Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsy (HNPP)

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6.1 Autosomal dominant CMT1 and HNPP

6.1.1 Clinical features

Charcot-Marie-Tooth disease type 1 (CMT1), also called hereditary motor and sensory neuropathy type I (HMSN I), shows a classical CMT phenotype with progressive muscular atrophy of the distal muscles, foot deformities (pes cavus and claw toes) and absent deep tendon reflexes [29, 158]. In all cases, a varying degree of symmetrical sensory loss is present. Different types have been designated on the basis of genetic classification, i.e., by locus or by gene.

Therefore, the different CMT1 types do not necessarily reflect different clinical phenotypes.

In CMT1A, caused by a 1.4 Mb duplication on chromosome 17p11 or by point mutations in PMP22, the first overt symptoms usually appear in the second decade of life, but careful neurological examination reveals earlier neurologic deficits with a mean age of onset around four years [13]. The course is mild to moderately severe. In some cases, a visible and palpable hypertrophy of peripheral nerves, especially the great auricular nerve, can be found. CMT1A patients with point mutations are usually more severely affected than patients with a CMT1A duplication. Patients homozygous for the CMT1A duplication have a more severe phenotype and an earlier onset age (< 1 year) than the heterozygous CMT1A duplication patients [82]. In a few CMT1A patients, deafness and/or vocal cord dysfunction have been observed [20, 67, 129]. CMT1B, caused by point mutations in the MPZ gene, is usually more severe than CMT1A. The age of onset is very variable as well as the expressivity (severity of the disease) in different members of the same CMT1B family [136]. Some CMT1B patients have special features, such as significant sensory signs, early involvement of upper extremities [178], bulbar signs [28], auditory involvement [142], and pupillary abnormalities [36]. Patients with CMT1C, caused by point mutations in the LI- TAF gene, manifest characteristic CMT symptoms, including high-arched feet, distal muscle weakness and atrophy, depressed deep-tendon reflexes and sensory impairment [12]. Most patients with CMT1D, caused by point

(CMT1) and hereditary neuropathy with liability to pressure palsy (HNPP)

E. Nelis, P. De Jonghe, V. Timmerman

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mutations in EGR2, are severely affected. In some CMT1D patients, cranial nerve involvement occurs but it is not clear whether the palsies were of peripheral or central origin [19, 108]. CMT1F, caused by point mutations in NEFL, is in most cases characterized by an age of onset below 15years and in some cases neonatal or early childhood onset with delayed motor development [61].

Roussy-Levy syndrome (RLS) comprises clinical features of CMT1 combined with static tremor of the upper limbs and gait ataxia [127]. RLS usually begins in infancy or childhood and manifests as a delay in starting to walk with clumsiness and frequent falls. This condition resembles CMT1 in its dominant inheritance, foot deformity, weakness and atrophy of distal limb muscles, especially the peroneal muscles, absent tendon reflexes, and some distal sensory loss. There was a long-lasting debate whether or not RLS is a separate genetic entity (see section ªGenetics and pathomechanismº).

Djerine-Sottas syndrome (DSS), also referred to as HMSN III, shares considerable overlap with CMT1, although the symptoms of DSS have an earlier onset (< 3 years) and are more severe. The original patients [37]

showed clubfoot, kyphoscoliosis, generalized weakness and muscular atrophy with fasciculations beginning first in the leg muscles, decreased reac- tivity to electric stimulation, areflexia, marked distal sensory loss in all four extremities, in-coordination in the arms, Romberg sign, miosis, decreased pupillary reaction to light, and nystagmus. About one third of the patients diagnosed as DSS remain ambulant for walking distances of at least one kilometer into the fourth decade of life despite the early and severe initial presentation [44]. Some patients with multiple cranial nerve hypertrophy were described [139, 175]. The mode of inheritance can be autosomal dominant or recessive (see section ªGenetics and pathomechanismº).

In many patients with a seemingly recessive mode of inheritance, dominant de novo mutations have been identified [44].

Congenital hypomyelination (CH) is characterized clinically by congenital onset of hypotonia, areflexia and distal muscle weakness. There has been some controversy and difficulty in differentiating congenital hypomyelination from DSS since there is considerable overlap in the clinical presentation. Since in most CH and DSS patients mutations in the same genes associated with CMT1 have been identified, DSS and CH can be considered as variants of the same group of demyelinating HMSN, but some reports suggest that CH improves during the first years of life, while DSS progresses [114].

Hereditary neuropathy with liability to pressure palsy (HNPP), also called tomaculous neuropathy, is autosomal dominantly inherited and often manifests between the 20^th and 40^th year of life. After minimal trauma or compression load, the patients suffer painless focal peripheral nerve lesions that are mostly located at sites where the nerve is exposed to pressure (e.g., ulnar sulcus, head of the fibula), or at physiologic entrapment sites like the carpal tunnel. The symptoms improve over days to months [169].

Painless brachial plexus lesions and involvement of the hypoglossal nerve

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have also been described as symptoms of HNPP [104, 170]. Some patients develop a slowly progressive neuropathy clinically indistinguishable from CMT1, while others show electrophysiological, but not clinical signs of a generalized neuropathy [84]. Atypical manifestations of the disorder include pes cavus, scoliosis, and deafness [45]. HNPP can mimic the symptoms of carpal tunnel syndrome, since the peripheral nerve entrapment can include median nerve compression at the carpal canal. A family with dominantly inherited carpal tunnel syndrome was associated with the deletion in chromosome 17p11 that causes HNPP [117].

6.1.2 Electrodiagnostic and laboratory features

Slowed motor nerve conduction velocities (MNCVs) in CMT patients were first described in the 1950s [71, 72]. In CMT1, MNCVs are uniformly slowed to less than 38 m/s for the motor median nerve [54]. Because of secondary axonal damage in CMT1, compound muscle action potentials (CMAP) are often reduced. The degree of neurological dysfunction correlates much better with the degree of CMAP reduction (secondary axonal damage) than with the degree of MNCV slowing [68]. The amplitudes of the sensory nerve action potentials (SNAP) are severely reduced or the potentials are not recordable.

In a study of 105CMT1A duplication patients belonging to 45unrelated families, the median MNCV ranged from 10 to 42 m/s. The majority of the values fell between 16 and 30 m/s [34]. In a series of 119 patients with the CMT1A duplication [17], the MNCV was found to be uniformly reduced in all nerves. The SNAPs were consistently abnormal. An early age of onset and more severely reduced MNCVs were predictive of a more severe disease course. Although the disease is clinically progressive, median MNCV and CMAP amplitudes did not change during the course of the disease. A long- itudinal study of children with the CMT1A duplication showed that all subjects had abnormal motor and sensory NCV from the age of 2 [14]. The CMAP from extensor digitorum brevis was reduced or absent in 50% and 100% at the ages of 5and 10 years, respectively. CMT1A patients with point mutation in PMP22 have slower NCVs than CMT1A duplication patients:

some patients with PMP22 point mutations have been described as DSS or even CH patients. The MNCVs of CMT1B patients are often very slow (< 20 m/s) and the electrophysiological delineation from DSS is not clearly defined. Some CMT1B patients have near normal NCVs [88]. The two original CMT1C pedigrees have a mean median MNCV of 23 m/s and 26 m/s, respectively. There was prominent temporal dispersion and evidence for conduction block, especially in the tibial nerves [12, 144]. In CMT1 patients with NEFL mutations, NCVs are usually within the CMT1 range [35, 61], while some patients may have NCVs that approach the normal range [91].

