Mutations in four different genes— PMP22 , MPZ , Cx32 , and EGR2 —have been associated with myelinopathies. The proteins encoded by these genes are distinct, but each is expressed by myelinating Schwann cells. PMP22 and MPZ appear to encode compact myelin structural proteins (recent evidence suggests an association between these two proteins in peripheral nervous system (PNS) myelin197a); Cx32 encodes a noncompact myelin gap junction protein that is responsible for direct transport of small molecules across the myelin sheath (Fig. 227-5);198 and EGR2 encodes a transcription factor required for myelin gene expression that appears to be important for myelin development and maintenance. A comprehensive mutation database for myelinopathy associated mutations is available (http://molgen-www.uia.ac.be/CMTMutations/).
Myelin proteins and myelinopathies. A Schwann cell and the structure of compact myelin with the intraperiod and major dense lines (above left). The detailed blow-up depicts the structure of the compact myelin membrane proteins P0 and PMP22, as well as the noncompact myelin protein Cx32, with their major associated disease phenotypes in rectangles. A side view of the peripheral nerve with axon and myelin demonstrating the distribution of myelin protein P0 (B), PMP22(A) in the compact myelin and Cx32(X) in the noncompact myelin (below). (Adapted from Roa and Lupski.198 Used with permission.)
PMP22 Gene and Protein Structure.
The PMP22 (GenBank L03203) cDNA was isolated as an axon-regulated Schwann cell transcript, encoding a myelin membrane protein, that is down-regulated after sciatic nerve injury199–202 and independently from a cDNA library of human fetal spinal cord.203 It was recognized to be identical to gas-3, a growth arrest-specific gene.202,204 It has also been designated CD25 and SR13. The PMP22 gene contains four coding exons and is regulated by two alternatively used promoters.205,206 It is expressed predominantly in the peripheral nerve,162,205 but also in other nonneural tissues during development and in the adult.207
The PMP22 protein is an integral membrane glycoprotein with four proposed transmembrane domains. It has a mass of ≈22 kDa (an 18-kDa core polypeptide and 4 kDa of carbohydrates linked to an asparagine residue). The glycosylated portion contains an L2/HNK-1 epitope.208,209 This epitope is implicated in intercellular recognition, interaction, and adhesion processes. PMP22 comprises 2 to 5 percent of PNS myelin210 and is found uniformly in compact, but not noncompacted, regions of myelin.200,210,211 Although ultrastructural studies of uncompacted myelin lamellae in nerves obtained from HNPP patients suggest a structural role for PMP22, the exact function of PMP22 remains unknown.212 An alternative proposed function is regulation of Schwann-cell growth and differentiation,213,214 as suggested by the initial identification of PMP22 as a growth-arrest specific gene. Some investigators suggest a dual role as a structural protein important to myelin compaction and as a regulatory protein important in myelin development and maintenance.28 Whether PMP22 has a regulatory role in addition to its structural function remains elusive. Nevertheless, mutation studies of other myelin structural proteins ( MPZ , Cx32 ) reveal dramatic effects on myelin development and maintenance.
PMP22 Mutations and Myelinopathies.
PMP22 point mutations have been associated with CMT1, DSS, and HNPP.166–169,215–229 These mutations include mostly missense amino acid substitutions, but deletion of a single amino acid and a few frameshift mutations have also been reported (Fig. 227-6). Codon position 72 has been suggested to be a mutation hotspot because of four independent reports of the same mutation. This mutation occurs at a CpG dinucleotide. Most mutations occur in amino acids comprising the putative transmembrane domains. Eleven of the 24 mutations are de novo and associated with sporadic disease. All alterations except one are associated with disease in the heterozygous state and thus are dominant alleles. Most PMP22 mutations result in a more severe neuropathy than that associated with the CMT1A duplication: 13 of 26 with DSS and many of the CMT1 mutations convey a more severe CMT phenotype.
Myelinopathy associated PMP22 mutations. The four predicted transmembrane domains of PMP22 are shown with individual amino acids represented as open circles. Specific mutations are delineated below with the associated phenotype shown by symbols depicted in the upper right. Essentially all mutations are in transmembrane domains. Identical mutations to the spontaneous mouse models Tr (mutation 3) and Tr
J (mutation 23) have been identified in a severe CMT1 family and DSS family respectively.
