European CMT Consortium, 5th Workshop Report FULL ARTICLE

[Guest guest]

Workshop report 5th Workshop of the European CMT Consortium,

69th ENMC International Workshop:

Therapeutic approaches in CMT neuropathies and related disorders

23±25 April 1999, Soestduinen, The Netherlands

Rudolf ia, ¬ Bercianob, Van Broeckhovenc,*

aBayerische Julius-Maximilians-Universita»t, Neurologische Klinik im

Kopfklinikum, Experimentelle Entwicklungsneurobiologie,

f Schneider Strasse 11, 97080 Wu»rzburg, Germany

bUniversity Hospital ``Marque¬s de Valdecilla'', Service of Neurology,

39008 Santander, Spain

cLaboratory of Molecular Genetics, Flanders Interuniversity Institute

for Biotechnology (VIB), Born-Bunge Foundation (BBS),

University of Antwerpen (UIA), Department of Biochemistry,

Universiteitsplein 1, B-2610 Antwerpen, Belgium

Received 18 August 1999

1. Introduction

Inherited demyelinating neuropathies are chronic disorders of the

peripheral nervous system that cause muscle weakness, clumsiness in

walking and sensory dysfunction.

The disorders are mostly of dominant inheritance with a prevalence of

1/2.500. So far, four genes have been identiÆed that are related to

these disorders, including the peripheral myelin protein 22 (PMP22), the

myelin protein zero (MPZ, P0), the gap junction protein connexin 32

(Cx32,GJB1), and the early growth response 2 transcription factor

(EGR2).Dependent on the mutated gene and on the severity of the

resulting disorder, different subforms can be distinguished.A

duplication of or mutations in PMP22 causes the demyelinating

Charcot±Marie±Tooth disorder type 1A (CMT1A), whereas some other

mutations in this gene are associated with a particularly severe

dysmyelinating neuropathy, the Dejerine±Sottas syndrome (DSS).

Mutations in P0 can cause three different inherited neuropathies, CMT1B,

DSS

or congenital hypomyelination (CH). Cx32 is the culprit gene for CMTX,

the X-chromosome linked, dominant form of the CMT disorders. Mutations

in EGR2 have recently been identiÆed to be associated with CMT1, DSS and

CH. Although

recent years have seen a profound increase in the knowledge about the

culprit genes and their putative roles [1], the inherited neuropathies

are still untreatable. Since most of the diseases are not only

signiÆcantly disabling, but also lead to irreversible degenerative

processes, such as muscle atrophy, treatment strategies are urgently

needed.

A common histopathological feature in nerve biopsies is the presence of

abnormal myelin sheaths and reduced numbers of myelin proÆles.

Electrophysiologically, the disrupted myelin formation or myelin

degeneration is reØected by lowered nerve

conductions, increased muscle response latencies and dispersed compound

action potentials. In addition, the disorders are often associated with

reduced amplitudes of

compound action potentials, a feature that is rather indicative of

compromised axon properties than of myelin disruption.

Axonal abnormalities or damage is of particular interest, since

dysfunction of axons will have robust clinical consequences.

Particularly in lower limbs, irreversible degenerative processes such as

muscle atrophy are the typical consequence of axon damage and the

increasing loss of muscle strength results in malformation of the

skeleton. In severe cases, such changes manifest in wheel-chair

dependency,

scoliosis and reduced life span. Presently, a series of spontaneous or

engineered animal models are available that very well mimic the genetic

and histopathological characteristics of the human disorders.

These models are not only instrumental in understanding the pathogenesis

of the disorders, but may also help to develop treatment strategies.

There might be at least two principle strategies for therapeutic

approaches. One possibility is to consider gene therapeutic strategies.

Alternatively, strategies in treating the symptomatic features might be

promising as well. Such strategies could include to preserve

degenerating myelin and axons with trophic factors. Reducing the

activities of

immune cells that might foster the degenerative events Neuromuscular

Disorders 10 (2000) 69±74 0960-8966/00/$ - see front matter q 2000

Elsevier Science B.V. All rights reserved.PII: S0960-8966(99)00095-4

www.elsevier.com/locate/nmd * Corresponding author. Tel.:

132-3-820-2601; fax: 132-3-820-2541. E-mail address:

cvbroeck@... (C. Van Broeckhoven) - could be another promising

way to go.

