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.     

Peripheral myelin protein 22: Facts and hypotheses

Mutations affecting the peripheral myelin protein 22 (PMP22) gene are associated with inherited motor and sensory neuropathies in mouse (Trembler and Trembler-J) and human (Charcot-Marie-Tooth disease type 1A and Dejerine-Sottas syndrome). Although genetic studies have established a critical role of PMP22 in the formation and/or maintenance of myelin in the peripheral nervous system, the biological function of PMP22 in myelin and in non-myelin forming cells remains largely enigmatic. In this Mini Review, we will summarize the current knowledge about PMP22 and discuss its hypothetical function(s) in a broad context. © 1995 Wiley-Liss, Inc.     

Hereditary neuropathy with liability to pressure palsies associated with central nervous system myelin lesions

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder most commonly caused by a 1.5-Mb deletion in chromosome 17p11.2 which contains the peripheral myelin protein-22 (PMP22) gene. Mutations resulting in functional loss of one PMP22 gene copy are less frequent. We present a 51-year-old patient with a l.5-Mb deletion in chromosome 17p11.2 who exhibited signs of peripheral as well as central nervous system lesions. He gave a history of recurrent episodes of limb numbness and weakness with spontaneous but incomplete recovery since age 20. His father and two brothers had similar symptoms. Neurological examination revealed signs of multiple mononeuropathy associated with frontal lobe, corticospinal tract and cerebellar dysfunction, as well as signs of initial cognitive impairment. Electrophysiological investigations showed a demyelinating peripheral nerve disease with multiple conduction blocks and conduction disturbances in both optic nerves. Magnetic resonance imaging of the brain revealed multiple subcortical and periventricular foci of myelin lesions. The association of central and peripheral nervous system lesions in this patient indicates a possible role of PMP22 not only in peripheral but also in central nervous system myelin structure.     

Accumulation of peripheral myelin protein 22 in onion bulbs and Schwann cells of biopsied nerves from patients with Charcot-Marie-Tooth disease type 1A

Peripheral myelin protein 22 (PMP-22) is a glycoprotein expressed in the myelin sheath of myelinated Schwann cells. Duplication of the PMP-22 gene and its gene dosage effect have been postulated to be involved in the pathogenesis in the majority of individuals with Charcot-Marie-Tooth disease type 1A (CMT1A). Northern blot analysis has demonstrated that the mean relative ratio of PMP-22 mRNA/β-actin mRNA in biopsied nerves of patients with CMT1A is significantly higher than that in disease controls. To investigate whether the elevated expression of PMP-22 mRNA is reflected in the amount and the localization of PMP-22, we analyzed PMP-22, myelin basic protein (MBP), protein zero (P0), and S-100 immunoreactivities in biopsied nerves from six patients with CMT1A, five patients with other types of CMT, five patients with acquired demyelinating neuropathies, and two normal subjects. In all patients with CMT other than CMT1A and acquired demyelinating neuropathy, as well as in normal subjects, the myelin sheath was immunoreactive for PMP-22, MBP, and P0, while the Schwann cell cytoplasm was immunoreactive only for S-100. In five out of six patients with CMT1A, however, the PMP-22 immunoreactivity was present not only on the myelin sheath but also in the Schwann cell cytoplasm and onion bulbs (OBs). Although OBs are nonspecific and also seen in other inherited or acquired demyelinating neuropathies, the PMP-22-positive OBs were seen exclusively in CMT1A.The finding suggested that the expression of PMP-22 was abnormal for its localization and probably for the amount in patients with CMT1A carrying duplication of the PMP-22 gene. Received: 5 February 1996 / Revised, accepted: 20 May 1996     

Heterosis for myelin content is limited to the central nervous system

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post-natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.     

