Nonmyelinating Schwann cell involvement with well-preserved unmyelinated axons in Charcot-Marie-Tooth disease type 1A

Electron microscopic examination was performed to compare morphologic changes of nonmyelinating Schwann cells and unmyelinated axons in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) with peripheral myelin protein 22 duplication (n = 27) and normal control individuals (n = 14). Complete transverse sural nerve cross-sections were obtained in 16 patients and the total number of axons and Schwann cells in each cross-section was estimated. In patients with CMT1A, the number of myelinated axons was significantly decreased, whereas unmyelinated axons were well-preserved and did not show any marked changes. The numbers of nuclei, subunits, and profiles of nonmyelinating Schwann cells were all increased significantly in patients with CMT1A, whereas the numbers of axons per unmyelinated axon-containing subunit were significantly decreased. Schwann cell subunits consisted of layers of flattened cytoplasmic profiles wrapped around unmyelinated axons in the patient with CMT1A. The numbers of nonmyelinating Schwann cell profiles were increased and the numbers of axons per unmyelinated axon-containing subunit were reduced even in young patients with well-preserved myelinated fibers. In conclusion, there is marked alteration of the population and morphology of nonmyelinating Schwann cells, and axon-Schwann cell interactions seem to be regulated differently between myelinated and unmyelinated fibers in CMT1A.     

LONG-TERM EFFECT OF INTRAVENOUS IMMUNOGLOBLIN THERAPY (IVIg) IN MULTIFOCAL MOTOR NEUROPATHY (MMN)

Peripheral myelin protein 22 (PMP22) plays a dual role in regulating cells growth and peripheral myelin compaction. Charcot‐Marie‐Tooth 1A neuropathy (CMT1A) and Hereditary Neuropathy with liability to Pressure Palsies (HNPP) are respectively due to a duplication and a deletion of the PMP22 gene, leading to over and underexpression of the protein. It is unknown how the altered PMP22 dosage impairs peripheral myelination. By means of transmission electron microscopy (TEM) morphometry we found that myelin periodicity is significantly increased in CMT1A patients (9.79 ± 0.9 nm) and compared to normal sural nerves (9.1 ± 0.7 nm). Conversely, myelin periodicity was slightly reduced in HNPP patients (8.9 ± 0.7 nm). Interestingly, the difference between CMT1A and HNPP patients was highly significant. Moreover, we found an increased myelin periodicity in sciatic nerves of a transgenic rat model of CMT1A (8.9 ± 0.08) and in organotypic dorsal root ganglia cultures (10.1 ± 0.1 nm) established from this rat line, compared to control animals (8.6 ± 0.1) and cultures (9.6 ± 0.1 nm). Since TEM studies suffer considerably from radiation damage, we also used small‐angle X‐ray scattering (SAXS) with a synchrotron radiation microbeam (5 m diameter) for in‐situ experiments on single sural nerves of CMT 1A and HNPP patients and of normal controls. For comparison with the TEM‐data, the periodicities were divided by two. Myelin periodicities ranged from 8.4 to 8.9 nm (mean: 8.7 ± 0.3) in CMT 1A nerves, from 8.0 to 8.4 nm (mean: 8.1 ± 0.3) in normal controls, and from 6.9 to 7.2 nm (mean 7.1 ± 0.3) in HNPP. In conclusion, altered PMP22 levels induce significant changes in myelin lamellae spacing, which may be recorded by appropriate morphometric techniques, and in particular by microbeam SAXS. Differences in term of myelin periodicity between TEM morphometry and x‐ray microbeam analysis may be due to the higher resolution and accuracy of the latter technique to measure periodic structures. Having an altered myelin period could lead to demyelination and subsequent remyelination. Therefore, the pathomechanism of both CMT1A and HNPP may be also related to a dysfunction of PMP22 as a structural protein of peripheral myelin.     