Three mutations (L6fs, V30M, and G94fs) are associated with HNPP. L6fs is a null allele that causes a classic HNPP phenotype.189 G94fs results in a mixed HNPP and CMT1 phenotype190,230 because 23 patients from 6 families228 showed clinical, electrophysiological and morphologic characteristics of HNPP but neuropathologic features of CMT1. The V30M mutation, which causes a mild HNPP phenotype and HNPP in nerve xenograft studies, is a conservative substitution at the junction of the first transmembrane domain and the extracellular loop.229
The T118M alteration, also located at the border of an extracellular loop and transmembrane domain, has been proposed to be either a recessive pathogenic mutation132 or a benign variant.231 It was identified as a recessive pathogenic mutation due to a severe CMT1 phenotype in a patient who was a compound heterozygote for this allele and an HNPP deletion. This patient was clinically distinct from her HNPP-affected children who inherited only the HNPP deletion, and from her son who had normal electrophysiological studies and who was heterozygous for the T118M allele.132 The same alteration was alternatively interpreted as a benign variant after its identification in two unaffected parents of sporadic CMT1 cases while being absent in one of the affected individuals.231 Interestingly, overexpression of the T118M mutation in NIH3T3 cells showed a reduced ability to trigger apoptosis as compared to wild-type PMP22 , but apoptosis occurred when coexpressed with wild-type PMP22.232 In contrast, other disease-associated PMP22 alleles have a dominant effect because their reduced ability to trigger apoptosis is not abrogated by coexpression of wild-type PMP22. These data support the hypothesis that the T118M variant is recessive and disease causing, because like other disease-causing PMP22 alleles, it has an effect on the regulation of susceptibility to apoptosis. Recent in vitro experiments suggest that impaired intracellular trafficking is a common disease mechanism for PMP22 point mutations causing peripheral neuropathies.232a These experiments provide independent evidence that T118M affects PMP22 function and is not a benign alteration.232a Nevertheless, identification of a recessive CMT1 patient or unaffected individual who is homozygous for the T118M variant is required to definitively answer the question of whether the T118M amino acid substitution is a recessive mutation or benign variant.
A recessive R157W mutation of PMP22 was associated with DSS, in three affected siblings from consanguineous parents.233 Heterozygous parents with this mutation were asymptomatic with normal NCV. A different missense amino acid substitution at the identical position, R157G, was also reported in the hemizygous state associated with CMT1.234 The compound heterozygous CMT1 individual had the R157G allele on one chromosome and the HNPP deletion on the other homologous chromosome. Molecular modeling localizes this mutation to the intracellular domain of PMP22. The genotype R157G/def thus appears to convey a more severe phenotype than wt/def (CMT1 vs. HNPP) while R157W/wt and R157G/wt have normal phenotypes consistent with a recessive gain-of-function mechanism. This recessive gain-of-function mechnanism is consistent with the PMP22 protein potentially having multimeric interactions.
PMP22 Mutations and Disease Mechanisms.