Molecular alterations resulting from frameshift mutations in peripheral myelin protein 22: implications for neuropathy severity

Alterations in peripheral myelin protein 22 (PMP22) expression are associated with a heterogeneous group of hereditary demyelinating peripheral neuropathies. Two mutations at glycine 94, a single guanine insertion or deletion in PMP22, result in different reading frameshifts and, consequently, an extended G94fsX222 or a truncated G94fsX110 protein, respectively. Both of these autosomal dominant mutations alter the second half of PMP22 and yet are linked to clinical phenotypes with distinct severities. The G94fsX222 is associated with hereditary neuropathy with liability to pressure palsies, whereas G94fsX110 causes severe neuropathy diagnosed as Dejerine-Sottas disease or Charcot-Marie-Tooth disease type IA. To investigate the subcellular changes associated with the G94 frameshift mutations, we expressed epitope-tagged forms in primary rat Schwann cells. Biochemical and immunolabeling studies indicate that, unlike the wild-type protein, which is targeted for the plasma membrane, frameshift PMP22s are retained in the cell, prior to reaching the medial Golgi compartment. Similar to Wt-PMP22, both frameshift mutants are targeted for proteasomal degradation and accumulate in detergent-insoluble, ubiquitin-containing aggregates upon inhibition of this pathway. The extended frameshift PMP22 shows the ability to form spontaneous aggregates in the absence of proteasome inhibition. On the other hand, Schwann cells expressing the truncated protein proliferate at a significantly higher rate than Schwann cells expressing the wild-type or the extended PMP22. In summary, these results suggest that a greater potential for PMP22 aggregation is associated with a less severe phenotype, whereas dysregulation of Schwann cell proliferation is linked to severe neuropathy.     

Mutational analysis of GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in Serbian Charcot-Marie-Tooth patients

We report the results of mutational analysis in the following genes: GJB1 , MPZ , PMP22 , EGR2 , and LITAF/SIMPLE in 57 Charcot-Marie-Tooth (CMT) patients of Serbian origin without the PMP22 duplication. We found 10 different mutations in 14 CMT patients: 6 mutations in GJB1 , 3 in MPZ , and 1 in PMP22 . Five of six GJB1 mutations are reported for the first time, and the most frequent one appears to be a founder mutation in the Serbian population. No mutations were found in EGR2 or LITAF . Thus, GJB1 mutation analysis should be done in patients without the PMP22 duplication and male-to-male transmission of CMT.     

Peripheral myelin protein 22 and protein zero: a novel association in peripheral nervous system myelin.

Mutations found in the two major glycosylated transmembrane proteins of the PNS myelin, the peripheral myelin protein zero (P0) and peripheral myelin protein 22 (PMP22), have been independently associated with the most common hereditary demyelinating peripheral neuropathies. Genotype-phenotype correlations in humans and transgenic animals have provided functional evidence that P0 and PMP22 are involved in formation and maintenance of compact myelin. Here, we demonstrate for the first time that P0 and PMP22 proteins form complexes in the myelin membrane, as shown by coimmunoprecipitation experiments, and that glycosylation is not involved in mediating these interactions. Complex formation was also detected when the two proteins were coexpressed in heterologous cells. In transfected cells, P0 and PMP22 are recruited and colocalize at the apposed plasma membranes of expressors as shown by confocal microscopy. These findings provide a new basis for a better understanding of myelin assembly and of the pathomechanisms involved in demyelinating peripheral neuropathies. Furthermore, these results propose a possible explanation why alterations in either of these molecules are sufficient to destabilize the myelin structure and cause a similar disease phenotype.     