Investigating the inØuence of steroid hormones and studying epigenetic

factors might be important as well. All these strategies could be tested

in the appropriate animal models for the disorders. This workshop was

designed to present and to discuss critically data or theoretical

backgrounds of possible approaches that might be instrumental in

ameliorating the disorders in human. Leading physicians and scientists

from different disciplines working in this Æeld have evaluated

critically the putative treatment strategies for the still incurable

disorders. The workshop consisted of Æve sessions: (1) Genetic,

clinical, pathological and physiological aspects; (2) Current strategies

of rehabilitation and surgery; (3)Rescue of axons and Schwann cell

phenotype; (4) Immune system and epigenetic factors as modulators of

pathology;

(5) Gene therapy approaches. 2. Genetic, clinical, pathological and

physiological

aspects Timmerman (Antwerpen, Belgium) reviewed the genetic

basis of Charcot±Marie±Tooth (CMT) disorders, to date including 18 loci

on 11 different chromosomes.

However, only four genes have been identiÆed so far:

MPZ/P0 on chromosome 1q22-23, PMP22 on 17p11.2,Cx32/GJB1 on Xq13.1, and

more recently EGR2 on 10q21.1±q22.1. A large number of mutations

involving these genes have been reported and are listed in the Mutation

Database of Inherited Peripheral Neuropathies at http://

molgen-www.uia.ac.be/CMTMutations/ [2]. Interestingly,distinct mutations

at the same gene can result in distinct phenotypes, such as classical

peroneal muscular atrophy (PMA), DSS, CH or hereditary neuropathy with

liability to pressure palsies (HNPP), while mutations in distinct genes

can result in the same phenotype. The most common mutation is the 1.5 Mb

tandem duplication in CMT1 patients and the reciprocal deletion in HNPP

patients.

Two particular mutations involving Cx32 and EGR2 were presented. In the

CMT1X pedigree a missense mutation (Asn205Ser) in the 4th transmembrane

domain of Cx32 was found [3]. The patients showed typical CMT syndrome

but electrophysiological studies revealed, besides the peripheral

neuropathy, also evidence of subclinical involvement

of the central nervous system (CNS). Therefore, careful CNS evaluation

is worthwhile in patients with suspected Cx32 mutation. Among 50

unrelated CMT patients without

mutations in the known myelin genes, a de novo missense mutation

(Arg359Trp) in the a-helix of the Ærst zinc-Ænger domain of the EGR2

transcription factor was found in a DSS patient [4]. Finally,

Timmerman outlined that experimental in vitro (cellular) and in vivo

(rodent) models of inherited myelinopathies will provide the tools to

elucidate the pathogenesis of CMT syndromes.

De Jonghe (Antwerpen, Belgium) carried out a clinical overview of

CMT disorders. CMT is the most common inherited sensori-motor

neuropathy. CMT patients usually

have gait problems and impaired hand function due to distal paresis.

Contrariwise sensory symptoms are seldom prominent. CMT can be divided

into two main subtypes: CMT1

and CMT2. CMT1 is a demyelinating neuropathy characterised by severely

slowed nerve conduction velocities (NCV), whereas CMT2 is an axonal

neuropathy with normal or slightly reduced NCV. In both types initial

symptoms are stereotyped; weakness invariably starts in the intrinsic

foot muscles progressing to weakness and atrophy

of the extensors of the toes and feet. This stereotyped evolution might

be helpful in monitoring therapeutic measurements.

Inheritance of CMT disorders may be autosomal dominant or recessive, or

X-linked. Genotype±phenotype correlations have shown that there exist

considerable differences

between groups of patients carrying the same mutation or distinct

mutations within the same gene. There is also substantial overlap in

NCVs between different CMT

subgroups. In CMT1 there is poor correlation between the degree of

slowing of NCV and clinical severity.