DSS patients have by definition markedly reduced MNCVs (less than 10±12 m/s) and usually absent SNAPs [44]. Elevated protein content of the cerebrospinal fluid is not a mandatory feature of DSS as suggested in the

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past. On the contrary, it might be a feature of very early onset chronic inflammatory demyelinating neuropathy (CIDP) and should be followed up with a nerve biopsy in order to detect inflammatory infiltrates. CH patients have extremely reduced MNCVs (3±8 m/s) or no CMAPs are detectable at all. No conduction blocks are present.

Electrophysiological examination in HNPP patients shows prolonged distal latencies, slowing of MNCVs and conduction blocks, especially at physiolog- ical pressure or entrapment sites, in nearly all patients as well as in asymptomatic carriers, disclosing a mild underlying generalized neuropathy [2, 9].

6.1.3 Pathological features

Pathological examination of peripheral nerves in CMT1 shows a marked decrease in the density of myelinated fibers and de- and remyelination with formation of so called ªonion bulbsº [151]. Onion bulbs are altered myelin structures generated by repeated de- and remyelination. They usually con- sist of Schwann cell processes, and more rarely of basal membranes.

In CMT1A duplication patients, the onion bulbs are small to moderate in size, appear with a moderate frequency and are more prominent in nerves with less severe axonal loss. In the early stage, the myelin sheaths are thicker than normal, later the axons become thinly myelinated. A large reduction in the number of myelinated axons is observed. In CMT1A patients with PMP22 point mutations, onion bulbs are large and present on most axons. The myelin sheaths are thinner than normal. In some patients, uncompacted myelin or tomaculae (lat. ªsausagesº), focal thickenings of the myelin sheath, are observed. In CMT1B patients, sural nerve biopsies often show demyelination, onion bulbs and uncompacted myelin but other myelin features, e.g., focally folded myelin, have also been observed [49].

In rare CMT1B cases, segmental demyelination/remyelination in combina- tion with axonal degeneration/regeneration is observed [74]. A sural nerve biopsy of a CMT1C patient demonstrated onion bulb hypertrophy typical of demyelinating CMT [144]. In CMT1D, nerve biopsy showed a severe loss of myelinated fibers with numerous onion bulbs [108, 177]. A single nerve biopsy of a CMT1 patient with a NEFL mutation showed axonal pathology with axonal regeneration clusters and onion bulb formations [61].

In DSS (also called HMSN III), hypertrophic nerves and abundant onion bulbs are noted on nerve biopsies. The onion bulbs are sometimes composed of basal lamina material [26]. CH is characterized by hypomyelination of most or all fibers, without signs of demyelination, and is considered to result from a congenital impairment in myelin formation. In contrast, DSS is thought to be due to aberrant demyelination and subsequent remyelination of the peripheral nerve. A correlation of morbidity and mortality was found with the presence/absence of onion bulbs: patients with few onion bulbs died in early infancy, usually because of difficulty in swallow-

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ing and respiration after birth. Patients with atypical onion bulbs survived but developed severe motor and sensory impairment [53].

Biopsies of the sural nerve of HNPP patients show characteristic tomaculae, signs of de- and remyelination with onion bulbs and variable axonal loss [150]. Tomaculae are sausage-shaped enlargements consisting of focal thickenings of the myelin sheath composed of redundant loops of myelin with normal periodicity. These are not absolutely specific for HNPP but may also infrequently be present in other hereditary peripheral neuropathies and in CIDP [130].

6.1.4 Genetics and pathomechanism

Molecular genetic studies have shown that autosomal dominant CMT1 is genetically heterogeneous and four loci have been mapped: CMT1A to chromosome 17p11.2 [164], CMT1B to 1q22-q23 [16], CMT1C to 16p13.1 [144] and CMT1D to 10q21.1-22.1 [166]. The CMT1 causing genes for all these loci have been identified over the last decade and are PMP22, MPZ, LITAF and EGR2, respectively. Furthermore, mutations in NEFL, originally associated with CMT2, are also found in autosomal dominant CMT1 patients [61]. In the following paragraphs, we will discuss how positional cloning strategies and candidate gene approaches have contributed to the identification of the loci and genes for autosomal dominant CMT1.

z The CMT1A duplication and HNPP deletion

The CMT1A locus was assigned to chromosome 17p by Vance et al. [164]

in an American CMT family. At that time detailed genetic maps were not available and the genetic markers ± restriction fragment length polymorphisms ± used in positional cloning strategies had a low informativity.

However, thanks to the availability of multigenerational pedigrees, others confirmed linkage to the CMT1A locus in several large families, and re- fined it to a large region (30cM) on chromosome 17p11.2-p12 [90, 92, 111, 120, 155]. In 1991, two research groups independently identified a tandem duplication of a chromosomal segment in 17p11.2-p12 in CMT1A patients [82, 121]. This duplication occurred de novo via an unequal crossing-over event and was subsequently transmitted to the next generations. The de novo appearance of the CMT1A duplication is a frequent finding in isolated CMT1 patients [55]. These de novo duplications are usually of pater- nal origin and arise from unequal crossing-over events during male sper- matogenesis [105]. In addition, a few duplications of maternal origin have been described [18, 85]. Large genetic epidemiological studies estimated that the frequency of the CMT1A duplication in dominant CMT1 cases is about 71% CMT1 [102, 171]. Physical mapping experiments were performed and the size of the CMT1A duplication region was estimated to be 1.5Mb [113, 122]. The unequal crossing-over event in CMT1A occurs dur-

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ing meiosis via a chromosomal misalignment of large repeat elements, called the ªCMT1A-REPsº, flanking the CMT1A region (Fig. 6.1) [113].

With the exception of a few rare cases, the CMT1A duplication always has the same size, i.e., 1.5Mb, suggesting that the repeat sequences in this region influence this constant DNA rearrangement. A patient, mosaic for the CMT1A duplication, was reported to have a reversion of the 1.5Mb CMT1A duplication in several somatic tissues [79].

Interestingly, Chance et al. [27] reported patients affected with HNPP to have a reciprocal deletion of the 1.5Mb CMT1A region on 17p11.2-p12.

Subsequent studies confirmed that the large majority of HNPP patients car- ried the 1.5Mb deletion [102]. At the molecular level this implies that three copies of the CMT1A-REP sequence are located on the CMT1A duplication chromosomes while only one copy is present on the HNPP deletion chromosomes. It is unknown why HNPP seems to be much rarer than CMT1A. It has been hypothesized that a significant proportion of HNPPs are never diagnosed because the course is mild and patients may present only once in their life with a pressure induced palsy.