Studies of mouse models and of cell culture systems have shown that PMP22 mutations function as gain-of-function mutations, dominant-negative mutations, or both. The fact that the Tr mutant, Pmp22 Tr/+ mouse (GenBank M32240 for mouse Pmp22), displays severe hypomyelination while the heterozygous Pmp22 +/− knockout mice show focal hypermyelination suggests that Tr-PMP22 behaves as a gain-of-function or dominant-negative mutation and not as a null allele.235 Consistent with this gain-of-function hypothesis, Pmp22 Tr/− mice show severe hypomyelination, while Pmp22 −/− mice have focal hypermyelination.235 However, an overlapping dominant-negative effect is suggested by the increasing myelin deficiency with increasing numbers of Tr alleles; that is, Pmp22 Tr/Tr has a more severe phenotype than Pmp22 Tr/−.235 The hypomyelination observed in the Tr Ncnp allele, an in-frame deletion of the second transmembrane domain and a part of the third transmembrane domain, also supports a gain-of-function mechanism.236
Two contrasting observations have been made with Pmp22 mutations. In the first situation, the Tr protein alters protein trafficking and exhibits a dominant-negative effect on wild-type Pmp22237 in Tr animals and transfected cells.232 In the second situation, the distribution of Tr and TrJ mutant proteins differ from that of wild-type Pmp22, but neither Tr nor TrJ proteins have a dominant-negative effect on the cellular distribution of wild-type Pmp22238 suggesting that impaired trafficking of mutated Pmp22 effects Schwann cell physiology leading to myelin instability and loss.238
Three observations support altered trafficking and a dominant-negative effect of mutant Pmp22 protein. First, Pmp22 immunoreactive proteins accumulate in the ER/Golgi of Schwann cells of Tr mice.237,238 Second, Tr protein is retained in the ER of transfected COS-7 and cultured Schwann cells.237 The retention is hypothesized to be due to protein misfolding and/or impaired processing. Third, the cotransfection of wild-type Pmp22 and the TrJ mutant Pmp22, each with different epitope tags, demonstrated that TrJ protein, unlike Pmp22, does not reach the cell membrane but accumulates in the intermediate compartment,239 and that TrJ protein causes some, but not all, of the wild-type Pmp22 to be diverted from its normal trafficking pathway and retained in the intermediate compartment.239 An alternative cellular mechanism suggests that mutant Pmp22 protein is incorporated into the plasma membrane, decreasing myelin stability.240 This hypothesis is supported by the findings that the endosomal/lysosomal pathway is up-regulated and there is an accumulation of myelin proteins (PMP22, MBP and P0) in the endosomes/lysosomes.240
The human PMP22 loss-of-function point mutations result in HNPP while presumably gain-of-function or dominant-negative alleles result in CMT1 or DSS. Regardless of the exact mechanism by which the latter mutations produce a severe neuropathy, it is obvious that the stoichiometry of PMP22 is crucial for myelin function and maintenance. The major genetic mechanism for disease is not aberrant protein, but altered PMP22 dosage.10
MPZ Gene and Protein Structure.
The cDNA for the major structural protein of the peripheral myelin sheath, P0, was isolated by differential screening and hybrid selection241 (GenBank NM000530). The MPZ gene encoding human P0 contains six coding exons and is regulated by a single promoter.242 The MPZ promoter is often utilized as a Schwann cell-specific expression promoter in cells and transgenic mice.243 Multiple regulatory elements control transcription of MPZ. 244 The gene maps to 1q22-q23 (242) where the CMT1B locus (Table 227-3) was assigned.
P0 is a 28-kDa protein expressed exclusively by myelinating Schwann cells and accounts for >50 percent of the total PNS myelin protein.245 P0 is an integral membrane protein, and is localized to compact myelin. It consists of a single membrane-spanning region, a large hydrophobic glycosylated immunoglobulin-like extracellular domain, and a smaller basic intracellular domain241 (Fig. 227-5). The immunoglobulin-like extracellular domain is necessary for cell adhesion246,247 and plays a significant role in the formation and compaction of the intraperiod line.246,248 It contains an asparagine-linked glycosylation site that carries the L2/HNK-1 adhesive carbohydrate epitope.249 Membrane juxtaposition in the extracellular space of PNS myelin is thought to be mediated by homophilic interactions of the P0 extracellular domain (Fig. 227-5), while apposition of the cytoplasmic faces of the membrane is thought to be mediated by the intracellular domains.241 The intracellular domain is extremely basic and has been shown to participate in electrostatic interactions with the opposing anionic lipid bilayer to help in the formation of the major dense line250,251 (Fig. 227-5).
The crystal structure of the rat P0 extracellular domain (97 percent identical to human P0) consists of alternatively oriented cyclic tetramers, which are comprised of P0 protomers arranged head-to-tail about a fourfold axis.252 Each tetramer is related to four others of opposite orientation through protomer-protomer interfaces, which generates a network of molecules with half emanating from one membrane surface and the other half from the opposite surface.252 The interactions between opposing membrane faces have some similarities that have been likened to a “molecular velcro.”10 Outwardly pointing tryptophan side chains extend from the apices of each tetramer and may intercalate into the opposing membrane surface.252 Molecular modeling predicted some amino acids critical to homophilic interactions even prior to the availability of crystal structure information.253 The dimensions of the crystal model correspond with the dimensions of PNS myelin as determined by independent methods.
MPZ Mutations and Myelinopathies.