Schwann cell differentiation in Charcot-Marie-Tooth disease type 1A (CMT1A): normal number of myelinating Schwann cells in young CMT1A patients and neural cell adhesion molecule expression in onion bulbs

Charcot-Marie-Tooth disease type 1A (CMT1A) is a common hereditary demyelinating neuropathy caused by a duplication of the gene for the myelin protein PMP22, resulting in overexpression of PMP22 in young patients. Although genetically well defined, the pathogenesis of the hereditary demyelinating neuropathy CMT1A is still unclear. Homology of PMP22 cDNA to the growth arrest-specific gene gas3 and experiments in vitro showing decreased proliferation in PMP22-overexpressing Schwann cells suggest a role of PMP22 in Schwann cell differentiation. Furthermore, overexpression of PMP22 in fibroblasts induces programmed cell death. In this report we applied morphometrical methods using electron micrographs and immunohistochemistry to further characterise Schwann cells in CMT1A nerve biopsy samples from CMT1A patients. We show that the total number of PMP22-expressing Schwann cells, i.e. Schwann cells that are in a 1:1 relationship with axons, was not reduced in sural nerve biopsy samples from six young CMT1A patients. We excluded non-specific secondary Schwann cell proliferation. Thus, in young CMT1A patients with increased PMP22 overexpression there seems to be no evidence for altered initial Schwann cell proliferation in achieving a 1:1 relationship to axons prior to the process of de- and remyelination. Further, using electron microscopy we found no evidence for apoptosis of Schwann cells in CMT1A . However, we provide additional support for an abnormal Schwann cell phenotype in CMT1A by showing the expression of neural cell adhesion molecule immunoreactivity in onion bulbs. Thus, the role of PMP22 in cell growth and differentiation does not lead to an altered number of myelinating Schwann cells but to altered Schwann cell differentiation in CMT1A. Received: 23 September 1996 / Revised: 28 November 1996, 31 January 1997, 2 April 1997 / Accepted: 3 April 1997     

Peripheral Myelin Protein 22 gene duplication with atypical presentations: A new example of the wide spectrum of Charcot-Marie-Tooth 1A disease

Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are both autosomal-dominant disorders linked to peripheral myelin anomalies. CMT1A is associated with a Peripheral Myelin Protein 22 (PMP22) duplication, whereas HNPP is due to a PMP22 deletion on chromosome 17. In spite of this crucial difference, we report three observations of patients with the 1.4 megabase CMT1A duplication and atypical presentation (electrophysiological, clinical or pathological): a 10 year-old girl with tomaculous lesions on nerve biopsy; a 26 year-old woman with recurrent paresthesiae and block conduction on the electrophysiological study; a 46 year-old woman with transient recurrent nerve palsies mimicking HNPP. These observations highlight the wide spectrum of CMT1A and the overlap between CMT1A and HNPP (both linked to the PMP22 gene), and finally illustrate the complexity of the genotype–phenotype correlations in Charcot-Marie-Tooth diseases.     

Effects of PMP22 duplication and deletions on the axonal cytoskeleton

Axonal loss in Charcot-Marie-Tooth type 1A (CMT1A) is an important feature correlated with the functional disability in affected individuals. It is not known, however, how the most common genetic defect in Schwann cells (PMP22 duplication) causes the CMT1A phenotype and results in axonal loss. In this study, sural nerve segments from individuals with PMP22 duplications or deletions, causing the reciprocal disorder hereditary neuropathy with pressure palsies (HNPP), were grafted into the cut ends of the sciatic nerve of nude mice. The xenografts and host segments were studied at 2, 4, 6, 8, 12, and 16 weeks after grafting and compared with the controls from healthy volunteers. Within the CMT1A xenografts, the nude mice axons in the proximal part of the graft showed a significant increase in axonal area with an increase in the neurofilament and membranous organelle (mitochondria) density, compared with distal graft and distal host segments. A preferential distal axonal loss, associated with a perpetual axonal atrophy, degeneration, and axonal sprouting was observed over time, with increasing intensity at 8 to 16 weeks. These alterations were seen to a lesser extent in HNPP xenografts and were not observed in controls. In addition, the onset of regeneration-associated myelination was delayed, more significantly in HNPP xenografts than those of CMT1A. Our findings indicate that the PMP22 duplication in Schwann cells results in an impairment in the normal axonal cytoskeletal organization, resulting in distal axonal degeneration and fiber loss, and the affect of PMP22 deletion on axonal cytoskeleton is less deleterious. Ann Neurol 1999;45:16–24     

