X-linked Charcot Marie Tooth Disease Research Articles

X-linked Charcot Marie Tooth mutations alter CO2 sensitivity of connexin32 hemichannels.

Connexin32 (Cx32) is expressed in myelinating Schwann cells. It forms both reflexive gap junctions, to facilitate transfer of molecules from the outer to the inner myelin layers and hemichannels at the paranode to permit action potential-evoked release of ATP into the extracellular space. Loss of function mutations in Cx32 cause X-linked Charcot Marie Tooth disease (CMTX), a slowly developing peripheral neuropathy. The mechanistic links between Cx32 mutations and CMTX are not well understood. As Cx32 hemichannels can be opened by increases in PCO2, we have examined whether CMTX mutations alter this CO2 sensitivity. By using Ca2+ imaging, dye loading and genetically encoded ATP sensors to measure ATP release, we have found 5 CMTX mutations that abolish the CO2 sensitivity of Cx32 hemichannels (A88D, 111-116 Del, C179Y, E102G, V139M). Others cause a partial loss (L56F, R220Stop, and R15W). Some CMTX mutations have no apparent effect on CO2 sensitivity (R15Q, L9F, G12S, V13L, V84I, W133R). The mutation R15W alters multiple additional aspects of hemichannel function including Ca2+ and ATP permeability. The mutations that abolish CO2 sensitivity are transdominant and abolish CO2 sensitivity of co-expressed Cx32WT. We have shown that Schwannoma RT4 D6P2T cells can release ATP in response to elevated PCO2 via the opening of Cx32. This is consistent with the hypothesis that the CO2 sensitivity of Cx32 may be important for maintenance of healthy myelin. Our data, showing a transdominant effect of certain CMTX mutations on CO2 sensitivity, may need to be taken into account in any future gene therapies for this condition.

A novel splicing mutation in 5'UTR of GJB1 causes X-linked Charcot-Marie-tooth disease.

Charcot-Marie-Tooth (CMT) disease is the most frequent hereditary motor sensory neurological disease. GJB1 gene is the second most frequent cause of CMT, accounting for approximately 10% of CMT cases worldwide. We identified a large Han family with X-linked CMT disease. In this study, the probands and his mother underwent electrophysiological examinations and other family members were assessed retrospectively. Whole-exome sequencing, Sanger sequencing, and SNP array linkage analysis were performed to find and confirm the variant. The functional effect of the identified variant was further investigated in HEK293 cells and MCF-7 cells by minigene splicing assay. The affected individuals had some clinical symptoms including symmetric atrophy and progressive weakness of the distal muscles in their twenties. Electrophysiological examinations result in peripheral nerve injury of the upper and lower limbs. Whole-exome sequencing identified a novel hemizygous deletion mutation (NM_000166: c.-16-8_-14del) in the GJB1 gene. SNP array linkage analysis and co-segregation analysis confirmed this mutation. Minigene splicing assay verified that this mutation leads to the activation of cryptic splicing sites in exon 2 which results in the deletion of exon 2. Our study provides theoretical guidance for prenatal diagnosis and subsequent fertility of this family. This result expands the spectrum of mutations in GJB1 known to be associated with CMTX and contributes to the diagnosis of CMT and clinical genetic counseling.

Connexin Mutations and Hereditary Diseases.

Inherited diseases caused by connexin mutations are found in multiple organs and include hereditary deafness, congenital cataract, congenital heart diseases, hereditary skin diseases, and X-linked Charcot–Marie–Tooth disease (CMT1X). A large number of knockout and knock-in animal models have been used to study the pathology and pathogenesis of diseases of different organs. Because the structures of different connexins are highly homologous and the functions of gap junctions formed by these connexins are similar, connexin-related hereditary diseases may share the same pathogenic mechanism. Here, we analyze the similarities and differences of the pathology and pathogenesis in animal models and find that connexin mutations in gap junction genes expressed in the ear, eye, heart, skin, and peripheral nerves can affect cellular proliferation and differentiation of corresponding organs. Additionally, some dominant mutations (e.g., Cx43 p.Gly60Ser, Cx32 p.Arg75Trp, Cx32 p.Asn175Asp, and Cx32 p.Arg142Trp) are identified as gain-of-function variants in vivo, which may play a vital role in the onset of dominant inherited diseases. Specifically, patients with these dominant mutations receive no benefits from gene therapy. Finally, the complete loss of gap junctional function or altered channel function including permeability (ions, adenosine triphosphate (ATP), Inositol 1,4,5-trisphosphate (IP3), Ca2+, glucose, miRNA) and electric activity are also identified in vivo or in vitro.