MPZ mutations were originally identified in families that showed linkage to chromosome 1,254,255 the first CMT locus mapped in the human genome.70 Subsequently, MPZ mutations were also found in patients with DSS 256 and CHN,257 and in some patients having a phenotype consistent with CMT2.257,258 Interestingly, a MPZ mutation, N102K, has been identified in the original family studied by Roussy and Lévy suggesting this represents a clinical variant of CMT1.258a More than 50 different myelinopathy-associated mutations in MPZ have been described259–285 (Fig. 227-7). The majority of the myelinopathy-associated mutations are missense mutations, but amino acid deletions, frameshift, and nonsense mutations also occur. Many mutations are de novo and all of the mutations that have been reported to date are dominant. Germ line mosaicism for one MPZ point mutation was reported in association with a DSS phenotype in two sisters.258 Most mutations occur in the extracellular domain, in contrast to PMP22 in which the majority of mutations affect the four putative transmembrane domains.
Myelinopathy-associated MPZ mutations. The single predicted transmembrane domain, extracellular immunoglobulin-like domain and intracellular domain are shown for the protein P0. Single amino acid positions are shown as open circles. Specific mutations are delineated below with the mutation type and manifested phenotype shown in the key in the upper left. Note that most mutations occur in the extracellular domain. However, the severity of the phenotype conveyed by the majority of mutations in the intracellular domain suggest an important function for this part of the P0 molecule (amino acid numbering based on processed protein; * = phenotype seen when homozygous for mutation).
Some mutations of MPZ suggest an underlying genetic mechanism. The codon for Arg69 (codon 98 if one includes the signal peptide in the numbering scheme) is proposed to have a high frequency of mutation based on the identification of three different mutations in 4 of 20 unrelated French CMT1 families without the CMT1A duplication.272 This high frequency of mutations occurs at a CpG dinucleotide. It is of interest that four different missense amino acid substitutions (R69H, R69S, R69P, R69C) and three different clinical phenotypes have been reported with mutations at this codon. The identical missense amino acid substitution R69C has been alternatively reported with DSS,271 CHN,283 and a severe CMT1B276 phenotype. The R69C, R69S,257 and R69H mutations271 have clinically distinct phenotypes.
Some MPZ mutations involve more than one amino acid; usually these amino acids are adjacent, but in one case of DSS, three separate de novo mutations were identified on the same chromosome.280 These three mutations did not occur in CpG dinucleotides, there is no significant secondary structure, which argues against correction of a quasipalindrome as a mechanism for multiple point mutations, and there are no known pseudogenes to participate in a gene conversion event. One potential mechanism for multiple point mutations is defective repair of a deleted region.
Selected mutations provide insights into the role of P0 in myelin structure, glycosylation requirements for function, and survival of the organism. The identification of a heterozygous P0 mutation associated with congenital hypomyelination suggests that a severe allele of a major myelin protein component can have dramatic effects on myelin structure and formation.257 The identical mutation, Q186X, was also identified in another case of CHN.281 This study used a polyclonal antibody directed against the entire P0 peptide to show that almost all myelinated fibers expressed the protein normally.281 Mutation of the single glycosylation site in P0 resulted in a relatively mild CMT1B phenotype,265 but this may not be too surprising because homophilic adhesive interactions of P0 are not dependent on glycosylation as shown by studies in heterologous cell systems.246,248,286,287 Homozygous mutation of MPZ has been reported twice (F35del, G74fs) and in both instances caused DSS;257,270 the early frameshift mutation, G74fs, is likely a null allele suggesting that absence of P0 is compatible with survival.