Histochemical demonstration of cytoplasmic glycosaminoglycans in the macroneurons of the human central nervous system

The presence of glycosaminoglycans (GAG) has been histochemically demonstrated in the CNS of various mammalian species. They have been related with some nerve functions as neurotransmitters storage and synaptic transmission. In the present paper, the histochemical properties of nerve cell cytoplasmic GAG were studied in several regions of adult human CNS. Samples of brain cortex, pons, upper medulla, and cerebellar cortex obtained by autopsy from subjects not dying after neurological diseases were fixed by immersion in glutaraldehyde, dehydrated with ethanol, and embedded in paraffin. The sections were stained with Alcian blue solutions adjusted to pH 2.5, 4.0, and 5.7. To the latter solution MgCl₂ was added in increasing concentration from 0.05 to 1.2 M. Testicular hyaluronidase, neuraminidase, and ribonuclease were applied on simultaneous sections with their respective controls. The sequence of these reactions allowed us to demonstrate the presence of hyaluronic acid along chondroitin-4- and/or 6-sulphate in the cytoplasm of most nerve cells. The sulphated GAG showed certain variability in the various regions studied related specially with their grade of sulphation.     

Distribution of the iron-regulating protein hepcidin in the murine central nervous system

Iron serves as an essential trace element for all body tissues, including the central nervous system (CNS). Because iron deficiency as well as iron overload is known to cause damage to the mammalian brain, the maintenance of iron homeostasis is crucial. It has been discovered recently that hepcidin plays an essential role in iron metabolism outside the CNS. A defect in hepcidin expression is responsible for iron accumulation and mice over-expressing hepcidin die postnatally by a severe anemia. We have used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the cellular distribution of hepcidin mRNA and protein in brain, spinal cord, and dorsal root ganglia. Our results show a wide-spread distribution of hepcidin in different brain areas, including the olfactory bulb, cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon, cerebellum, pons, spinal cord, as well as in dorsal root ganglia of the peripheral nervous system. Hepcidin immunoreactivity is not restricted to neurons, but can be detected in both neurons and GFAP-positive glia cells. Because hepcidin action in organs outside the CNS is linked to iron homeostasis, we speculate that it is also involved in such processes in the CNS, putatively together with other iron regulating proteins. Cellular mechanisms and functions of hepcidin in the CNS remain to be elucidated.     

A novel homozygous variant extending the peripheral myelin protein 22 by 9 amino acids causes early‐onset Charcot‐Marie‐Tooth disease with predominant severe sensory ataxia

Peripheral myelin protein 22 (PMP22) related neuropathies account for over 50% of inherited peripheral neuropathies. A gene copy variation results in CMT1A (duplication) and hereditary neuropathy with liability to pressure palsies (HNPP; single deletion). Point mutations comprise both phenotypes. The underlying pathological mechanisms are incompletely understood and biallelic mutations of PMP22 are very rare. We describe a 9‐year‐old girl who presented before the age of 1 year with severe locomotor delay. She now requires support for standing and walking in view of her severe sensory ataxia. Strikingly, her muscle power and bulk are close to normal in all segments. Nerve conduction studies showed sensory‐motor velocities below 5 m/s. Genetic analysis revealed a homozygous sequence change in the PMP22 gene causing the loss of termination codon (c.483A > G; p.[*161Trpext*10]), extending the protein by 9 amino acids. Both heterozygous parents have neurophysiological abnormalities consistent with HNPP, consistent with this being a loss‐of‐function mutation. PMP22‐deficient human models are rare but important to decipher the physiological function of the PMP22 protein in vivo. The predominance of large fiber sensory involvement in this and other rare similar cases suggests a pivotal role played by PMP22 in the embryogenesis of dorsal root ganglia in humans.     

Early onset demyelinating Charcot‐Marie‐Tooth disease caused by a novel in‐frame isoleucine deletion in peripheral myelin protein 2

Peripheral myelin protein 2 (PMP2) is a small protein located on the cytoplasmic side of compact myelin, involved in the lipids transport and in the myelination process. In the last years few families affected with demyelinating Charcot‐Marie‐Tooth neuropathy (CMT1), caused by PMP2 mutations, have been identified. In this study we describe the first case of a PMP2 in‐frame deletion. PMP2 was analyzed by direct sequencing after exclusion of the most frequent CMT‐associated genes by using a next generation sequencing (NGS) genes panel. Sanger sequencing was used for family's segregation analysis. Molecular modeling analysis was used to evaluate the mutation impact on the protein structure. A novel PMP2: p.I50del has been identified in a child with early onset CMT1 and in three affected family members. All family members show an early onset demyelinating neuropathy without other distinguish features. Molecular modeling analysis and in silico evaluations do not suggest a strong impact on the overall protein structure, but a most likely altered protein function. This study suggests the importance to add PMP2 in CMT NGS genes panels or, at most, to test it after major CMT1 genes exclusion, due to the lack of diagnostic‐addressing additional features.     