A case of X-linked Charcot-Marie-tooth disease type 1 manifesting as recurrent alternating hemiplegia with transient cerebral white matter lesions

X-linked Charcot Marie Tooth disease type 1 (CMTX1) is a clinically heterogenous X-linked hereditary neuropathy caused by mutation of the gene encoding gap junction beta 1 protein (GJB1). Typical clinical manifestations of CMTX1 are progressive weakness or sensory disturbance due to peripheral neuropathy. However, there have been some CMTX1 cases with accompanying central nervous system (CNS) manifestations. We report the case of a genetically confirmed CMTX1 patient who presented recurrent transient CNS symptoms without any symptom or sign of peripheral nervous system involvement.

Effects of early crush on aging wild type and Connexin 32 knockout mice: Evidence for a neuroprotective state in CMT1X mouse nerve.

The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury.

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The cover image is based on the Original Article A novel GJB1 mutation associated with X-linked Charcot–Marie–Tooth disease in a large Chinese family pedigree by Yingdi Liu et al., https://doi.org/10.1002/mgg3.1127.

Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

BackgroundThe gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot–Marie–Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells.ResultsWe report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation.ConclusionTaken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions.

Substitution impact of highly conserved arginine residue at position 75 in GJB1 gene in association with X-linked Charcot–Marie-tooth disease: A computational study

X-linked Charcot-Marie-Tooth type 1 X (CMTX1) disease is a subtype of Charcot-Marie-Tooth (CMT), which is mainly caused by mutations in the GJB1 gene. It is also known as connexin 32 (Cx32) that leads to Schwann cell abnormalities and peripheral neuropathy. CMTX1 is considered as the second most common form of CMT disease. The aim of this study is to computationally predict the potential impact of different single amino acid substitutions at position 75 of Cx32, from arginine (R) to proline (P), glutamine (Q) and tryptophan (W). This position is known to be highly conserved among the family of connexin. To understand the structural and functional changes due to these single amino acid substitutions, we employed a homology-modeling technique to build the three-dimensional structure models for the native and mutant proteins. The protein structures were further embedded into a POPC lipid bilayer, inserted into a water box, and subjected to molecular dynamics simulation for 50 ns. Our results show that the mutants R75P, R75Q and R75W display variable structural conformation and dynamic behavior compared to the native protein. Our data proves useful in predicting the potential pathogenicity of the mutant proteins and is expected to serve as a platform for drug discovery for patients with CMT.

Mutation Analysis of Gap Junction Protein Beta 1 and Genotype-Phenotype Correlation in X-linked Charcot-Marie-Tooth Disease in Chinese Patients.

Background:Among patients with Charcot–Marie–Tooth disease (CMT), the X-linked variant (CMTX) caused by gap junction protein beta 1 (GJB1) gene mutation is the second most frequent type, accounting for approximately 90% of all CMTX. More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 (CX32). CX32 is thought to form gap junctions that promote the diffusion pathway between cells. GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin, and novel mutations are continually discovered.Methods:We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People's Liberation Army General Hospital from December 20, 2012, to December 31, 2015. Clinical examination, nerve conduction studies, and molecular and bioinformatics analyses were performed to identify patients with CMTX1.Results:Nine GJB1 mutations (c.283G>A, c.77C>T, c.643C>T, c.515C>T, c.191G>A, c.610C>T, c.490C>T, c.491G>A, and c.44G>A) were discovered in nine patients. Median motor nerve conduction velocities of all nine patients were < 38 m/s, resembling CMT Type 1. Three novel mutations, c.643C>T, c.191G>A, and c.610C>T, were revealed and bioinformatics analyses indicated high pathogenicity.Conclusions:The three novel missense mutations within the GJB1 gene broaden the mutational diversity of CMT1X. Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations.

Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function.

G.P.326 - Deletion of the P2 promoter of the GJB1 gene confirms X-linked Charcot–Marie–Tooth disease in a large family

G.P.326 - Deletion of the P2 promoter of the GJB1 gene confirms X-linked Charcot–Marie–Tooth disease in a large family

A novel mutation in GJB1 (c.212T>G) in a Chinese family with X-linked Charcot–Marie–Tooth disease