The crystal structure of the P0 extracellular domain allows predictions of how mutations might affect processing, structure, and interactions, and enables attempts at correlation with disease severity. Different P0 mutations at specific amino acid positions can lead to distinct phenotypes, and these provide an excellent model to examine how disease severity may correlate with mutation effect.252,257 At positions 34 and 69, changes to cysteine are associated with a DSS phenotype while other substitutions produce CMT1. Outwardly pointing thiols hypothetically produce a dominant-negative effect through the formation of disulfide aggregates in the extracellular space, and disrupt myelin structure by forming abnormal P0 complexes.257 Less severe CMT1-associated mutations at positions 34 and 69 may constitute loss-of-function alleles or weaker dominant-negative alleles.257 Furthermore, the identification of nonsense mutations at positions 125 and 152 in patients with a CMT1 phenotype supports the postulate that alleles conveying a milder phenotype may represent loss-of-function alleles, because the truncated proteins may be unstable and never reach the membrane.257 This model of disease severity associated with degree of dominant-negative effect has been supported by functional studies of MPZ mutations in cell surface localization and adhesion studies using Drosophila S2 and mammalian CHO (Chinese hamster ovary) cells.286,287
No crystal structure information is available for the P0 intracellular domain but the severity of the phenotype associated with intracellular mutations suggests that this domain of P0 plays an important functional role for myelin. Five of seven intracellular mutations cause a severe myelinopathy phenotype. In these cases, the mutant P0 apparently exerts a dominant-negative effect. Supporting this contention is the observation that a P0 protein truncated within the intracellular domain reaches the cell surface and inhibits extracellular domain adhesion in a cell adhesion assay.288 The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain.289
In summary, MPZ mutations are a less common cause of myelinopathy than the CMT1A duplication and the HNPP deletion, but they appear to be more common than PMP22 point mutations. Different MPZ point mutations may behave as loss-of-function, gain-of-function, or dominant-negative alleles. De novo point mutations have been identified in cases of sporadic neuropathy.
Mouse Models for P0 Deficiency.
A murine knockout of MPZ demonstrates that P0 is essential for normal compaction and maintenance of the peripheral myelin sheath and the continued integrity of the associated axon.290 P0-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.291 P0 −/− animals show hypomyelination by day 4 with subsequent demyelination and impaired nerve conduction. P0 +/− mice show normal myelination but develop progressive demyelination after 4 months of age. This demonstrates that only half the normal dose of P0 is sufficient to begin myelination, but apparently is not enough to support myelin maintenance at a later age. The pathology of homozygous and heterozygous P0 mutants resembles that of severely affected DSS and the CMT1B patients, respectively.291 Motor NCV also resemble those observed in humans with MPZ mutations.292 Interestingly, P0 +/− mice develop a peripheral neuropathy that resembles chronic inflammatory demyelinating polyneuropathy (CIDP). By 1 year of age, the mice develop severe, asymmetric slowing of motor nerves with temporal dispersion or conduction block, that are features of acquired demyelinating neuropathies including chronic inflammatory demyelinating polyneuropathy.293
Cx32 Gene and Protein Structure.
The Cx32 gene encoding connexin 32, also known as GJB1 encoding gap junction protein β1, has two exons. The entire coding sequence and 3′ untranslated region are contained within the second exon21 (GenBank NM-000166). In humans, Cx32 maps to Xq13.1. There are two alternative Cx32 promoters but only one is active in the peripheral nerve.294,295
Connexin 32 is a gap junction protein that belongs to a large family of at least 13 different mammalian genes. Six connexins form a hemichannel (connexon) in the cell membrane and two connexons between two apposed cells produce a functional channel that allows the rapid transport of ions and small molecules.296 Cx32 is expressed in myelinating Schwann cells and is localized to noncompact myelin in the paranode and Schmitt-Lanterman incisures.295 Cx32 gap junctions connect the layers of the Schwann cell cytoplasm and shorten the diffusion pathway for nutrients and ions between the Schwann cell body and the periaxonal cytoplasm by more than one thousandfold.297 Video microscopy of intracellularly injected dyes demonstrate that functional gap junctions are present within the myelin sheath.298 Cx32 expression is linked to myelin formation and is dependent on axonal signals.299,300 Although Cx32 is expressed in the liver, pancreas, stomach, kidney, and uterus,297 no phenotypic abnormalities have been detected in these organs in Charcot-Marie-Tooth disease (CMTX) patients.
Cx32 Mutations and X-Linked Neuropathy.
