Competitive binding of triplex-forming oligonucleotides in the two alternate promoters of the PMP22 gene

Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.     

Mutational analysis of the MPZ, PMP22 and Cx32 genes in patients of Spanish ancestry with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and hereditary neuropathy with liability to pressure palsies (HNPP) are two inherited peripheral neuropathies. The most prevalent mutations are a reciprocal 1.5-Mb duplication and 1.5-Mb deletion, respectively, at the CMT1A/HNPP locus on chromosome 17p11.2. Point mutations in the coding region of the myelin genes, peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) or connexin 32 (Cx32) have been reported in CMT patients, including CMT type 1 (CMT1), CMT type 2 (CMT2) and Déjérine-Sottas neuropathy (DS) patients, and only in the coding region of PMP22 in HNPP families lacking a deletion. We have investigated point and small mutations in the MPZ, PMP22 and Cx32 genes in a series of patients of Spanish ancestry: 47 CMT patients without duplications, and 5 HNPP patients without deletions. We found 15 different mutations in 16 CMT patients (34%). Nine different mutations in ten patients were detected in the Cx32 gene, this being the most frequently involved gene in this series, whereas five mutations involved the MPZ gene and only one the PMP22 gene. Six out of nine nucleotide substitutions in the Cx32 gene involved two codons encoding arginine at positions 164 and 183, suggesting that these two codons may constitute two Cx32 regions prone to mutate in the Spanish population. Analysis of HNPP patients revealed a 5′ splicing mutation in intron 1 of the PMP22 gene in a family with autosomal dominance, which confirms allelic heterogeneity in HNPP. Ectopic mRNA analysis on leukocytes suggests that this mutation might behave as a null allele. Received: 25 July 1996 / Revised: 15 November 1996     

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.     

Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol’s effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT.     

The PMP22 Gene and Its Related Diseases

Peripheral myelin protein-22 (PMP22) is primarily expressed in the compact myelin of the peripheral nervous system. Levels of PMP22 have to be tightly regulated since alterations of PMP22 levels by mutations of the PMP22 gene are responsible for >50 % of all patients with inherited peripheral neuropathies, including Charcot–Marie–Tooth type-1A (CMT1A) with trisomy of PMP22, hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of PMP22, and CMT1E with point mutations of PMP22. While overexpression and point-mutations of the PMP22 gene may produce gain-of-function phenotypes, deletion of PMP22 results in a loss-of-function phenotype that reveals the normal physiological functions of the PMP22 protein. In this article, we will review the basic genetics, biochemistry and molecular structure of PMP22, followed by discussion of the current understanding of pathogenic mechanisms involving in the inherited neuropathies with mutations in PMP22 gene.     

Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain.

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc-IVLH(Bom)VAVLVLLFVSTIV-OMe (1) and its Pro16 mutant Boc-IVLH(Bom)VAVPVLLFVSTIV-OMe (2) were synthesized. Circular dichorism (CD)-spectral analysis suggested that peptide 1 adopts a stable alpha-helical conformation in membrane-mimetic solvent,1-BuOH/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) system. On the contrary, the mutant 2 favors beta-sheet conformation in the same solvent system. Interestingly, alpha-helix to beta-sheet transition of 2 was observed at higher contents of 1-BuOH than 70%.     

Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A

Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin–proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.     

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.     