Gap junction protein beta 1 (GJB1) gene mutations lead to X-linked Charcot–Marie–Tooth (CMTX) disease. We investigated a Chinese family with CMTX and identified a novel GJB1 point mutation. Clinical and electrophysiological features of the pedigree were examined, and sequence alterations of the coding region of GJB1 that encode connexin32 were determined by direct sequencing. Sequence alignment of the mutation site was performed using Clustal W. Mutation effects were analysed using PolyPhen-2, SIFT and Mutation Taster software. The three-dimensional structures of the mutant and wild-type proteins were predicted by modeling with SWISS MODEL online software. The affected family members displayed typical Charcot–Marie–Tooth phenotypes, but phenotypic heterogeneity was observed. Nerve conduction velocities of all affected patients were slow. Sequencing of GJB1 revealed a heterozygous T>G missense mutation at nucleotide 212 in the proband, the proband’s mother and the proband’s daughter. The affected male sibling of the proband displayed a hemizygous missense mutation with T>G transition at the identical position on the GJB1 gene. This mutation resulted in an amino acid change from isoleucine to serine that was predicted to lead to tertiary structural alterations that would disrupt the function of the GJB1 protein. A novel point mutation in GJB1 was detected, expanding the spectrum of GJB1 mutations known to be associated with CMTX.

Gene expression profiling studies in regenerating nerves in a mouse model for CMT1X: Uninjured Cx32-knockout peripheral nerves display expression profile of injured wild type nerves

X-linked Charcot–Marie–Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the human gene for Connexin32 (Cx32). This present study uses Ilumina Ref8-v2 BeadArray to examine the expression profiles of injured and uninjured sciatic nerves at 5, 7, and 14days post-crush injury (dpi) from Wild Type (WT) and Cx32-knockout (Cx32KO) mice to identify the genes and signaling pathways that are dysregulated in the absence of Schwann cell Cx32. Given the assumption that loss of Schwann cell Cx32 disrupts the regeneration and maintenance of myelinated nerve leading to a demyelinating neuropathy in CMT1X, we initially hypothesized that nerve crush injury would result in significant increases in differential gene expression in Cx32KO mice relative to WT nerves. However, microarray analysis revealed a striking collapse in the number of differentially expressed genes at 5 and 7dpi in Cx32KO nerves relative to WT, while uninjured and 14dpi time points showed large numbers of differentially regulated genes. Further comparisons within each genotype showed limited changes in Cx32KO gene expression following crush injury when compared to uninjured Cx32KO nerves. By contrast, WT nerves exhibited robust changes in gene expression at 5 and 7dpi with no significant differences in gene expression by 14dpi relative to uninjured WT nerve samples. Taken together, these data suggest that the gene expression profile in uninjured Cx32KO sciatic nerve strongly resembles that of a WT nerve following injury and that loss of Schwann cell Cx32 leads to a basal state of gene expression similar to that of an injured WT nerve. These findings support a role for Cx32 in non-myelinating and regenerating populations of Schwann cells in normal axonal maintenance in re-myelination, and regeneration of peripheral nerve following injury. Disruption of Schwann cell–axonal communication in CMT1X may cause dysregulation of signaling pathways that are essential for the maintenance of intact myelinated peripheral nerves and to establish the necessary conditions for successful regeneration and remyelination following nerve injury.

Four novel connexin 32 mutations in X-linked Charcot–Marie–Tooth disease. Phenotypic variability and central nervous system involvement

Charcot–Marie–Tooth (CMT) disease, the most common hereditary neuropathy, is clinically and genetically heterogeneous. X-linked CMT (CMTX) is usually caused by mutations in the gap junction protein b 1 gene (GJB1) coding for connexin 32 (Cx32). The clinical manifestations of CMTX are characterized by significant variability, with some patients exhibiting central nervous system (CNS) involvement. We report four novel mutations in GJB1, c.191G>A (p.Cys64Tyr), c.508G>T (p.Val170Phe), c.778A>G (p.Lys260Glu) and c.300C>G (p.His100Gln) identified in four unrelated Greek families. These mutations were characterized by variable phenotypic expression, including a family with the Roussy–Lévy syndrome, and three of them were associated with mild clinical CNS manifestations.

Axonal excitability in X-linked dominant Charcot Marie Tooth disease

ObjectiveWe investigated peripheral nerve function in X-linked Charcot–Marie–Tooth disease type 1 (CMTX1), and considered the functional consequences of mutant connexin-32. MethodsTwelve subjects (9 female, 3 male) were assessed clinically, by nerve conduction and excitability studies. A model of myelinated axon was used to clarify the contributing changes. ResultsAll subjects had abnormal nerve conduction. Excitability studies on median nerve axons showed greater threshold changes to hyperpolarising currents, with “fanning out” in threshold electrotonus, and modest changes in the recovery cycle. Modelling suggested shortening of internodal length, increase in nodal fast potassium currents, shift of the voltage activation hyperpolarisation-activated cyclic-nucleotide-gated channels, and axonal hyperpolarisation. Plotting threshold versus extent of hyperpolarising threshold change in threshold electrotonus distinguished the CMTX1 patients from other chronic demyelinating neuropathies reported in the literature except hereditary neuropathy with pressure palsies (HNPP). ConclusionsSome measures of axonal excitability are similar in CMTX1 and HNPP (though not the recovery cycle), but they differ from those in other chronic demyelinating neuropathies. The findings in CMTX1 are consistent with known pathology, but are not correlated to neuropathy severity. SignificanceThe findings in CMTX1 could be largely the result of morphological alterations, rather than plasticity in channel expression or distribution.