Cx32 was identified as the gene responsible for X-linked dominant CMTX by a positional candidate approach.297 Mutations of Cx32 were identified in seven of the original eight X-linked CMT families examined.297 To date, over 200 different Cx32 mutations have been described.301–323 The majority of Cx32 mutations are missense mutations, but amino acid deletions, frameshift, and nonsense mutations also occur. The mutations occur throughout the entire Cx32 protein structure (Fig. 227-8), and unlike PMP22 and P0, are not concentrated in transmembrane domains or extracellular domains. Two noncoding region mutations have also been reported in the nerve-specific Cx32 promoter310 and in the 5′ untranslated region of exon 1b.310,322 Each mutation results in a CMT phenotype that is usually more severe in males than in females. Nevertheless, the severity of the CMTX phenotype in males can vary markedly. The severity of the phenotype has not been correlated with the effect a Cx32 mutation has on protein function.
Myelinopathy-associated Cx32 mutation. The four transmembrane domains of Cx32 are shown with individual amino acids represented as open circles. Note that all types of mutations occur (missense > frameshift > nonsense > amino acid deletions), these mutations occur throughout the Cx32 molecule, and each results in a CMT1 phenotype.
Functional Consequences of Cx32 Mutations.
One kindred has been identified with a deletion of the Cx32 gene.324 How other Cx32 mutations cause demyelination is unknown. This issue has been investigated in a variety of expression systems and four distinct effects of Cx32 mutations have been identified: (a) altered intracellular trafficking of mutant Cx32 protein causing accumulation in a cellular compartment; (b) failure to synthesize stable protein; (c) inability to form functional channels; and (d) abnormalities of pore size or gating.
Altered trafficking and localization to the cytoplasm, as determined by indirect immunofluorescence using laser confocal microscopy, was shown for the mutants G12S, E186K, S208K, and R142W.325 The cytoplasmic immunoreactivity colocalized with the Golgi apparatus and/or the ER. This could result in a potentially toxic cytoplasmic accumulation of Cx32 in cells. Toxic interactions with other proteins, such as chaperones or proteins of the trafficking machinery, might also occur in compartments in which mutant Cx32 accumulates. Mutant Cx32 could have dominant-negative effects on other connexins, but dominant-negative interactions between normal and mutant Cx32 are not likely to exist in myelinating Schwann cells, even in women, because Cx32 is subject to X-inactivation.325 Cx32 accumulation and evidence of cytoplasmic accumulation of other mutated myelin proteins237 suggested that diseases affecting myelinating cells may share a common pathophysiology.325 The immunocytochemical localization technique defined two other classes of mutants. One which expressed mRNA but no protein, and the other in which at least some Cx32 is properly routed to the cell surface (R15Q, V63I, V139M, R220X).325 It was proposed that the mutant Cx32 is incorporated into the plasma membrane but is not capable of forming functional gap junctions.325
The ability of CMTX mutant proteins to form homotypic channels (i.e., both connexons are composed of the same connexin) has been tested using the paired Xenopus oocyte expression system. Three mutations (R142W, E186K, and 175fs) behaved as loss-of-function alleles and did not produce functional channels.326 In a subsequent study, 7 of the 11 mutations (R22G, R22P, L90H, V95M, P172S, E208L, and Y211X) did not induce the formation of homotypic channels and thus behaved as loss-of-function mutations.327 Four mutations (L56F, E102G, 111del6, and R220X) assembled homotypic channels efficiently and induced conductance levels of the same order of magnitude as those developed by homotypic pairs expressing wild-type Cx32,327 but exhibited altered gating properties when compared to wild-type controls.327
In a HeLa cell system, the function of Cx32 mutations found in X-linked CMT were tested for their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant-negative mechanism.328 Four mutations (C60F, V139M, R215W, and R220X) restored gap junctional intercellular communication. When transfected with wild-type Cx32, each mutant Cx32—except R220X—inhibited the gap junctional intercellular communication suggesting a dominant-negative effect.328
The analysis of the channel properties of mutations was analyzed in mouse Neuro-2A cell lines. Several mutations (V38M, P87A, E102G, 111del6, S26L, I30N, M34T, V35M) form functional channels when expressed either homotypically or heterotypically with Cx32 in pairs of Xenopus oocytes. The observation that five mutations (S26L, I30N, P87A, E102G, and 111del6) formed functional gap junctions whose voltage dependence did not differ substantially from wild-type Cx32 suggested that alterations in junctional permeability might be responsible for the loss of Cx32 function underlying CMTX. Single-channel studies of two mutations in Neuro-2A cells demonstrated reduced junctional permeability caused by a decrease in either pore size (S26L) or open channel probability (M34T).329 It was proposed that the permeation of second messengers such as cAMP through reflexive gap junctions between adjacent cytoplasmic loops of myelinating Schwann cells was likely to be reduced in these channels. Thus, these mutations could impair the transduction of signals arising from normal glial-neuronal interactions and thereby cause demyelination and axonal degeneration.329
Functional abnormalities of CMTX caused by Cx32 mutations (C53S and P172R) were examined in transfected C6 glioma cells. Immunocytochemical and dye-transfer studies showed a lack of cell-to-cell communication because of failure to incorporate mutant Cx32 protein in the cell membrane330 but the mutations did not interfere with cell proliferation or the expression of a myelin-specific gene (PLP).