Real-time quantitative polymerase chain reaction

In Charcot-Marie-Tooth type 1A disease (CMTIA), heterozygosity for the peripheral myelin protein 22 (PMP22) duplication increases the gene dose from two to three, whereas, in hereditary neuropathy with liability to pressure palsies (HNPP), heterozygosity for the PMP22 deletion reduces the gene dose from two to one. Thirty-eight Norwegian patients with CMT1, 4 patients with HNPP, 15 asymptomatic family members, and 45 normal controls were studied using the ABI 7700 sequence detection system and the TaqMan method of real-time quantitative polymerase chain reaction (PCR). Using a comparative threshold cycle (Ct) method and albumin as reference gene, the gene copy number by PMP22 gene duplication or deletion on chromosome 17p11.2-12 was quantified. The PMP22 duplication ratio ranged from 1.50 to 2.21, the PMP22 deletion ratio ranged from 0.44 to 0.55, and the PMP22 ratio in normals ranged from 0.82 to 1.27. All samples were run in triplicate, with a mean standard deviation of 0.07 (range 0.01-0.17). Thirty-four of thirty-eight CMT1 patients (89.6%) had the PMP22 duplication and the four HNPP patients had the PMP22 deletion. This was not found in any of the asymptomatic family members or the controls. Real-time quantitative PCR is a sensitive, specific, and reproducible method for diagnosing PMP22 duplication and deletion. The method is fast, allowing 13 patients to be diagnosed in 2 h. It involves no radioisotopes and requires no post-PCR handling. In our opinion, real-time quantitative PCR is the first method of choice in diagnosing PMP22 duplication and deletion.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

A unique point mutation in the PMP22 gene is associated with Charcot-Marie-Tooth disease and deafness.

Charcot-Marie-Tooth disease (CMT) with deafness is clinically distinct among the genetically heterogeneous group of CMT disorders. Molecular studies in a large family with autosomal dominant CMT and deafness have not been reported. The present molecular study involves a family with progressive features of CMT and deafness, originally reported by Kousseff et al. Genetic analysis of 70 individuals (31 affected, 28 unaffected, and 11 spouses) revealed linkage to markers on chromosome 17p11.2-p12, with a maximum LOD score of 9.01 for marker D17S1357 at a recombination fraction of .03. Haplotype analysis placed the CMT-deafness locus between markers D17S839 and D17S122, a approximately 0.6-Mb interval. This critical region lies within the CMT type 1A duplication region and excludes MYO15, a gene coding an unconventional myosin that causes a form of autosomal recessive deafness called DFNB3. Affected individuals from this family do not have the common 1.5-Mb duplication of CMT type 1A. Direct sequencing of the candidate peripheral myelin protein 22 (PMP22) gene detected a unique G-->C transversion in the heterozygous state in all affected individuals, at position 248 in coding exon 3, predicted to result in an Ala67Pro substitution in the second transmembrane domain of PMP22.     

The peripheral neuropathy-linked Trembler and Trembler-J mutant forms of peripheral myelin protein 22 are folding-destabilized

Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D ( Trembler phenotype) and L16P ( Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the three-dimensional structures of these disease-linked mutants are similar to that of the folded wild-type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild-type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild-type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild-type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant.     

A Mutation in PMP2 Causes Dominant Demyelinating Charcot-Marie-Tooth Neuropathy

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral neuropathies with diverse genetic causes. In this study, we identified p.I43N mutation in PMP2 from a family exhibiting autosomal dominant demyelinating CMT neuropathy by whole exome sequencing and characterized the clinical features. The age at onset was the first to second decades and muscle atrophy started in the distal portion of the leg. Predominant fatty replacement in the anterior and lateral compartment was similar to that in CMT1A caused by PMP22 duplication. Sural nerve biopsy showed onion bulbs and degenerating fibers with various myelin abnormalities. The relevance of PMP2 mutation as a genetic cause of dominant CMT1 was assessed using transgenic mouse models. Transgenic mice expressing wild type or mutant (p.I43N) PMP2 exhibited abnormal motor function. Electrophysiological data revealed that both mice had reduced motor nerve conduction velocities (MNCV). Electron microscopy revealed that demyelinating fibers and internodal lengths were shortened in both transgenic mice. These data imply that overexpression of wild type as well as mutant PMP2 also causes the CMT1 phenotype, which has been documented in the PMP22. This report might expand the genetic and clinical features of CMT and a further mechanism study will enhance our understanding of PMP2-associated peripheral neuropathy.     

