate the mutational spectrum of CMT2 in a cohort of patients of Han Chinese.

Methodology and Principal Findings

Genomic DNA from 36 unrelated Taiwanese CMT2 patients of Han Chinese descent was screened for mutations in the coding regions of the MFN2, RAB7, TRPV4, GARS, NEFL, HSPB1, MPZ, GDAP1, HSPB8, DNM2, AARS and YARS genes. Ten disparate mutations were identified in 14 patients (38.9% of the cohort), including p.N71Y in AARS (2.8%), p.T164A in HSPB1 (2.8%), and p.[H256R]+[R282H] in GDAP1 (2.8%) in one patient each, three NEFL mutations in six patients (16.7%) and four MFN2 mutations in five patients (13.9%). The following six mutations were novel: the individual AARS, HSPB1 and GDAP1 mutations and c.475-1G>T, p.L233V and p.E744M mutations in MFN2. An in vitro splicing assay revealed that the MFN2 c.475-1G>T mutation causes a 4 amino acid deletion (p.T159_Q162del). Despite an extensive survey, the genetic causes of CMT2 remained elusive in the remaining 22 CMT2 patients (61.1%).

Conclusions and Significance

This study illustrates the spectrum of CMT2 mutations in a Taiwanese CMT2 cohort and expands the number of CMT2-associated mutations. The relevance of the AARS and HSPB1 mutations in the pathogenesis of CMT2 is further highlighted. Moreover, the frequency of the NEFL mutations in this study cohort was unexpectedly high. Genetic testing for NEFL and MFN2 mutations should, therefore, be the first step in the molecular diagnosis of CMT2 in ethnic Chinese.     

Adaptation by alternative RNA splicing of slow troponin T isoforms in type 1 but not type 2 Charcot-Marie-Tooth disease

Slow troponin T (TnT) plays an indispensable role in skeletal muscle function. Alternative RNA splicing in the NH₂-terminal region produces high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms of slow TnT. Normal adult slow muscle fibers express mainly HMW slow TnT. Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral polyneuropathies caused by various neuronal defects. We found in the present study that LMW slow TnT was significantly upregulated in demyelination form type 1 CMT (CMT1) but not axonal form type 2 CMT (CMT2) muscles. Contractility analysis showed an increased specific force in single fibers isolated from CMT1 but not CMT2 muscles compared with control muscles. However, an in vitro motility assay showed normal velocity of the myosin motor isolated from CMT1 and CMT2 muscle biopsies, consistent with their unchanged myosin isoform contents. Supporting a role of slow TnT isoform regulation in contractility change, LMW and HMW slow TnT isoforms showed differences in the molecular conformation in conserved central and COOH-terminal regions with changed binding affinity for troponin I and tropomyosin. In addition to providing a biochemical marker for the differential diagnosis of CMT, the upregulation of LMW slow TnT isoforms under the distinct pathophysiology of CMT1 demonstrates an adaptation of muscle function to neurological disorders by alternative splicing modification of myofilament proteins. muscle adaptation; demyelination; force and velocity     

Therapy of Niemann-Pick disease, type C

Niemann-Pick disease, type C (NPC) is a progressive autosomal recessive neurodegenerative disease, characterized by late endosomal-lysosomal accumulation of multiple lipid molecules in association with abnormal tubulovesicular trafficking. The major gene product, NPC1 protein, is not suitable for transduction therapies, and gene replacement or repair is not yet practicable for NPC and related disorders. Attempts at therapy to date have focused on reduction of the accumulating molecules that are presumed to have direct or indirect toxic effects. More recent insights into the pathophysiology of NPC raise the possibility of small molecule therapies to interdict pathways triggering apoptosis and related routes to cell death and dysfunction.     

Charcot-Marie-Tooth type 1A disease caused by a novel Ser112Arg mutation in thePMP22 gene, coexisting with a slowly progressive hearing impairment

Among 57 mutations in the peripheral myelin protein 22 gene (PMP22) identified so far in patients affected by Charcot-Marie-Tooth disease (CMT), only 8 have been shown to segregate with a mixed phenotype of CMT and hearing impairment. In this study, we report a new Ser1 12Arg mutation in thePMP22 gene, identified in a patient with early-onset CMT and slowly progressive hearing impairment beginning in the second decade of life. We suggest that the Ser1 12Arg mutation in thePMP22 gene might have a causative role in the early-onset CMT with hearing impairment. Thus, our study extends the spectrum of CMT phenotypes putatively associated withPMP22 gene mutations.     

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.