Further detailed analysis of the CMT1A duplication/HNPP deletion mutations demonstrated a ªhotspotº region of frequent unequal crossing over within the CMT1A-REP elements in a cohort of unrelated CMT1A and HNPP patients of North American [124], European [81, 156] and Japanese [174] origin. The sequence within the hotspot of recombination showed 98% identity between the proximal and distal CMT1A-REP elements [66, 123]. Sequence comparison of the low copy CMT1A-REPs revealed a mari- ner transposon-like element (MITE) near the hotspot of unequal recombination. However, it is unlikely that the MITE codes for an actively tran- scribed transposase since the open reading frame contains several frame- Fig. 6.1. Model of unequal crossing over between misaligned CMT1A-REPs leading to the CMT1A duplication (1) and HNPP deletion (2). The blue boxes represent the proximal CMT1A- REP, the bright blue boxes the distal CMT1A-REP flanking the 1.4 Mb region, whereas the dark blue boxes represent the PMP22 gene

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shift mutations [124]. Physical mapping strategies using large insert clone contigs (P1 and bacterial artificial chromosomes: PACs and BACs) were performed to delineate the complete nucleotide sequence of the CMT1A/

HNPP genomic segment to 1,421,129 bp of DNA. This latter study did not only allow the identification of several genes in the region, but also ex- plains an evolutionary mechanism relevant for the formation of the CMT1A-REP by DNA rearrangement during primate speciation [59].

z CMT1A and related disorders ± peripheral myelin protein 22 gene (PMP22) (OMIM 118220, 118300)

One year after the identification of the CMT1A duplication, PMP22 was assigned to the CMT1A region [89, 112, 154, 162]. Importantly, the smaller duplications and deletions reported in the literature, still contained the PMP22 gene [105, 161], supporting a gene dosage effect as the disease mechanism [83]. Furthermore, over- and underexpression of PMP22 has been confirmed at the transcript and protein level in CMT1A and HNPP patient nerve biopsies, respectively [163]. The dosage sensitivity of PMP22 transcripts and protein was nicely illustrated by the genotype-phenotype correlations in man and rodents over- and underexpressing PMP22 (reviewed in [15, 101]).

Beside the CMT1A duplication and HNPP deletion, point mutations in the PMP22 gene can result in the following distinct phenotypes: classical CMT1 (CMT1A), HNPP and the more severe DSS and CH (IPNMDB;

http://www.molgen.ua.ac.be/CMTMutations/) (Table 6.1). Mutations that truncate or severely alter the protein sequence and are rapidly degraded, mimicking underexpression resulting from the deletion, are predicted to lead to an HNPP phenotype. Indeed, most loss-of-function mutations result in HNPP, while gain-of-function mutations lead to CMT1 or DSS, either by an increased dosage of a normal PMP22 protein or by a toxic effect of the mutated PMP22 molecule. In vitro studies have demonstrated that missense mutations lead to impaired intracellular trafficking of PMP22 resulting in an accumulation of the mutant protein in the endoplasmic reticulum and Golgi apparatus. The mutant protein also traps normal PMP22 resulting in a decreased amount of PMP22 available for incorpora- tion in the myelin membrane [95].

The PMP22 gene was first cloned as the human homologue of the mouse growth arrest-specific 3 gene (Gas3) [87]. The gene is located on mouse chromosome 11, a syntenic region to human chromosome 17p11.2. It encodes a membrane protein comprising 2±5% of total peripheral myelin protein content [107]. PMP22 expression in the peripheral nervous system (PNS) is most likely regulated by axonal contact [141]. The PMP22 gene has two tissue-specific promoters, one being nerve-specific [145]. The PMP22 protein has four transmembrane domains, two extracellular loops, and cytoplasmic amino and carboxy termini. After synthesis in the rough endoplasmic reticulum, the majority of PMP22 becomes rapidly degraded

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and only a small fraction is processed in the Golgi apparatus and trans- ported to the cell membrane [106]. Although PMP22 has been known for almost two decades, its function is still unclear. Initial in vitro studies showed that PMP22 is a growth arrest and apoptosis-specific protein [42, 86]. Mouse pmp22 has been detected during development and in distinct adult neural and non-neural tissues. The zebrafish orthologue of PMP22 also shows expression in embryonic neural crest cells, suggesting a role in the early development of the PNS [172]. Moderate overexpression of PMP22 can induce susceptibility to apoptosis in some cell types. When this apoptotic response is counteracted, PMP22 can still modulate cell shaping and cell spreading. Therefore, PMP22 may have an important role in Schwann cell differentiation and myelination [25]. In the adult PNS, PMP22 most likely functions as an integral membrane protein since it is confined to the compact myelin of Schwann cells [69, 140]. Co-immunopre- cipitation and confocal microscopy experiments demonstrated that PMP22 and myelin protein zero (MPZ/P0), the major component of the peripheral myelin membrane, form complexes suggesting a complementary role of both proteins in cell adhesion of compact myelin [33]. Mutated or overex- pressed PMP22 causes a ªgain-of-functionº endoplasmatic reticulum reten- tion phenotype. Recently, it was demonstrated that PMP22 associates in a specific, transient, and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX) [39]. In the Tr-j mouse, a prolonged association of mutant PMP22 with CNX was found. Since CNX and PMP22 co-localize in large intracellular myelin-like figures, sequestration of CNX in intracellular myelin-like figures may be relevant for the CMT pathology.

z CMT1B and related disorders ± myelin protein zero gene (MPZ) (OMIM 118200)

The myelin protein zero gene (MPZ) is located on chromosome 1q22-q23. It encodes a 219 amino acid, 28±30 kDa glycoprotein that accounts for more than 50% of total PNS myelin protein. The protein called P0 has one transmembrane domain, an extracellular aminoterminus and an intracellular carboxy-terminus. Crystallographic 3-D structural analysis of the extracellular domain shows similarity to an immunoglobulin variable domain [137]. Dur- ing Schwann cell development, P0 is simultaneously induced with genes encoding other myelin proteins, such as PMP22, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) [78]. P0 is upregulated at the onset of myelination. As a compact myelin protein, P0 most likely acts as a `dou- ble adhesive protein'. It holds myelin together at the intraperiod line through interactions of its extracellular, selfadhesive immunoglobulin domain [137]

and at the major dense line via interactions of its cytoplasmic domain [40]. Apart from its structural role in myelination, P0 plays a regulatory role as well. P0-overexpressing mice show failure in axon sorting and a myelination arrest at early mesaxon formation. In early developing Schwann cells, high P0 overexpression inhibits polarization of Schwann cell membranes into