De novo Ser72Leu mutation in the peripheral myelin protein 22 in two Polish patients with a severe form of Charcot-Marie-Tooth disease

To date, 12 cases of heterozygous Ser72Leu mutations in the peripheral myelin protein 22 have been reported in patients suffering from severe demyelinating form of Charcot-Marie-Tooth disease (CMT1) and congenital hypomyelinating neuropathy (CHN) [MIM# 605253]. In the present study we report two cases of de novo S72L mutations in the PMP22 gene detected in patients of Polish origin suffering from CMT1 disease.     

A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies

Peripheral myelin protein 22 (PMP 22) is a component of compact myelin in the peripheral nervous system. The amount of PMP 22 in myelin is tightly regulated, and PMP 22 over or under‐expression cause Charcot‐Marie‐Tooth 1A (CMT 1A) and Hereditary Neuropathy with Pressure Palsies (HNPP ). Despite the importance of PMP 22 , its function remains largely unknown. It was reported that PMP 22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT 1A or HNPP . Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP 22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP . In contrast, ablation of integrin β4 worsens nerve conduction velocities and non‐compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers.

     

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca²⁺]_i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca²⁺]_i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca²⁺ into CMT1A SC. Correction of the altered [Ca²⁺]_i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca²⁺]_i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.Charcot-Marie-Tooth disease type 1 (CMT1)³ is a progressive hereditary motor and sensory neuropathy, characterized by distal muscle wasting and weakness, foot deformities, and severe slowing of nerve conduction, because of progressive demyelination (1). With a prevalence of 1 case in 2500, CMT1 is the most common hereditary neurologic disorder, and in the majority of cases (CMT1A) the disease is associated with a duplication on chromosome 17p11.2 of the gene for PMP22 (peripheral myelin protein 22) (2). PMP22 is a 22-kDa glycoprotein mainly expressed by myelinating Schwann cells (SC) and localized in compact myelin (3). The transgenic rat model of CMT1A, obtained by overexpression of PMP22 (4), confirms a role of PMP22 in the pathogenesis of CMT1A. Both PMP22 overexpression because of gene duplication and point mutations of PMP22 are associated with a CMT1A phenotype.The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear. Some reports indicate that a perturbed homeostasis of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) might be causally involved in the demyelination process. Conditions inducing an increased [Ca²⁺]_i in SC impair cell differentiation and myelination (5, 6), similarly to what occurs in CMT1A. Incubation of intact rat nerves with Ca²⁺ and ionophores causes a progressive demyelination, spreading from the paranodes and invading regions of formerly compact myelin, which is dependent upon a rise in the [Ca²⁺]_i of SC (5).Additional evidence for the detrimental effect of a [Ca²⁺]_i elevation on myelin production by SC comes from application of ATP to murine SC monocultures, inducing an immediate and large increase in the [Ca²⁺]_i. As a result of ATP treatment, maturation and differentiation of SC, as well as expression of the myelin basic protein and production of compact myelin, are completely prevented (6). Taken together, the above observations indicate that abnormally elevated Ca²⁺ levels are causally related to impairment of myelin production by SC.In this study, we addressed the possible correlation between PMP22 overexpression and alteration of the [Ca²⁺]_i homeostasis in SC from a rat model of CMT1A. We recorded higher levels of basal [Ca²⁺]_i in affected than in control cells, and we identified the mechanisms responsible for the perturbation of the [Ca²⁺]_i levels in CMT1A SC. Experiments with pharmacological inhibitors and with small interfering RNA (siRNA) unequivocally demonstrated a correlation in CMT1A SC between overexpression of the purinergic receptor P2X7 and influx of extracellular [Ca²⁺]_i across this plasma membrane receptor/channel. In addition, correction of the abnormally elevated [Ca²⁺]_i levels by the use of a P2X7 antagonist or through down-regulation of the expression of P2X7 by transfection with siRNA or with short hairpin RNA-expressing plasmid (shRNA) restored the normal phenotype in CMT1A SC. These findings suggest that CMT1A should be considered as a “calcium disease.” Identification of P2X7 activation as the pathogenetic mechanism underlying demyelination may provide the rationale for a new therapeutic strategy for CMT1A, a disease with no currently available treatment.     