Improved culture methods to expand schwann cells with altered growth behaviour from CMT1A patients

A duplication of the gene for myelin protein PMP22 is by far the most common cause of the hereditary demyelinating neuropathy CMT1A. A role for PMP22 in cell growth in addition to its function as a myelin protein has been suggested because PMP22 is homologous to a gene specifically upregulated during growth arrest. Furthermore, transfected rat Schwann cells overexpressing PMP22 show reduced growth. In addition, abnormal Schwann cell differentiation has been described in nerve biopsies from CMT1A patients. To analyse whether the duplication of the PMP22 gene in CMT1A neuropathy primarily alters Schwann cell differentiation and to exclude nonspecific secondary responses, we improved human Schwann cell culturing. This allowed us long-term passaging of human Schwann cells with unchanged phenotype, assessed by expression of different Schwann cell markers. Subsequently we established Schwann cell cultures from CMT1A nerve biopsies. We find decreased proliferation of Schwann cells from different CMT1A patients in all passages. We also demonstrate PMP22 mRNA overexpression in cultured CMT1A Schwann cells. We conclude that decreased proliferation in cultured Schwann cells that carry the CMT1A duplication indicates abnormal differentiation of CMT1A Schwann cells. The identification of an abnormal phenotype of CMT1A Schwann cells in culture could possibly lead to an in vitro disease model. GLIA 23:89–98, 1998. © 1998 Wiley-Liss, Inc.     

Peripheral myelin protein-22 expression in charcot-marie-tooth disease type 1a sural nerve biopsies

Peripheral myelin protein-22 (PMP22) is expressed in myelinating Schwann cells and shows significant homology to murine growth arest-specific gene gas3. Charcot-Marie-Tooth disease type 1a (CMT1a) is a common hereditary demyelinating neuropathy. Recently it was demonstrated that the gene for PMP22 is duplicated in CMT1a patients. A gene dosage mechanism has been postulated to cause CMT1a. According to this hypothesis, the increase in copy number of PMP22 gene would lead to an elevated expression of PMP22 and thereby cause the demyelinating phenotype of CMT1a. In the present communication we analyzed PMP22 mRNA and protein expression in sural nerve biopsies from CMT1a patients and normal controls. We show that PMP22 mRNA expression in CMT1a is not uniform. We found both elevated as well as normal PMP22 mRNA levels in patients. Interestingly, the highest PMP22 mRNA level was found in the least affected patient. In contrast to the mRNA levels, PMP22 was clearly reduced in all CMT1a patients as shown by immunohistochemistry. Thus the CMT1a phenotype may not be strictly correlated with increased PMP22 mRNA and protein expression. Possible roles of PMP22 in the pathogenesis of CMT1a are discussed. © 1994 Wiley-Liss, Inc.     

Studies on the effects of altered PMP22 expression during myelination in vitro

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot-Marie-Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over- or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT-PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation. J. Neurosci. Res. 48:31–42, 1997. © 1977 Wiley-Liss, Inc.     