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appropriate functional domains, dynamic axonal interaction and Schwann cell membrane expansion required for appropriate axonal sorting and myelination [176]. Most MPZ mutations cause a classical CMT1 phenotype (CMT1B). However, some MPZ mutations lead to a more severe DSS or CH phenotype (reviewed in [99]). Specific MPZ mutations are also associated with CMT2, the axonal form of CMT (Table 6.1). The Thr124Met mutation is associated with a distinct CMT2 phenotype with pupillary abnormalities and deafness [28, 36, 93, 135]. Mutant P0 could affect myelin formation in three ways: (1) by not reaching the myelin membrane, (2) by reaching the myelin membrane but having lost its adhesive properties, or (3) by reaching the myelin membrane and having a dominant negative effect on the wildtype P0. In vitro studies have shown that mutated P0 can indeed reduce adhesion [41, 173]. The complex-formation between PMP22 and P0 might clarify the remarkable similarity between the CMT1A and CMT1B phenotypes [33]. Al- terations in either protein may interfere with the normal association of P0 and PMP22 into one functional complex. The disturbed interaction would subsequently result in demyelination as a common pathological pathway in CMT1A and CMT1B.

z CMT1C ± lipopolysaccharide-induced tumor necrosis factor gene (LITAF) (OMIM 601098)

Two autosomal dominant CMT1 families were reported to have a locus mapping to 16p13.1-p12.3 (CMT1C) [144]. Only recently, three missense mutations in LITAF were identified in the two previously known CMT1C families and confirmed in a third family of different origin [143]. The LITAF protein (also known as SIMPLE; small integral membrane protein of lysosome/late endosome) is widely expressed and encodes a 161-amino acid protein that might be involved in protein degradation pathways. The missense mutations (G112S, T115N, W116G) cluster in a domain of the LITAF protein relevant for peripheral nerve function [12]. The function of LITAF is currently unknown, but the subcellular localization and putative domains point to a ubiquitin- mediated lysosomal degradation protein. LITAF is located in the lysosome/

late endosome [94], its murine orthologue interacts with NEDD4 an E3-ubiquitin ligase [60], and it contains a conserved motif known to interact with another protein involved in sorting of ubiquitinated proteins to the multi-ve- sicular body [157].

Further mutation analysis of LITAF in CMT1 patients is needed to esti- mate the frequency of LITAF mutations in the total CMT1 population, and to delineate the associated phenotype.

z CMT1D ± early growth response element 2 gene (EGR2) (OMIM 607678) The EGR2 gene is located on chromosome 10q21.1-q22.1 [62] and encodes a 51 kDa protein of 475 amino acids. EGR2 is the human homologue of the mouse Krox20 gene [30], with an overall amino acid identity of 89%

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(100% in the zinc finger domain) [167]. EGR2 is a member of the EGR family. The EGR proteins encode transcription factors containing Cys2His2 zinc finger domains, which bind a GC-rich consensus binding site [147].

Analysis of homozygous and heterozygous Krox20 knockout mice has shown that Krox20 is important in the development and segmentation of the hindbrain [131, 146]. Surviving homozygous Krox20 knockout mice have hypomyelination of the PNS with Schwann cells blocked at an early stage of differentiation, causing a trembling phenotype [159]. Krox20 expression is activated before onset of myelination in the PNS and is essential for the final differentiation of myelinating Schwann cells [181]. These data suggest that Krox20 and its human homologue EGR2 are transcription factors required for the transactivation of PNS myelination-specific genes. In- deed, using microarray expression profiling, 98 known genes were identified that were induced by Egr2 in Schwann cells [96]. The putative Egr2 target genes included myelin proteins and enzymes required for synthesis of normal myelin lipids. RT-PCR to monitor Schwann cell gene expression confirmed that Egr2 is sufficient for induction of target genes including MPZ, PMP22, MBP, MAG, GJB1 and PRX. Furthermore, a dominant-negative inhibition of wildtype Egr2-mediated induction of essential myelin genes using EGR2 DNA-binding domain mutants was demonstrated [96]. A recent study, however, provides evidence that myelin-related signaling in Schwann cells can be independent of Krox20 [109].

Several mutations in EGR2 have been described in patients with different phenotypes, i.e., classical CMT1, DSS and CH [153, 166]. Some of these mutations were present in the homozygous and others in the heterozygous state (Table 6.1). One mutation causes a CMT1 phenotype with cranial nerve deficits [108]. Clinical involvement of cranial nerves is unusual for CMT1 and may demonstrate a similar role for Krox20 and EGR2 in brainstem and cranial nerve development [108]. The effect of some mutations on the DNA binding capacities of EGR2 has been studied in order to correlate the residual DNA binding capacities to the clinical severity. The results confirm that the severity of CMT1D correlates with the residual amount of DNA binding of the mutated EGR2 [167]. The dominant nature of these mutations seems to be in contrast with the Krox20 heterozygous knockout mouse, which shows no phenotypical abnormalities [131]. This suggests that these mutations do not cause a loss of function, but rather have a dominant negative or a gain-of-function effect. Another mutation was shown to interfere with the binding of NGFI-A binding proteins, possible co-repressors of EGR2, probably leading to increased transcription of EGR2 [167].

z CMT1F ± neurofilament light polypeptide gene (NEFL) (OMIM 607734) Mutations in NEFL were recently reported as a cause for autosomal dominant axonal CMT linked to chromosome 8p21 (CMT2E) [91]. Further analysis of NEFL in CMT patients showed that patients with NEFL mutations have an early disease onset and are usually severely affected, with moder-

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ately to severely slowed NCVs. Based on the clinical characteristics, some of them were diagnosed with DSS [35, 61].

NEFL encodes a 62 kDa structural protein, which is one of the most abundant cytoskeletal components of neuronal cells [43]. NEFL assembles with neurofilaments of higher molecular mass, medium (NEFM) and heavy (NEFH) chain polypeptides, into intermediate filaments and forms an ex- tensive fibrous network in the cytoplasm of the neuron. Neurofilament accumulation tightly correlates with radial growth of axons during myelination. Neurofilaments determine the axonal diameter and, hence, the conduction velocity of peripheral nerves [4, 75]. Nefl knockout mice do not have a CMT-like phenotype [180], while transgenic mice have a severe peripheral neuropathy with massive motor neuron death [76].

z Roussy-Levy syndrome ± myelin protein zero gene (MPZ), peripheral myelin protein 22 gene (PMP22) (OMIM 180800)

Plant-Bordeneuve et al. [115] identified a point mutation in MPZ in the original family studied by Roussy and Levy. This finding shows that the family belongs to the CMT1B subtype. Other RLS patients, however, showed the CMT1A duplication [5, 152] (Table 6.1). These findings provide evidence against the RLS as a distinct genetic entity, but suggest a close re- Table 6.1. Genes associated with CMT1 and related peripheral neuropathies

