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Historical perspective and nomenclature
In 1886 Drs Charcot and Marie of France and Dr Tooth of England described patients with an inherited form of peroneal muscular atrophy, characterised by a progressive weakness and atrophy of distal muscles, usually originating in the feet and lower legs and progressing to the hands and forearms—a disorder now known as Charcot-Marie-Tooth (CMT) disease. Early literature attested to the existence of several distinct disorders in addition to CMT disease, including Dejerine-Sottas syndrome (DSS) and Roussy-Lévy syndrome, but histopathological evidence combined with the advent of electrodiagnostic testing and molecular investigation have demonstrated that these syndromes are clinical variants of CMT disease.
The duplication of a 1.5 Mb DNA fragment encompassing the PMP22 gene on chromosome 17p11.2 is associated with over 70% of CMT1 cases
Point mutations of the PMP22,P0, and Cx32 genes are also associated with the CMT1 phenotype
PMP22 and P0 encode myelin proteins which have important roles in the compaction of myelin. Cx32 encodes a gap junction protein which is considered to allow trafficking of metabolites and secondary messengers between the non-compact layers of myelin. Murine models have validated the hypothesised roles of these proteins
CMT disease is extremely heterogeneous, with at least eight additional loci identified. No genes have as yet been identified at these loci
The term CMT disease is now applied to a range of hereditary peripheral neuropathies, with a population prevalence of approximately one in 2500, which are able to be differentiated at several levels. None the less, the nosology surrounding CMT disease remains confusing and is further confused by the term hereditary motor and sensory neuropathy (HMSN), introduced to describe a broad range of neurological disorders with both motor and sensory involvement.
Classification of CMT disease
NERVE CONDUCTION STUDIES AND PATHOLOGY
A major differentiating factor between the different forms of CMT disease is identified by electrophysiological examination and nerve pathology. The combined strengths of these assessments allow the subdivision of CMT disease into two major groups, termed CMT disease type 1 and type 2 (CMT1 and CMT2).1 2
CMT1(HMSNI) is the more common of the two and characterised by diffusely low nerve conduction velocities (NCVs), typically <38m/s, and the appearance of “onion bulbs” on peripheral nerve biopsy due to demyelination and remyelination of the nerve. CMT2 (HMSNII) on the other hand is characterised by normal or near normal nerve conduction velocities and a decreased number of myelinated axons. While there is no evidence of demyelination and remyelination in CMT2, there are signs of marked axonal degeneration.
Patients with Dejerine-Sottas syndrome (HMSNIII) have markedly reduced NCVs1 and at peripheral nerve biopsy demyelination and onion bulb formation, similar to that in CMT1, is found in addition to evidence of hypomyelination.
The two major forms of CMT disease (CMT1/CMT2) are clinically very similar, with both motor and sensory nerve function impaired. The distal muscle weakness described above may be obvious from an early age as an abnormality of gait or clumsiness in running. Other typical features include weakness at the ankle, steppage gait, and the characteristic pes cavus deformity of the foot. Generally, the clinical features of DSS overlap with a severe CMT1 phenotype with patients demonstrating a chronic and progressive motor and sensory neuropathy, but with an onset usually in infancy or early childhood.
Hereditary neuropathy with liability to pressure palsies (HNPP) describes a neuropathy which causes nerve palsies and sensory dysfunction following minor nerve trauma. These palsies dissipate but a slowly progressive neuropathy is evident in patients. Nerve pathology demonstrates the typical demyelination and remyelination observed in CMT1 in addition to multiple sausage shaped focal thickenings (tomaculi) of the myelin sheath.3
Molecular basis of CMT disease
The clinical heterogeneity of CMT disease is paralleled at a genetic level, with autosomal dominant and recessive and X linked dominant and recessive families documented. Recent molecular investigations have given further insight into the heterogeneity of CMT disease and, in some instances, allowed the localisation of defective genes (table 1).
CMT1 is acknowledged as the most common form of these peripheral neuropathies and consequently genetic linkage studies have focused predominately on these families, resulting in the identification of three subgroups of families, each with a distinct genetic locus.4 In each of these groups the disorder is transmitted in an autosomal dominant manner. The first subgroup, CMT1A, maps to chromosome 17p11.2 and is caused, in the majority of cases, by a duplication of 1.5 Mb DNA, encompassing the peripheral myelin protein 22 ( PMP22) gene. The duplication is caused by unequal crossing over via two repeat sequences located at the proximal and distal end of the duplicated segment. This duplication event associated with CMT1A accounts for 70–85% of CMT1 cases. The reciprocal product of this crossover (deletion of a copy ofPMP22) has been shown to be the molecular defect in the majority of HNPP cases.3 Point mutations withinPMP22 may also account for a small proportion of CMT1 or HNPP cases, rather than duplication or deletion. A proportion of the remainder of CMT1 cases is accounted for by point mutations in a second myelin protein, peripheral myelin protein zero(P 0), which maps to 1q21-q23. This locus has been designated CMT1B. Heterozygous mutations within either of these genes have been identified in DSS patients, leading to DSS now being classed as a severe CMT1 variant.4
In addition CMT1 families shown not to be linked to either the CMT1A or CMT1B loci indicate that a third CMT1 gene (CMT1C) exists. No candidate region has yet been identified in these patients, but the identification of a DSS locus on chromosome 85 leads one to speculate that this may represent the CMT1C locus.