PMP22‐Related neuropathies and other clinical manifestations in Chinese han patients with charcot‐marie‐tooth disease type 1

Introduction: Most cases of Charcot‐Marie‐Tooth (CMT) disease are caused by mutations in the peripheral myelin protein 22 gene (PMP22), including heterozygous duplications (CMT1A), deletions (HNPP), and point mutations (CMT1E). Methods: Single‐nucleotide polymorphism (SNP) arrays were used to study PMP22 mutations based on the results of multiplex ligation‐dependent probe amplification (MLPA) and polymerase chain reaction–restriction fragment length polymorphism methods in 77 Chinese Han families with CMT1. PMP22 sequencing was performed in MLPA‐negative probands. Clinical characteristics were collected for all CMT1A/HNPP probands and their family members. Results: Twenty‐one of 77 CMT1 probands (27.3%) carried duplication/deletion (dup/del) copynumber variants. No point mutations were detected. SNP array and MLPA seem to have similar sensitivity. Fifty‐seven patients from 19 CMT1A families had the classical CMT phenotype, except for 1 with concomitant CIDP. Two HNPP probands presented with acute ulnar nerve palsy or recurrent sural nerve palsy, respectively. Conclusions: The SNP array has wide coverage, high sensitivity, and high resolution and can be used as a screening tool to detect PMP22 dup/del as shown in this Chinese Han population. Muscle Nerve 52 : 69–75, 2015     

Impairment of PMP22 transgenic Schwann cells differentiation in culture: implications for Charcot-Marie-Tooth type 1A disease

Charcot-Marie-Tooth type 1A (CMT1A) is a hereditary demyelinating neuropathy due to an increased genetic dosage of the peripheral myelin protein 22 (PMP22). The mechanisms leading from PMP22 overexpression to impairment of myelination are still unclear. We evaluated expression and processing of PMP22, viability, proliferation, migration, motility and shaping properties, and ability of forming myelin of PMP22 transgenic (PMP22(tg)) Schwann cells in culture. In basal conditions, PMP22(tg) Schwann cells, although expressing higher PMP22 levels than control ones, show normal motility, migration and shaping properties. Addition of forskolin to the media induces an additional stimulation of PMP22 expression and results in an impairment of cells migration and motility, and a reduction of cell area and perimeter. Similarly, co-culturing transgenic Schwann cells with neurons causes an altered cells differentiation and an impairment of myelin formation. In conclusion, exposure of PMP22(tg) Schwann to the axon or to axonal-mimicking stimuli significantly affects the transition of transgenic Schwann cells to the myelinating phenotype.     

Variable phenotypes are associated with PMP22 missense mutations

Charcot-Marie-Tooth disease (CMT) is the commonest hereditary neuropathy encompassing a large group of clinically and genetically heterogeneous disorders. The commonest form of CMT, CMT1A, is usually caused by a 1.4 megabase duplication of chromosome 17 containing the PMP22 gene. Mutations of PMP22 are a less common cause of CMT. We describe clinical, electrophysiological and molecular findings of 10 patients carrying PMP22 missense mutations. The phenotype varied from mild hereditary neuropathy with liability to pressure palsies (HNPP) to severe CMT1. We identified six different point mutations, including two novel mutations. Three families were also found to harbour a Thr118Met mutation. Although PMP22 point mutations are not common, our findings highlight the importance of sequencing the PMP22 gene in patients with variable CMT phenotypes and also confirm that the PMP22 Thr118Met mutation is associated with a neuropathy albeit with reduced penetrance.     

Report of a novel mutation in the PMP22 gene causing an axonal neuropathy

Introduction: Point mutations in the peripheral myelin protein 22 (PMP22) gene rarely cause the hereditary neuropathies Charcot–Marie–Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), both of which show a demyelinating phenotype. Methods: In this study we characterized a family with an axonal neuropathy. Results: Three family members carried a heterozygous point mutation of the PMP22 gene, resulting in amino acid substitution R159C. Screening of 185 healthy controls did not reveal the R159C allele in any case. Discussion: The novel R159C mutation represents a very rare case of a dominant PMP22 mutation causing an axonal neuropathy. Muscle Nerve, 2011     

A Family Harboring CMT1A Duplication and HNPP Deletion

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion.     