Anneke Gabree»ls-Festen (Nijmegen, The Netherlands) analysed

pathological processes in demyelinating types of CMT1. At present,

defects of three myelin genes are known

to play a role in the demyelinating forms of CMT. Although primarily

defects of myelin, they show a variable but usually marked involvement

of myelinated axons. In young patients with the PMP22 duplication the

myelinated Æbre size histogram shows a remarkable lack of the smallest

Æbres. The mean g-ratio is lower than normal, probably resulting from a

general hypermyelination. A secondary large Æbre loss increases with

age. This is an extremely important Ænding as there is good correlation

with electrophysiological Ændings in CMT1A children (vide infra).

Hypomyelination is

the pathological hallmark in patients with PMP22 missense mutations.

Young patients with a PMP22 deletion show normal histogram and normal

mean g-ratio; in later stages

secondary axonal loss becomes apparent. Pathology in P0 mutations is

markedly different depending on the site and type of mutation, but a

secondary and varying loss of large myelinated axons is a general

phenomenon. Axonal degeneration

and regeneration with cluster formation are the pathological hallmark of

Cx32 mutations, demyelination being exceeded by axonal pathology. The

processes by

which the myelin disorders are leading to axonal degeneration are an

important Æeld of research.

¬ Berciano (Santander, Spain) presented the electrophysiological

abnormalities accounting for the appearance and progression of extensor

digitorum brevis (EDB) atrophy in CMT1A children. The study was based on

a longitudinal study of 12 CMT1A children. EDB atrophy was observed in

17% of patients by age 5, in 80% by age 9, and in all 8 patients who had

reached the second decade at the end.

Nerve conduction maturation was systematically abnormal, but by age 5

the mean values of NCV of peroneal nerve did not differ from those in

older patients. Compound muscle

action potentials (CMAP) amplitudes of EDB were reduced R. i et

al. / Neuromuscular Disorders 10 (2000) 69±74 70 in 42% of cases

initially and 100% upon last exam. Furthermore, a constant Ænding

throughout the study was progressive

attenuation of CMAPs, these becoming unobtainable in four cases. It is

concluded that EDB atrophy in CMT1A children is an age-dependant sign

which is accounted for

by gradual attenuation of the distal peroneal nerve CMAP amplitudes,

this most probably being correlated with axonal loss (vide supra).

3. Current strategies of rehabilitation and surgery Paolo Vinci and

Perelli (Aricci, Italy) presented their programme of

rehabilitation inCMTdisease. This relies

on improvement of muscle strength, prevention of deformities,good

ambulation, prevention of falls, improvement of hand abilities,

psychological support, and prevention and treatment of pain. Muscle

strengthening should focus on both weak muscles and their antagonists,

stretching these probably being the best strategy. Leg muscle fatigue

should be avoided by wearing appropriate foot orthoses (AFOs).

Functional stretching of foot plantar Øexor and supinator muscles,

obtained by wearing shoes with a lateral edge and with no heel, is

essential in preventing foot deformities.

Treatment of gait difÆculties varies according to severity of disease.

The authors have designed a new plastic AFO consisting of a properly

shaped strip of very thin polypropylene between the lining and the vamp

of 15 cm high boot.

Finally, Aycart (Madrid, Spain) presented a carefully planned

surgical approach of foot deformities in CMT.These could be divided into

three stages. In the initial stage, foot deformity is restricted to the

metatarsal, metatarsophalangeal,

and digital segments of the foot. Here the surgical approach is focused

on digital and metatarsophalangeal joints. The next stage encompasses

rigid plantarØexed Ærst

metatarsal and associated varus deformity. Surgical correction now

includes a series of surgical procedures, namely: dorsiØexory wedge

osteotomy of the Ærst metatarsal, Dwyer calcaneal osteotomy, and split

tibialis anterior tendon transfer.

The last and advanced stage is a severe foot structural disorder usually

accompanied by signiÆcant muscular weakness and atrophy of leg muscles.

Realignment of foot

deformity may require triple arthrodesis or Cole osteotomy and major

tendon transfers. In any case, surgical procedures should be

individualised. It is crucial to inform the patient that CMT is a

dynamic process, so there may be potential deterioration of the surgical

correction in the long run.

4. Rescue of axons and Schwann cell phenotype Kristjan n (London,

UK) was focussing on two issues including the involvement of growth

factors in

Schwann cell survival and the signalling between Schwann cells and the

prospective connective tissue sheath during nerve development. In the

Ærst part, it was shown that

Schwann cell progenitor cells switch from axon-dependency to an

axon-independent state during their development.