Hereditary motor and sensory neuropathy (HMSN) type X1 in an Argentinean family reveals independent GJB1/Cx32 mutations at the identical nucleotide position

X-linked Charcot–Marie-Tooth disease (CMT Type X1, OMIM: 302800) represents a frequent cause of hereditary peripheral motor and sensory neuropathies and is associated with mutations in GJB1 encoding the gap junction beta 1 protein connexin 32 (Cx32). Studying an Argentinean family of Italian origin with seven affected males in three generations exhibiting clinical signs of CMT, eight obligate female carriers were identified genealogically. DNA sequencing of exon 2 and adjacent regions of the GJB1 gene in two symptomatic males whose respective maternal grandfathers, both affected, were brothers, revealed mutations in GJB1/Cx32. Surprisingly, each of the two affected patients had a different mutation in hemizygous state at the same nucleotide position: c.383C>T (p.S128L) and c.383C>A (p.S128X). In both cases, the identified mutation was present in heterozygous state in the corresponding maternal genomic DNA. Furthermore, X-chromosomal microsatellite analysis showed identical marker alleles in both patients. Together with the genealogical information, these molecular data imply that a primarily mutated allele mutated for a second time. In conclusion, two different mutations at the same nucleotide position in this Argentinean family represent a finding with a very low probability of occurrence.

Genetic epidemiology of Charcot-Marie-Tooth disease

Genetic epidemiology of Charcot-Marie-Tooth disease

G.P.86 X-linked Charcot–Marie–Tooth disease with c.22A&gt;C missense mutation in the GJB1 gene in Koreans

<h2>Abstract</h2> X-linked Charcot–Marie–Tooth disease (CMT1X) is caused by gap junction beta 1 (GJB1) gene mutations, which encodes the connexin 32. Connexin 32 is an integral transmembrane protein expressed in peripheral nerve myelin. The frequency of CMT1X varies among different ethnic groups. The frequency in Koreans is low compared with those in Europeans. Herein we present one CMT1X patient with missense mutation in the GJB1 gene. The 27-year-old male presented with complaints of slowly progressive weakness beginning in the distal leg muscles. He had walked with waddling gait ever since he had started walking at 2years of age. He felt a weak hand grip around 8years of age. He could not run and have numbness on toe tips after 18years old. The family history revealed a more severe disease phenotype in affected males compared with affected females. Physical examination showed pes cavus, wasting of muscles below knee, and intrinsic hand muscle atrophy. Neurological examination demonstrated predominantly distal muscle weakness in the lower and upper limbs, decreased pinprick sensation in stocking-glove pattern, and areflexia. Electrophysiological study revealed sensori-motor polyneuropathy, but did not suggest sufficient evidence of demyelination or axonal degeneration. Gene analysis identified c.22A>C Missense Mutation in the GJB1 gene. We report a CMT1X patient having typical X-linked dominant inheritance and missense mutation in the GJB1 gene.

How do mutations in GJB1 cause X-linked Charcot–Marie–Tooth disease?

The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive weakness, atrophy, and sensory abnormalities that are most pronounced in the distal extremities. Some patients have CNS manifestations. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and length-dependent axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. Mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32) cause CMT1X; more than 400 different mutations have been described. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. Effective therapies remain to be developed. This article is part of a Special Issue entitled Electrical Synapses.

X inactivation in females with X-linked Charcot–Marie–Tooth disease

X-linked Charcot–Marie–Tooth disease (CMT1X) is the second most common inherited neuropathy, caused by mutations in gap junction beta-1 (GJB1). Males have a uniformly moderately severe phenotype while females have a variable phenotype, suggested to be due to X inactivation. We aimed to assess X inactivation pattern in females with CMT1X and correlate this with phenotype using the CMT examination score to determine whether the X inactivation pattern accounted for the variable phenotype in females with CMT1X. We determined X inactivation pattern in 67 females with CMT1X and 24 controls using the androgen receptor assay. We were able to determine which X chromosome carried the GJB1 mutation in 30 females. There was no difference in X inactivation pattern between patients and controls. In addition, there was no correlation between X inactivation pattern in blood and phenotype. A possible explanation for these findings is that the X inactivation pattern in Schwann cells rather than in blood may explain the variable phenotype in females with CMT1X.