Genes associated with the axonal form of CMT disease have, to date, been assigned to at least three loci.6 Again, in each group the mode of inheritance is autosomal dominant. CMT2 has been mapped on the basis of linkage studies to chromosomes 1p35-36 (CMT2A), 3q13-22 (CMT2B) and 7p14 (CMT2D). A fourth CMT2 form, distinguished clinically on the basis of diaphragm and vocal cord weakness, in addition to an axonal neuropathy, has also been reported. This group, designated CMT2C, has been shown to be genetically distinct from CMT2A and CMT2B.
Clinically, X linked dominant CMT disease resembles the CMT1 phenotype—reduced NCVs and a demyelinating pathology. However, expression of the disease in carrier females is variable ranging from asymptomatic with normal NCVs to disabling manifestations with reduced NCVs.
Linkage studies placed the X linked dominant CMTX locus in a 1 cM interval at Xq12-13.7 Several candidate genes from this region were proposed and sequence changes were identified in the coding region of connexin32 (Cx32) in seven out of eight CMTX patients.8 The demonstration ofCx32 expression in the peripheral nerve8 and identification of additional mutations in other CMTX families,9 confirmed Cx32 as the causal gene in this form of CMTX, designated CMTX1.
OTHER CMT DISEASE LOCI
The recessive forms of CMT disease have received less attention than the dominant forms due, in part, to the observed heterogeneity and subsequent difficulties in defining a clinical phenotype. Several distinct loci have been indicated by linkage studies, but there is currently no evidence to suggest that any of the genes involved in CMT1 or CMTX1 are responsible for either X linked or autosomal recessive cases of CMT disease.
Schwann cell biology
The CMT disease genes identified to date are expressed in Schwann cells, which associate with axonal elements and wrap layer after layer of themselves around the axon to form the myelin sheaths, thereby enhancing NCVs.
SCHWANN CELL PROLIFERATION AND DIFFERENTIATION
Proper Schwann cell development and myelination necessitates Schwann cell proliferation and differentiation, events which have been shown to be induced by axonal contact—an effect mimicked by raised cAMP levels.10 Recent evidence has attributed this mitogenic effect of axons to a family of Neu differentiation factors.11 Following association with axonal segments, Schwann cell differentiation, that is the expression of myelin genes such as P 0, PMP22, andCx32 occurs. However, the key factors involved in the timing of their expression are unknown.
Interestingly PMP22 and Cx32 are expressed in both neural and non-neural cells, facilitated by alternative promoters.12 13 Each promoter drives the expression, although not exclusively, of a particular transcript in different cell types. In vitro studies of PMP22 gene expression have shown that both neural and non-neural transcripts are induced to an equal extent by an increase in cAMP. Yet in vivo, myelination is allied with the upregulation of the neural transcript, out of proportion to the non-neural form.12 The intrinsic factors involved in this selective control are as yet undefined, although transcription factors similar in effect to Krox-20 andOct-6, which have been suggested to have a role in early myelinogenesis,11 may be involved.
The functional significance of these multiple promoters is unclear but conceivable that they are indicative of alternative functions—perhaps one being related to cell growth in early development, and a second in myelin structural integrity of mature Schwann cells.
FUNCTION OF THE MYELIN PROTEINS
The myelin proteins MBP, MAG, PMP22, P0, Cx32 all play a part in the maintenance of myelin integrity (fig 1), but for the sake of brevity in this article only those associated with CMT disease to date are discussed here. P0 is a major integral myelin membrane protein, traversing the membrane once, and mediates membrane adhesion in the concentric myelin wraps. The adhesive properties of the protein are attributed to the extracellular domain of P0which forms a tetrameric array that interlocks with similar arrangements in the opposing membranes, and a homophilic cytoplasmic interaction.14 P0 is also thought to partake in a heterophilic interaction with the transmembrane protein PMP22 via the L2/HNK-1 epitopes of their glycosidic links.15 The suggested role of these proteins in the compaction of myelin has been validated by the generation of various knockout mouse models that demonstrate poor myelin compaction, accompanied by axon degeneration and signs of demyelination.16-18
In addition to its structural role it has also been suggested that PMP22 plays a part in cellular growth, primarily due to its down regulation after nerve damage.19 Whether this is directly related to a cell growth phenomenon or the return to a “dedifferentiated” state of the cell is unknown. However, the role of PMP22 in cellular growth must still be considered, especially in view of the alternative transcript that predominates in non-neural cells.