PQBP-1 is expressed predominantly in the central nervous system during development

Mutations of PQBP-1 (polyglutamine binding protein-1) have been shown recently to cause human mental retardation accompanied by microcephaly at a high frequency. As a first step towards understanding the molecular basis of this developmental anomaly, we analysed developmental expression of PQBP-1 by in situ hybridization, immunohistochemistry and Western blot analysis. Although it had been shown by Northern blot analysis that PQBP-1 mRNA is expressed in multiple organs in adult mice, our present results revealed that PQBP-1 mRNA and protein are dominantly expressed in the central nervous system (CNS) in embryos and in newborn mice. The mean expression level of PQBP-1 reaches a peak around birth and is down-regulated in adulthood. Furthermore, the expression pattern in the CNS changes remarkably following birth. PQBP-1 mRNA in the cerebral cortex is high in embryos but it rapidly decreases after birth. PQBP-1 mRNA increases in external and internal granular cell layers of the cerebellum from postnatal day 1 (P1) to P5. In addition, expression in the subventricular zone, where neurogenesis occurs, was high from P5 to adulthood. Collectively, these findings suggest that PQBP-1 might be involved in neuronal proliferation and/or maturation. These ideas may be relevant to the insufficient growth of brain structure reported in PQBP-1-linked human mental retardation.     

Peripheral nervous system and central nervous system pathology in rapidly progressive lower motor neuron syndrome with immunoglobulin M anti-GM1 ganglioside antibody

Pathological studies, including novel teased peripheral nerve fiber studies, were performed in a patient who presented with a rapidly progressive, lower motor neuron syndrome and high titer of immunoglobulin M anti-GM1 ganglioside antibody. In the central nervous system, there was a severe loss of motor neurons and central chromatolysis with ubiquitin immunopositive cytoplasmic inclusions in residual motor neurons. In the peripheral nervous system, axonal degeneration of myelinated fibers in the anterior nerve roots was evident. Pathologic evidence of sensory nerve involvement was also found despite the absence of clinical or electrophysiological sensory abnormalities. Sectional studies of single myelinated nerve fibers from an antemortem sural nerve biopsy showed remyelination and globular paranodal swellings due to focal complex myelin folding and degeneration in 13% of fibers. Postmortem studies of the sural nerves 4 weeks later showed paranodal demyelination (90% of fibers), but no paranodal swellings and similar findings were present in samples of the ulnar, radial, median, tibial, and common peroneal nerves. Paranodal abnormalities of enlargement of the adaxonal space, myelin degeneration, and axonal compaction were found on cross-sectional studies of individual teased fibers, which on conventional light microscopic assessment appeared normal. These changes suggest a disturbance of paranodal axonal-myelin adhesion due to binding of the anti-GM1 ganglioside antibody to the common epitope known to be present on the myelin sheath and nodal axolemma in the paranodal region of both motor and sensory nerves.     

Injury reactive myelin/oligodendrocyte-derived axon growth inhibition in the adult mammalian central nervous system

Myelin inhibition is considered a constitutive, static, repulsive barrier not reactive to a central nervous system (CNS) lesion. However, recent evidence underlines the existence of considerable add-on axon growth inhibition upon CNS injury. This postlesional, reactive myelin/oligodendrocyte-derived inhibition will require the development of novel screening approaches and therapeutic reagents to promote axonal regeneration.