Gene Locus Inheritance Phenotype

zPMP22 (duplication) CMT1A AD CMT1, RLS

zPMP22 (duplication) CMT1A AR DSS

zPMP22 (deletion) HNPP AD HNPP

zPMP22 CMT1A, HNPP AD CMT1, HNPP, DSS, CH

zPMP22 CMT1A AR DSS

zMPZ CMT1B AD CMT1, CMT2, DSS, CH, RLS

zMPZ CMT1B AR DSS

zLITAF CMT1C AD CMT1

zEGR2 CMT1D AD CMT1, DSS

zEGR2 CMT4E AR CH

zNEFL CMT2E AD CMT1, CMT2, DSS

zGDAP1 CMT4A AR CMT1, CMT2, DSS

zMTMR2 CMT4B1 AR CMT1, CH

zSBF2/MTMR13 CMT4B2 AR CMT1

zKIAA1985 CMT4C AR CMT1

zNDRG1 CMT4D AR HMSN-L

zPRX CMT4F AR CMT1, DSS

zCTDP1 CCFDN AR CCFDN

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lation with CMT1. What causes the additional features of gait ataxia and essential tremor needs further clarification. Roussy-Levy syndrome (RLS) comprises clinical features of CMT1 combined with static tremor of the upper limbs and gait ataxia [127].

6.2 Autosomal recessive demyelinating CMT or CMT4

6.2.1 Clinical features

The autosomal recessive forms of demyelinating CMT are traditionally designated CMT4. These forms are rare. Every subform has been described in a few families only, each with particular ethnic, pathologic, or clinical characteristics: Many of these families are living in North Africa, or they belong to genetically isolated populations in Europe like the Bulgarian Sinti or Roma Gypsies. Many of these neuropathies are clinically severe and could also be classified as DSS.

CMT4A was identified in Tunisian families [10]. The disease usually manifests with delayed motor milestones in the second year of life. Patients develop a severe, predominant motor neuropathy and often become wheelchair-dependent in the first or second decade [10]. Foot deformities and scoliosis are often present. CMT4B1 is a relatively severe neuropathy with both distal and proximal paresis, foot deformities, scoliosis and onset in early childhood [119]. Patients become wheelchair-dependent in the third decade, and death occurs in the 4th to 5th decade [119]. Additional features, such as weakness of the jaw and of the facial and bulbar muscles and diaphragmatic weakness [56], have been noticed in some CMT4B1 patients. In CMT4B2, the onset of the neuropathy occurs in the first two decades and patients show a clinically typical but rather severe type of demyelinating CMT. One family features early-onset glaucoma as an additional clinical sign [6]. CMT4C patients show clinically a typical CMT1-like presentation in the first or second decade, which is complicated by a rather severe scoliosis in some patients [47, 77]. CMT4D, also known as HSMN-L, is a rare form of HMSN identified in Bulgarian Roma living close to the town of Lom [64, 65]. The disease manifests in childhood with weakness of the legs followed by palsies of the hands between 5and 15years of age.

Many patients between the age of 26 and 50 years are unable to walk. Deaf- ness is an invariant feature of the phenotype and usually develops in the third decade [65]. CMT4E patients present with a floppy infant syndrome at birth. Motor milestones are delayed, but the two patients described by Warner et al. eventually learned to walk with crutches [166]. Weakness and atrophy are distally accentuated but proximal weakness is also present. In CMT4F patients, the disease manifested with slightly delayed motor development (sitting at approximately 12 to 18 months, walking at approxi-

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mately 24 months). The gait was unsteady and wide-based. At the age of approximately 9 to 10 years the typical signs of CMT became apparent in the legs and at approximately 14 to 15years also in the arms [38]. Typical foot deformities and scoliosis were present. Marked sensory impairment ± sometimes more prominent than the motor deficits ± is a characteristic feature of CMT4F [22, 148]. HMSN-R, identified in Roman gypsies, is a steadily progressive neuropathy leading to total paralysis of the muscles of the lower legs in the fourth to fifth decade. All patients showed foot deformity, and most showed hand deformity. In addition, patients develop prominent sensory deficits [126]. Congenital cataracts facial dysmorphism neuropathy (CCFDN) is clinically characterized by a number of pathological features in addition to a rather severe predominantly demyelinating peripheral neuropathy: congenital cataracts with microcornea, facial dys- morphic features, mild cognitive deficit, short stature and hypogonadism, pyramidal signs and a number of additional CNS abnormalities [160].

6.2.2 Electrodiagnostic features

The motor and sensory NCVs in CMT4A are slowed to 25±35 m/s [10]. In CMT4B, MNCVs ranged from 15to 17 m/s in the upper limbs of the youngest patients to undetectable in the adult patients. Sensory action potentials were almost always absent. In all patients, auditory evoked potentials showed ab- normally delayed interpeak I±III latencies [11, 119]. In CMT4C patients the mean motor MNCV was 22.6 m/s [133]. MNCVs of CMT4D patients in the median, ulnar, tibial, and peroneal nerves are severely reduced in the youngest patients and unattainable after the age of 15years. Decreased conduction velocity and CMAP amplitude are also found proximally, in the axillary and facial nerves. Brainstem auditory evoked potentials (AEP) are markedly abnormal, with prolonged interpeak latencies consistent with demyelination [64, 65]. In CMT4E MNCVs are extremely reduced to 3±8 m/s [166]. The results of NCV studies performed on five CMT4F patients revealed the total absence of any sensory or motor evoked response in the upper and lower limbs.

EMG showed normal insertional activity, absence of spontaneous activity, and moderately reduced recruitment of motor-unit potentials in the distal muscles of four limbs [38]. Motor NCVs in HMSN-Russe was moderately reduced in the upper limbs but unobtainable in the legs. Sensory nerve action potentials were absent [149]. Motor and sensory conduction studies in CCFDN showed slowing to 20±34 m/s [160].

6.2.3 Pathological features

In CMT4A, nerve biopsies show typical features of a severe demyelinating neuropathy with onion bulb formation and loss of large myelinated axons [10]. In other CMT4A patients, loss of myelinated fibers without demyeli-

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nation is found. Focally folded redundant myelin sheaths are the histologi- cal hallmark of the CMT4B forms [46]. CMT4C is associated with a pecu- liar myelin pathology consisting of large cytoplasmic Schwann cell exten- sions and atypical multilayered wrapping of basal membranes around several axons [48]. Neuropathologic investigations in CMT4D showed that myelinated fibers were severely reduced in number and those that remained were of very small size. In CMT4E patients, sural nerve biopsies show virtual absence of myelin [166]. CMT4F is associated with a mixed demyelinating and axonal picture with multiple small onion bulbs [38]. Oc- casionally tomacula formation with focal myelin thickening was observed.

Furthermore, abnormalities of the paranodal myelin loops and focal absence of paranodal septate-like junctions between the terminal loops and axon were present [148]. In HMSN-Russe, there was loss of larger myelinated nerve fibers and profuse regenerative activity in the sural nerve [149]. Nerve biopsy examination of CCFDN cases indicated generalized hypomyelination superimposed upon which were demyelination and axonal degeneration in older subjects [126].