The α-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot–Marie–Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells

At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4–P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the α-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.     

Pharmacological induction of the heat shock response improves myelination in a neuropathic model

Misexpression and intracellular retention of peripheral myelin protein 22 (PMP22) is associated with hereditary neuropathies in humans, including Charcot-Marie-Tooth disease type 1A (CMT1A). Mice expressing extra copies of the human PMP22, termed C22, display morphologic and behavioral characteristics of CMT1A. In neuropathic Schwann cells, the turnover of the newly-synthesized PMP22 is decreased, leading to the formation of cytosolic protein aggregates. To aid the processing of PMP22 and alleviate the associated myelin defects, we pharmacologically stimulated the expression of protein chaperones by synthetic small-molecule inhibitors of heat shock protein 90 (HSP90). The exposure of Schwann cells to these compounds enhanced the levels of cytosolic chaperones in a time- and dose-dependent manner, with minimal cytotoxicity. Treatment of dorsal root ganglion (DRG) explants from neuropathic mice improved myelin formation and the processing of PMP22. These results warrant further studies with HSP90 inhibitors as potential therapeutic candidates for hereditary demyelinating neuropathies.     

Molecular diagnosis of PMP22-associated neuropathies using fluorescence in situ hybridization (FISH) on archival peripheral nerve tissue preparations

Charcot-Marie-Tooth (CMT) syndrome type 1 and tomaculous neuropathy, also called hereditary neuropathy with liability to pressure palsies (HNPP), represent two groups of neurological disorders with different subtypes, which can be distinguished at the molecular level. It is known that a 1.5-mb region on chromosome 17p11.2– 12, which includes the gene for the peripheral myelin protein 22 kDa (PMP22), is duplicated in more than 95% of patients with CMT type 1A (CMT1A; gene dosage 3) and is deleted in about 90% of subjects suffering from HNPP (gene dosage 1). This duplication/deletion can be detected reliably by interphase-two-color fluorescence in situ hybridization (FISH). We report here a technique for extraction of nuclei from paraffin-embedded and cryofixed sural nerve biopsies for precise molecular diagnosis, employing interphase-two-color FISH in clinically diagnosed CMT1 or HNPP patients. Following this technique we were able to identify six CMT1A duplications in 13 clinically diagnosed CMT1 cases and five HNPP deletions in 6 clinically diagnosed HNPP cases; 8 control persons were included in this study. This is the first report on the use of FISH in the detection of 17p11.2–12 duplication and deletion in archival biopsy material. Received: 10 January 1997 / Revised, accepted: 3 March 1997     

Evidence for impaired axonal regeneration in PMP22 duplication: studies in nerve xenografts

Whether axonal regeneration in Charcot-Marie-Tooth (CMT) neuropathies is impaired has not been addressed in detail. Our studies in nude mice harboring xenografts from patients with different primary Schwann cell (SC) genetic defects suggested an intimate association between the onset of myelination and impairment in the growth capacity of nude mice axons engulfed by the mutant SCs. To assess the effects of peripheral myelin protein 22 (PMP22) gene duplication on the regeneration process, we conducted morphometric studies to generate temporal growth profiles of myelinated axons within the xenografts obtained from CMT1A patients and from healthy controls. Axon size distribution histograms in controls at different time intervals revealed that size differentiation of myelinated fibers within the grafts is established as early as 2 weeks, and that the temporal pattern of myelination of different sized axons has striking similarities to myelination during development. In PMP22 duplication grafts, the onset of myelination is delayed and the regeneration capacity of all fiber sizes is impaired. This defect, however, is most pronounced for the large diameter axons. In addition, significant large fiber loss occurred after 12 weeks with a concomitant new cycle of regeneration of small size axons. These studies show that the PMP22 duplication in SCs have profound effects on the regeneration process, which might be a contributing factor to preferential distal axonal loss.