DNA rearrangements on both homologues of chromosome 17 in a mildly delayed individual with a family history of autosomal dominant carpal tunnel syndrome.

Disorders known to be caused by molecular and cytogenetic abnormalities of the proximal short arm of chromosome 17 include Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), Smith-Magenis syndrome (SMS), and mental retardation and congenital anomalies associated with partial duplication of 17p. We identified a patient with multifocal mononeuropathies and mild distal neuropathy, growth hormone deficiency, and mild mental retardation who was found to have a duplication of the SMS region of 17p11.2 and a deletion of the peripheral myelin protein 22 (PMP22) gene within 17p12 on the homologous chromosome. Further molecular analyses reveal that the dup(17)(p11.2p11.2) is a de novo event but that the PMP22 deletion is familial. The family members with deletions of PMP22 have abnormalities indicative of carpal tunnel syndrome, documented by electrophysiological studies prior to molecular analysis. The chromosomal duplication was shown by interphase FISH analysis to be a tandem duplication. These data indicate that familial entrapment neuropathies, such as carpal tunnel syndrome and focal ulnar neuropathy syndrome, can occur because of deletions of the PMP22 gene. The co-occurrence of the 17p11.2 duplication and the PMP22 deletion in this patient likely reflects the relatively high frequency at which these abnormalities arise and the underlying molecular characteristics of the genome in this region.     

P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors.

Protein zero (P0) and peripheral myelin protein 22 (PMP22) are most prominently expressed by myelinating Schwann cells as components of compact myelin of the peripheral nervous system (PNS), and mutants affecting P0 and PMP22 show severe defects in myelination. Recent expression studies suggest a role of P0 and PMP22 not only in myelination but also during embryonic development. Here we show that, in dorsal root ganglia (DRG) and differentiated neural crest cultures, P0 is expressed in the glial lineage whereas PMP22 is also detectable in neurons. In addition, however, P0 and PMP22 are both expressed in a multipotent cell type isolated from early DRG. Like neural crest stem cells (NCSCs), this P0/PMP22-positive cell gives rise to glia, neurons and smooth-muscle-like cells in response to instructive extracellular cues. In cultures of differentiating neural crest, a similar multipotent cell type can be identified in which expression of P0 and PMP22 precedes the appearance of neural differentiation markers. Intriguingly, this P0/PMP22-positive progenitor exhibits fate restrictions dependent on the cellular context in which it is exposed to environmental signals. While single P0/PMP22-positive progenitor cells can generate smooth muscle in response to factors of the TGF-(beta) family, communities of P0/PMP22-positive cells interpret TGF-(beta) factors differently and produce neurons or undergo increased cell death instead of generating smooth-muscle-like cells. Our data are consistent with a model in which cellular association of postmigratory multipotent progenitors might be involved in the suppression of a non-neural fate in forming peripheral ganglia.     

A rapid and definitive test for Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies using multiplexed real-time PCR

Alterations in gene copy number have been shown to cause disease in humans. Two of the most common inherited peripheral neuropathies, Charcot-Marie-Tooth 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), are two such diseases resulting from alteration in gene copy number of the dosage sensitive peripheral myelin protein 22 (PMP22) gene. Many complicated and laborious diagnostic tests exist for the diagnosis of these diseases. The aim of our study was to develop the first quantitative multiplex real-time PCR assay for the diagnosis of CMT1A and HNPP. A total of 160 individuals who were known to have CMT1A, HNPP, or were normal from previous testing were assayed by our multiplex real-time PCR method. The results confirmed the previously determined gene copy number of all patient and control individuals tested. The range of ratio values between the disease and control groups were easily defined. The assay is accurate, simple, and cost effective and can detect a 50% change in gene copy number. This represents an ideal assay for any small diagnostic laboratory.