6.2.4 Genetics and pathomechanism

Extremely rapid progress in the identification of the genetic defects causing the recessive types of CMT has been made in the last three years. This rapid progress is, on the one hand, due to technology improvement and the availability of the human genome sequence but, on the other hand, also caused by the fact that homozygosity mapping often permits the delineation of very small candidate regions. Currently only one genetically mapped subtype with unknown genetic cause remains.

z CMT4A ± ganglioside-induced differentiation associated protein 1 gene (GDAP1) (OMIM 214400)

CMT4A was identified in Tunisian families and maps to chromosome 8q13-q21 [10]. CMT4A is caused by point mutations in the ganglioside-induced differentiation associated protein 1 gene (GDAP1) and a Hispanic founder mutation for some families has been identified [8, 21]. Interest- ingly a locus for axonal AR CMT with pyramidal features was mapped to chromosome 8q21.3 [7]. This locus was also mapped in an inbred Tunisian family and overlaps with the CMT4A locus. This raises the possibility that this axonal AR CMT form and demyelinating CMT4A could be allelic. The finding of mutations in GDAP1 in families displaying an axonal type of CMT (CMT4C4) associated with vocal cord paresis supports this hypot- hesis [31]. Furthermore, GDAP1 mutations are also identified in CMT patients with both demyelinating and axonal features [1, 98, 132]. GDAP1 had been cloned in a screen for genes that are involved in ganglioside-induced differentiation of a mouse neuroblastoma cell line [80]. GDAP1 is

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expressed in brain and peripheral nerve. The exact function of GDAP1 in the PNS needs to be determined. It might be involved in neural differentiation [80]; however, the glutathione S-transferase domain points to a function in detoxification and protection against free radicals [128].

z CMT4B1 ± myotubularin related protein 2 gene (MTMR2) (OMIM 601382)

CMT4B1 is caused by point-mutations in the myotubularin related protein 2 gene (MTMR2) on chromosome 11q23 [23, 24, 56, 97]. Some mutations are found in patients with a very severe phenotype, diagnosed as CH. Mu- tations in the myotubularin gene (MTM1), which is the first cloned member of this gene family, cause X-linked recessive myotubulary myopathy [73]. The MTM1 and MTMR2 genes code for tyrosine phosphatases which influence transcription and cell proliferation [32]. MTMR2 physically interacts with the NEFL protein which is mutated in CMT2E, but the function of this interaction is yet unknown [118].

z CMT4B2 ± myotubularin related protein 13 gene (MTMR13 orSBF2) (OMIM 604563 and 607739)

CMT4B2 was found in Tunisian families and maps to chromosome 11p15 [11]. Two groups identified causative mutations in a novel gene of the MTMR family named MTMR13 or set binding factor 2 (SBF2) [6, 134].

Mutations in MTMR13 in the original family described by Ben Othmane have not yet been reported [11]. MTMR13 is a catalytically inactive member of the MTM gene family that might be involved in the regulation of active family members. MTMR13/SBF2 is expressed in multiple tissues, including brain and sciatic nerve [6].

z CMT4C ± unknown transcript KIAA1985 (OMIM 601596)

The CMT4C locus on chromosome 5q23-q33 was identified in two Algerian families [77]. Causative mutations have been identified in a novel transcript of yet unknown function called KIAA1985 [133]. Mutations in the originally linked families have not been reported. KIAA1985is strongly expressed in neural tissues, including peripheral nerve tissue. The translated protein defines a new protein family of unknown function with putative orthologues in vertebrates. Comparative sequence alignments indicate that members of this protein family contain multiple sarc homology (SH3) and tetratricopeptide repeat (TPR) domains that are likely involved in the formation of protein complexes.

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z CMT4D synonymous to HMSN-Lom ± N-myc downstream regulated gene 1 (NDRG1) (OMIM 601455)

In HMSN-L (CMT4D) families, linkage to chromosome 8q24 was found.

Conserved disease haplotypes suggested genetic homogeneity and a single founder mutation [64]. Mutation analysis of the N-myc downstream regulated 1 gene (NDRG1) revealed a homozygous nonsense mutation in all patients [63]. The exact function of NDRG1 is unknown. Computer analysis has predicted that the NDRGs belong to the a/b hydrolase superfamily;

however they do not appear to be active hydrolases [138]. NDRG1 is expressed in multiple tissues including Schwann cells and probably plays a role in growth control and cell differentiation. Only recently, a second NDRG1 mutation was identified [57].

CMT4E, often presenting as CH, is caused by recessive mutations in the EGR2 gene, which is discussed in detail in the section on ªCMT1Dº in this chapter.

z CMT4F ± periaxin gene (PRX) (OMIM 605725)

A CMT4F locus on chromosome 9p13 was found in a Lebanese family with a severe CMT1 phenotype [38], and a mutation in PRX was identified in this family [51]. PRX mutations were also identified in patients with a DSS or CMT1 phenotype [22, 148]. The pathogenesis of PRX mutations is not fully understood. However, progress is being made by studying both the periaxin-null mouse and the protein-protein interactions of periaxin. L- periaxin is a constituent of the dystroglycan-dystrophin-related protein-2 complex linking the Schwann cell cytoskeleton to the extracellular matrix.

Although periaxin-null mice myelinate normally, they develop a demyelinating peripheral neuropathy later in life. This suggests that periaxin is required for the stable maintenance of a normal myelin sheath. Sciatic nerve crushes in periaxin-null mice and showed that although the number of myelinated axons had returned to normal, the axon diameters remained smaller than in the contralateral uncrushed nerve. Not only do periaxin- null mice have more hyper-myelinated axons than their wildtype counter- parts but they also recapitulate this hypermyelination during regeneration.

Therefore, periaxin-null mice can undergo peripheral nerve remyelination, but the regulation of peripheral myelin thickness is disrupted [50, 168].

z Congenital cataract facial dysmorphism neuropathy (CCFDN) ± C-terminal domain phosphatase of RNA polymerase II gene (CTDP1) (OMIM 604186)

CCFDN was identified in 19 Wallachian Gypsy families and mapped to chromosome 18qter [3]. A conserved haplotype suggested a single founder mutation. CCFDN is caused by a single-nucleotide substitution in an anti- sense Alu element in intron 6 of CTDP1, an essential component of the eu-

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karyotic transcription machinery, resulting in a rare mechanism of aberrant splicing and an Alu insertion in the processed mRNA. CCFDN thus joins the group of `transcription syndromes' and is the first `purely' tran- scriptional defect identified that affects polymerase II-mediated gene expression. A clinically sometimes very similar disease is Marinesco-Sjogren syndrome, distinguished from CCFDN by its genetic locus on chromosome 5q31 [70].

z Recessive mutations in the peripheral myelin protein 22 gene (PMP22) and the myelin protein zero gene (MPZ)