The effect of the Cx32 missense mutation E102G was tested in vivo in a nerve myelination system by a nerve xenograft from humans to nude mice. Host mouse axons regenerate through the donor human graft and are remyelinated by human Schwann cells. Ultrastructural analysis showed that Schwann cells with the E102G mutation have a profound effect on nude mice axons, while the myelination did not appear to be affected.331 Those effects consisted of an increase in neurofilament density, a depletion of microtubules associated with fragmentation of smooth axonal reticulum, and increased vesicles and mitochondria. Thus, the Cx32 E1026 mutation appears to impair a modulatory function of Schwann cells on axons resulting in profound cytoskeletal alterations leading to distal axonal degeneration. These observations suggest a role of impaired Schwann cell-axon interactions in the pathogenesis of hereditary neuropathies.331
Mouse Models for Cx32 Deficiency.
Cx32 -deficient mice develop a late onset and progressive neuropathy with features of demyelination and remyelination, such as onion bulbs and abnormally thin myelin.332,333 Noncompacted aspects of myelin, such as enlarged periaxonal collars of Schwann cell cytoplasmic swelling, may represent pathological alteration caused by a compromised homeostasis of ions as a result of reduced “spatial buffering” of ions after neuronal activity.4,332 Interestingly, demyelination was much less pronounced in sensory nerves and dorsal roots, as has been described for P0 +/− and Pmp22+/− mice.332,333 However, the underlying cellular and molecular differences between sensory and motor fibers have not yet been determined. Similar to the findings in CMTX patients, electrophysiologic studies of Cx32-deficient mice demonstrated mild axonal loss, reflected by decreased amplitudes.332
A transgenic mouse was recently constructed with a frameshift mutation (175fs) that had been identified in a large CMTX family.44 Adult transgenic animals showed no pathologic features of the peripheral nerves indicating that the 175fs mutation results in a loss-of-function without additional toxic effects.334 In contrast, when transgenic mice expressing mutant (R142W) or wild-type human Cx32 were compared, the nerves from transgenic lines expressing R142W had decreased levels of Cx32 on western blots, aberrant localization of Cx32 protein by immunohistochemistry, and progressive demyelination with age.335 Thus, the R142W mutant protein had dominant effects that were distinct from overexpression. These transgenic experiments confirm in vitro studies that demonstrate that different Cx32 mutations may behave as loss-of-function or as dominant-negative alleles.
In summary, functional studies of disease-associated Cx32 mutations suggest that most alleles behave as loss-of-function mutation because (a) the mRNA or protein is unstable, or (b) the protein is made but does not form a functional channel, or (c) a functional channel is formed but it has altered gating properties. However, some mutant alleles can have a dominant-negative effect potentially by sequestering or interacting with other connexins.
EGR2 Gene and Protein Structure.
EGR2 is a member of a multigene family encoding Cys2His2 type zinc-finger proteins that play a role in the regulation of cellular differentiation and proliferation.336,337 The mouse orthologue, Egr2 (also known as Krox20), was initially identified as an immediate-early response gene encoding a protein that binds DNA in a sequence-specific manner and acts as a transcription factor.338–341 Stable expression of Egr2 is specifically associated with myelination in the peripheral nervous system.342 To date, three transcription factors have been implicated in the establishment of myelinating Schwann cells: PAX3, a paired domain-containing protein; SCIP (suppressed cAMP-inducible POU), a POU homeodomain protein; and KROX20/EGR2.343 PAX3 is involved in differentiating embryonic Schwann cells, whereas SCIP is produced at high levels only during a short phase of glial development in the central and peripheral nervous system. After the onset of myelination, Pax3 is expressed in nonmyelinating Schwann cells and Krox20 expression is restricted to myelinating Schwann cells.