The gap junction protein Cx32 has been localised to the paranodal regions and Schmidt-Lantermann incisures of myelin,8 and believed to function as a channel for the transport of electrolytes and metabolites between the myelin wraps of an individual cell. This trafficking of molecules is also considered to extend to the axon.
Effect of myelin gene defects at the phenotypic level
OVEREXPRESSION OF PMP22
The most common genotypic defect in CMT disease disease is the duplication of the PMP22 gene and its subsequent overexpression. On peripheral nerve biopsy these patients typically show evidence of demyelination and remyelination, onion bulb formation, and Schwann cell proliferation. Mouse models of the CMT1A phenotype have shown that overexpression is indeed involved, and demonstrate that with an increasing copy number of PMP22 the effects are exaggerated and the phenotype more severe.20 21 In these cases, it is hypothesised that myelination is initiated, but a breakdown in the process occurs, leading to the continued demyelination and remyelination typical of CMT1. This breakdown in the homoeostatic control may be caused by the inherent overexpression ofPMP22, which prevents the cells from differentiating further and in turn, lead to the apoptotic-like cells observed by Fabretti et al.22 Alternatively, the hypothesised increased proportion of PMP22 within the cell membrane may upset protein stoichiometry and hence the structural integrity of the myelin, leading to its degeneration. Both hypotheses would account for the observed Schwann cell proliferation as macrophage mediated myelin degeneration has been shown to have such a mitogenic effect.23 That no effects are noted in non-neural cells is interesting. Perhaps the effects of this overexpression are not detrimental to the cell, or alternatively, a functionally analogous protein compensates.
UNDEREXPRESSION OF PMP22
The exaggerated phenotype observed with increasing copy number is conversely duplicated, with a decreasing copy number. Knockout mouse models of PMP22 have shown that the double copy knockout model is more severely affected than the heterozygous mouse.18 Although no patients with the deletion of bothPMP22 copies have yet been observed, a similar effect would be envisaged.
POINT MUTATION WITHIN THE MYELIN GENES
The effect of this type of defect in PMP22 appears to be more severe than overexpression of the gene, both in human and animal models. Mutations of the P 0gene also appear to result in a severe phenotype. Even within this class, a spectrum of phenotypes is observed and considered to be due to the effect of the mutation—whether a loss of, or dominant negative gain of, function is afforded by the corresponding mutant protein, which in turn yields further phenotypic heterogeneity depending on the actual site of the mutation.
The mechanisms by which the defects in PMP22 andP0 lead to characteristic CMT1 phenotypes will be similar to that described above. Cx32 defects, on the other hand, have been shown to result in improper gating abilities of the connexin hemichannels,24 theoretically leading to deficiencies in intracellular transport of metabolites, and in turn to Schwann cell degeneration and possibly to the observed axonopathy evident in CMTX1 patients.
In contrast to coding region defects of myelin genes, the first evidence of mutations within the promoter sequences (ofCx32) has been reported.25 One of these mutations is thought to affect the rate of transcription initiation, while the second may create a new splice site motif or alternatively, a new translation start codon. However, the actual effect of these and similar promoter mutations requires further investigation.
Molecular diagnosis and prospects for the future
The molecular diagnosis of the peripheral neuropathies is a complex procedure and its success depends on various factors including knowledge of family history, availability of local clinical testing, and the resources of the diagnostic laboratory. Where possible, clinicians should provide full clinical details including nerve conduction velocities and a detailed family history. Unless there is evidence to suggest a type 2 phenotype, PMP22 duplication testing is the initial screen in CMT disease diagnosis as it accounts for over 70% of CMT1 cases. If no duplication is observed and there is no recorded male to male inheritance in the patient’s family history, then Cx32 screening follows as this represents the second most common genetic defect in CMT disease to date. Individuals referred with no obvious family history would be investigated likewise.
Mutation screening of P0 then PMP22, is the natural next step for patients shown to have no Cx32defect and/or no observed duplication. However, due to time and monetary constraints of diagnostic laboratories, combined with the somewhat low detection rate, these screens are often only performed within research programmes.
Now that the molecular basis of several neuropathies has been elucidated, research based on this new knowledge can aid in understanding the actual pathogenic mechanisms. Although to date, there is no effective treatment for alleviating the symptoms of CMT disease, these and similar studies will undoubtedly accelerate progress towards the goals of therapeutic medicine. Indeed, a canine model has shown that focally transplanted glial cells are capable of myelinating areas of the central nervous system,26 a situation comparable to the focal lesions observed in multiple sclerosis, thus inferring hope for the future in the treatment of multiple sclerosis and other disorders of myelination.