In a few cases, recessive mutations are found in genes for dominant forms of CMT1. In PMP22, a single homozygous missense mutation was found in 3 sibs with DSS. The parents, carrying the mutation in the heterozygous state, are clinically and electrophysiologically unaffected [110]. Two hemi- zygous PMP22 point mutations were described in CMT1 patients. Individ- uals heterozygous for the mutation are unaffected, while individuals with a PMP22 deletion on the other allele have a CMT1 phenotype [103, 125]. It is still controversial if these PMP22 mutations represent recessive CMT1 mutation or functionally irrelevant polymorphisms [100, 179]. Several MPZ mutations occur in the heterozygous as well as in the homozygous/compound heterozygous state. The heterozygous individuals are asymptomatic or have CMT1, while the homozygous/compound heterozygous individuals are severely affected, mostly they are diagnosed as DSS [58, 74, 116, 165].

z HMSN-Russe ± chromosome 10q22

CMT Russe (HMSN-R) is linked to chromosome 10q22 [52, 126]. Fine mapping reduced the critical candidate region to only 70 kb [52]. The fact that no sequence variant has been detected in the known genes in the critical region indicates that the HMSN-R mutation affects a novel gene that remains to be identified.

z Acknowledgements

Our research is funded by grants of the Fund for Scientific Research ± Flanders (FWO-Vlaanderen), the Special Research Fund of the University of Antwerpen, the Medical Foundation Queen Elisabeth, the Association Belge contre les Maladies Neuro-Musculaires, the Interuniversity Attraction Poles program P5/19 of the Belgian Federal Science Policy Office (Belgium) and the Muscular Dystrophy Association (USA). EN is a postdoctoral fellow of the FWO-Vlaanderen.

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References

1. Ammar N, Nelis E, Merlini L, Barisic N, Amouri R, Ceuterick C, Martin JJ, Tim- merman V, Hentati F, De Jonghe P (2003) Identification of novel GDAP1 mutations causing autosomal recessive Charcot-Marie-Tooth disease. Neuromusc Dis- ord 13:720±728

2. Andersson PB, Yuen E, Parko K, So YT (2000) Electrodiagnostic features of hereditary neuropathy with liability to pressure palsies. Neurology 54:40±44

3. Angelicheva D, Turnev I, Dye D, Chandler D, Thomas PK, Kalaydjieva L (1999) Congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome: a novel developmental disorder in Gypsies maps to 18qter. Eur J Hum Genet 7:560±566 4. Arbuthnott ER, Boyd IA, Kalu KU (1980) Ultrastructural dimensions of myeli-

nated peripheral nerve fibres in the cat and their relation to conduction velocity. J Physiol 308:125±157

5. Auer-Grumbach M, Strasser-Fuchs S, Wagner K, Korner E, Fazekas F (1998) Roussy-Levy syndrome is a phenotypic variant of Charcot-Marie-Tooth syndrome IA associated with a duplication on chromosome 17p11.2. J Neurol Sci 154:72±75 6. Azzedine H, Bolino A, Taieb T, Birouk N, Di Duca M, Bouhouche A, Benamou S,

Mrabet A, Hammadouche T, Chkili T, Gouider R, Ravazzolo R, Brice A, Laporte J, LeGuern E (2003) Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma. Am J Hum Genet 72:1141±1153

7. Barhoumi C, Amouri R, Ben Hamida C, Ben Hamida M, Machghoul S, Gueddiche M, Hentati F (2001) Linkage of a new locus for autosomal recessive axonal form of Charcot-Marie-Tooth disease to chromosome 8q21.3. Neuromusc Disord 11:27±34 8. Baxter RV, Ben Othmane K, Rochelle JM, Stajich JE, Hulette C, Dew-Knight S, Hentati F, Ben Hamida M, Bel S, Stenger JE, Gilbert JR, Pericak-Vance M, Vance JM (2002) Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot-Marie-Tooth disease type 4A/8q21. Nat Genet 30:21±22

9. Behse F, Buchthal F, Carlsen F, Knappeis GG (1972) Hereditary neuropathy with liability to pressure palsies. Electrophysiological and histopathological aspects.

Brain 95:777±794

10. Ben Othmane K, Hentati F, Lennon F, Ben Hamida C, Blel S, Roses AD, Pericak- Vance MA, Ben Hamida M, Vance JM (1993) Linkage of a locus (CMT4A) for autosomal recessive Charcot-Marie-Tooth disease to chromosome 8q. Hum Mol Genet 2:1625±1628

11. Ben Othmane K, Johnson E, Menold M, Graham FL, Hamida MB, Hasegawa O, Rogala AD, Ohnishi A, Pericak-Vance M, Hentati F, Vance JM (1999) Identification of a new locus for autosomal recessive Charcot-Marie-Tooth disease with focally folded myelin on chromosome 11p15. Genomics 62:344±349

12. Bennett CL, Shirk AJ, Huynh HM et al (2004) SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve. Ann Neurol 55:713±720

13. Berciano J, Garcia A, Combarros O (2003) Initial semeiology in children with Charcot-Marie-Tooth disease 1A duplication. Muscle Nerve 27:34±39

14. Berciano J, Garcia CA, Calleja J, Combarros O (2000) Clinico-electrophysiological correlation of extensor digitorum brevis muscle atrophy in children with Charcot- Marie-Tooth disease 1A duplication. Neuromusc Disord 10:419±424

15. Berger P, Young P, Suter U (2002) Molecular cell biology of Charcot-Marie-Tooth disease. Neurogenetics 4:1±15

16. Bird TD, Ott J, Giblett ER (1982) Evidence for linkage of Charcot-Marie-Tooth neuropathy to the Duffy locus on chromosome 1. Am J Hum Genet 34:388±394

(19)

17. Birouk N, Gouider R, LeGuern E, Gugenheim M, Tardieu S, Maisonobe T, Le For- estier N, Agid Y, Brice A, Bouche P (1997) Charcot-Marie-Tooth disease type 1A with 17p11.2 duplication. Clinical and electrophysiological phenotype study and factors influencing disease severity in 119 cases. Brain 120:813±823

18. Blair IP, Nash J, Gordon MJ, Nicholson GA (1996) Prevalence and origin of de novo duplications in Charcot-Marie-Tooth disease type 1A: First report of a de novo duplication with a maternal origin. Am J Hum Genet 58:472±476

19. Boerkoel CF, Takashima H, Bacino CA, Daentl D, Lupski JR (2001) EGR2 mutation R359W causes a spectrum of Dejerine-Sottas neuropathy. Neurogenetics 3:153±

20. Boerkoel CF, Takashima H, Garcia CA, Olney RK, Johnson J, Berry K, Russo P, Ken-157 nedy S, Teebi AS, Scavina M, Williams LL, Mancias P, Butler IJ, Krajewski K, Shy M, Lupski JR (2002) Charcot-Marie-Tooth disease and related neuropathies: mutation distribution and genotype-phenotype correlation. Ann Neurol 51:190±201 21. Boerkoel CF, Takashima H, Nakagawa M, Izumo S, Armstrong D, Butler I, Man-

cias P, Papasozomenos SC, Stern LZ, Lupski JR (2003) CMT4A: identification of a Hispanic GDAP1 founder mutation. Ann Neurol 53:400±405

22. Boerkoel CF, Takashima H, Stankiewicz P, Garcia CA, Leber SM, Rhee-Morris L, Lupski JR (2001) Periaxin mutations causes recessive Dejerine-Sottas Neuropathy.