Egr2 −/− (Krox20 −/−) mice display disrupted hindbrain segmentation and development344,345 and a block of Schwann cell differentiation at an early stage.346 Schwann cells ensheath individual axons but fail to form the spiral of membrane that becomes the myelin sheath.346 Based on these observations, Egr2 potentially activates genes required for peripheral nerve myelination, but none of these downstream genes have been identified. SCIP-null mice have a strikingly similar phenotype to that of Krox-20 null mice, with arrested Schwann cell differentiation.347
EGR2 Mutations and Myelinopathies.
The human EGR2 gene contains two coding exons (GenBank AF139463) and maps to chromosome 10q21q22. One apparent recessive and four different dominant missense mutations have been identified in EGR2 associated with myelinopathy phenotypes, including CHN, DSS, and CMT1348–350 (Fig. 227-9). In contrast to the homozygous knockout mouse, no hindbrain segmentation defects were noted. The differences in clinical severity and inheritance pattern can conceivably be explained by the location of the mutations and their differing effect on protein structure and function. The I268N mutation, which behaves as a recessive allele and causes CHN, occurs within an inhibitory domain that binds two repressors, NAB1351 and NAB2.352 Both repressors inhibit the transcriptional activity of multiple members of this immediate-early response gene family, including Egr1 (also known as Krox24/NGFIA/Zif268) and Egr2. Mutation of this conserved Ile residue to Phe in Egr1 abolishes the inhibitory effect of NAB1 and NAB2 and increases by fifteenfold the transcriptional activity of a reporter gene.353 Similarly, the I268N mutation disrupts repressor interactions.354 Thus, the loss of repression could result in increased expression of a dosage-sensitive PNS gene, which, in turn, might cause CHN in the same way that increased dosage of PMP22 expression by the CMT1A duplication results in CMT1.10
Myelinopathy associated EGR2 (Krox20) mutations. A, Structure of EGR2 (Krox20) Zn-finger domain showing the crucial amino acids (circles) comprising the protein fingers required for DNA contact. The cysteine and histidine amino acids chelating Zn to form the three fingers are shown. B, Shown are the amino acids that comprise the α-helix of each finger (indicated by rectangle in A). The positions of four different myelinopathy associated missense amino acid substitutions and their conveyed phenotypes are shown. C, Results of functional studies correlate residual binding with increasing myelinopathy severity suggesting dominant-negative effects.
Mutations within the region encoding the zinc-finger domain effect the DNA-binding properties of EGR2.348,354 These mutations cause a phenotype in the heterozygous state, thus act as dominant alleles. The Cys2His2 class of zinc-fingers usually has a conserved motif of 28 to 30 amino acids. Egr2 has two types of zinc-fingers that bind to different 3-bp DNA sequences. Type 1 fingers bind 5′-GCG-3′ and the type 2 fingers bind to 5′-GGG-3′, although some variability in the DNA binding sequence has been observed.355 The residues that make up the α-helix are important for DNA recognition within the major groove.356–359 Conserved amino acids within the α-helix of EGR2340 and Egr1360 have been shown by NMR modeling and x-ray crystallography to either hydrogen-bond with the donor sites on guanine or support DNA-binding interactions. The myelinopathy-associated mutations occur in these key amino acids348–350 (Fig. 227-9). Mutation of arginine in zinc finger 3 (R409W) of EGR2348 (Fig. 227-9), which causes a CMT1 phenotype, has been shown, in Egr1, to result in loss of DNA-binding ability.361 Intriguingly, alteration of the corresponding arginine in zinc-finger 2 of EGR2 also results in a CMT1 phenotype.350 The mutation in finger 1 (R359W) was found as a de novo mutation independently in a Belgian349 and an Italian350 family with DSS. The myelinopathy-associated EGR2 mutations alter DNA binding with the amount of residual binding directly correlating with disease severity.354 These observations are consistent with the hypothesis that dominant mutations cause a more severe myelinopathy (CHN and DSS) owing to dominant-negative effects, while a loss-of-function mutation, as evidenced by absence of DNA binding, results in a milder CMT1 phenotype.354