Am J Hum Genet 68:325±333

23. Bolino A, Brancolini V, Bono F, Bruni A, Gambardella A, Romeo G, Quattrone A, Devoto M (1996) Localization of a gene responsible for autosomal recessive demyelinating neuropathy with focally folded myelin sheaths to chromosome 11q23 by homozygosity mapping and haplotype sharing. Hum Mol Genet 5:1051±1054 24. Bolino A, Muglia M, Conforti FL, LeGuern E, Salih MA, Georgiou DM, Christo-

doulou K, Hausmanowa-Petrusewicz I, Mandich P, Schenone A, Gambardella A, Bono F, Quattrone A, Devoto M, Monaco AP (2000) Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet 25:17±19

25. Brancolini C, Marzinotto S, Edomi P, Agostoni E, Fiorentini C, Muller HW, Schneider C (1999) Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22. Mol Biol Cell 10:2441±2459

26. Ceuterick-de Groote C, De Jonghe P, Timmerman V, Van Goethem G, Læfgren A, Ceulemans B, Van Broeckhoven C, Martin JJ (2001) Infantile demyelinating neuropathy associated with a de novo point mutation on Ser72 in PMP22 and basal lamina onion bulbs in skin biopsy. Pathol Res Pract 197:193±198

27. Chance PF, Alderson MK, Leppig KA, Lensch MW, Matsunami N, Smith B, Swan- son PD, Odelberg SJ, Distsche CM, Bird TD (1993) DNA deletion associated with hereditary neuropathy with liability to pressure palsies. Cell 72:143±151

28. Chapon F, Latour P, Diraison P, Schaeffer S, Vandenberghe A (1999) Axonal phenotype of Charcot-Marie-Tooth disease associated with a mutation in the myelin protein zero gene. J Neurol Neurosurg Psychiatry 66:779±782

29. Charcot J-M, Marie P (1886) Sur une forme particuli re d'atrophie musculaire progressive, souvent familiale, dbutant par les pieds et les jambes et atteignant plus tard les mains. Rev Md 6:97±138

30. Chavrier P, Janssen-Timmen U, Mattei MG, Zerial M, Bravo R, Charnay P (1989) Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

Mol Cell Biol 9:787±797

31. Cuesta A, Pedrola L, Sevilla T, Garcia-Planells J, Chumillas MJ, Mayordomo F, Le- Guern E, Marin I, Vilchez JJ, Palau F (2002) The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie- Tooth type 4A disease. Nat Genet 30:22±25

(20)

32. Cui X, De V, I, Slany R, Miyamoto A, Firestein R, Cleary ML (1998) Association of SET domain and myotubularin-related proteins modulates growth control. Nat Genet 18:331±337

33. D'Urso D, Ehrhardt P, Mçller HW (1999) Peripheral myelin protein 22 and protein zero: a novel association in peripheral nervous system myelin. J Neurosci 19:3396±

34. De Jonghe P (1998) The inherited neuropathies of the peripheral nervous system3403 in the DNA era: genotype-phenotype correlations. University of Antwerpen 35. De Jonghe P, Mersiyanova IV, Nelis E, Del Favero J, Martin J-J, Van Broeckhoven

C, Evgrafov OV, Timmerman V (2001) Further evidence that neurofilament light chain gene mutations can cause Charcot-Marie-Tooth disease type 2E. Ann Neurol 49:245±249

36. De Jonghe P, Timmerman V, Ceuterick C, Nelis E, De Vriendt E, Læfgren A, Ver- cruyssen A, Verellen C, Van Maldergem L, Martin J-J, Van Broeckhoven C (1999) The Thr124Met mutation in the peripheral myelin protein zero (MPZ) gene is associated with a clinically distinct Charcot-Marie-Tooth phenotype. Brain 122:281±

37. Dejerine J, Sottas J (1893) Sur la nvrite interstitielle, hypertrophique et progres-290 sive de l'enfance. CR Soc Biol (Paris) 45:63±96

38. Delague V, Bareil C, Tuffery S, Bouvagnet P, Chouery E, Koussa S, Maisonobe T, Loiselet J, Megarbane A, Claustres M (2000) Mapping of a new locus for autosomal recessive demyelinating Charcot-Marie-Tooth disease to 19q13.1-13.3 in a large consanguineous Lebanese family: exclusion of MAG as a candidate gene. Am J Hum Genet 67:236±243

39. Dickson KM, Bergeron JJ, Shames I, Colby J, Nguyen DT, Chevet E, Thomas DY, Snipes GJ (2002) Association of calnexin with mutant peripheral myelin protein- 22 ex vivo: a basis for ªgain-of-functionº ER diseases. Proc Natl Acad Sci USA 99:9852±9857

40. Ding Y, Brunden KR (1994) The cytoplasmic domain of myelin glycoprotein P0 interacts with the negatively charged phospholipid bilayers. J Biol Chem 269:10764±10770

41. Ekici AB, Oezbey S, Fuchs C, Nelis E, Van Broeckhoven C, Schachner M, Rauten- strauss B (2002) Tracing Myelin Protein Zero (P0) in vivo by construction of P0- GFP fusion proteins. BMC Cell Biol 3:29

42. Fabretti E, Edomi P, Brancolini C, Schneider C (1995) Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev 9:1846±1856

43. Friede RL, Samorajski T (1970) Axon caliber related to neurofilaments and micro- tubules in sciatic nerve fibers of rats and mice. Anat Rec 167:379±387

44. Gabrels-Festen AA (2002) Dejerine-Sottas syndrome grown to maturity: overview of genetic and morphological heterogeneity and follow-up of 25patients. J Anat 200:341±356

45. Gabrels-Festen AA, Gabreels FJ, Joosten EM, Vingerhoets HM, Renier WO (1992) Hereditary neuropathy with liability to pressure palsies in childhood. Neuropedia- trics 23:138±143

46. Gabrels-Festen AA, Joosten EM, Gabreels FJ, Stegeman DF, Vos AJ, Busch HF (1990) Congenital demyelinating motor and sensory neuropathy with focally folded myelin sheaths. Brain 113 (Pt 6):1629±1643

47. Gabrels-Festen AA, van Beersum S, Eshuis L, LeGuern E, Gabreels F, van Engelen B, Mariman E (1999) Study on the gene and phenotypic characterisation of autosomal recessive demyelinating motor and sensory neuropathy (Charcot-Marie- Tooth disease) with a gene locus on chromosome 5q23-q33 [see comments]. J Neurol Neurosurg Psychiatry 66:569±574