The axon-independent survival of Schwann cells is mediated by an

autocrine circuit involving insulin growth factor-2 (IGF-2), platelet

derived growth factor (PDGF) and

neurotrophin-3 (NT-3) [5]. The survival of Schwann cells is relevant for

nerve injury as well as for demyelinating disorders when proliferating

Schwann cells loose contact with the axon and form onion bulbs. In the

second part, the phenotype of mice deÆcient in the Desert Hedgehog

protein was presented. The striking feature of peripheral nerves is the

formation of a highly abnormal and leaky perineurium so that the

protective function of the connective tissue sheath can no longer be

maintained.

Hans Werner Mu»ller (Du»sseldorf, Germany) was focussing on a novel

protein interaction between two important CMT-related myelin components,

PMP22 and P0. Both

myelin components co-precipitate in immune-precipitation assays [6]. The

interaction of both proteins appeared to be independent of

glycosylation. The signiÆcance of this interaction was further supported

by co-transfection experiments showing that both proteins are

co-localized at cell-contact sites. The interaction of P0 and PMP22

might have substantial impact on the pathogenesis of P0- and

PMP22-related forms of CMT.

Sereda (Berlin and Heidelberg, Germany) presented the phenotype

of a transgenic rat model expressing extra copies of mouse-PMP22. Low

overexpression

results in a CMT1A-like phenotype, higher overexpression in a much more

severe dysmyelinating phenotype [7]. In peripheral nerves of such high

overexpressors, protein metabolism is severely disturbed.

Brady's (Dallas, USA) presentation dealt with axon±glia

interactions in trembler and shiverer mutants.In trembler, a mouse

mutant with a substitution of aspartic

acid for glycine at codon 150 of PMP22, there is a clear impact of the

Schwann cell phenotype on axonal properties, such as phosphorylation of

neuroÆlaments, axonal transport, stability of neurotubuli and isoform

pattern of Tau-proteins [8]. Similar glial impact on axonal phenotype

might be found in various inherited neuropathies in human with high

clinical signiÆcance.

The following two presentations were closely related to this important

topic of disturbed axon±glia interactions that eventually lead to axon

damage. Ueli Suter (Zu»rich, Switzerland) presented data on

PMP22-deÆcient mice and mice overexpressing PMP22 with concern of myelin

deÆcits and the glial impact on axonal phenotype. Generally, myelin

abnormalities were more severe at the level of the spinal

roots, while axonopathic alterations were more severe in the peripheral

nerves proper, i.e. in more distal aspects of the axons [9].

Regula Frei and Rudolf i (Wu»rzburg, Germany) presented similar

Ændings of distal axonopathic alterations in homozygous P02/2 mice that

primarily suffer from severe dysmyelination. In proximal aspects of

nerves, the number of axons was not changed. By contrast, at very distal

sites of the R. i et al. / Neuromuscular Disorders 10 (2000) 69±74

71 nerves, the number of axons was dramatically reduced. This

degeneration of distal axons resulted in a loss of sensory Merkel cells

and in neurogenic muscle pathology [10].

Henning Schmalbruch's (Copenhagen, Denmark) contribution focused on

treatment strategies to prevent or retard axonal loss in mouse mutants

suffering from a genetically mediated progressive motor neuronopathy

(pmn). The pmn mice are characterized by a loss of motor axons that

starts at the distal sites and progresses retrogradely. This

``dyingback'' axonopathy can be reduced by infection of target

muscles with adenoviral vectors coding for NT-3 [11].

Even stronger rescue effects were seen when an additional vector coding

for ciliary neurotrophic factor (CNTF) was given. Although the primary

genetic causes leading to

axonopathy are different in pmn mice versus inherited neuropathies,

similar approaches might be promising for treatment of axonal loss in

inherited neuropathies.

Ignacio -Ale¬man (Madrid, Spain) presented data on the rescue

effect of IGF-I in neurotoxic damage of the olivo-cerebellar pathway

[12]. The beneÆcial effects of IGFI

might implicate axonal sprouting, neuroprotection and regulation of

nerve-growth-related genes. These observations might identify IGF-I as

another possible factor to treat some forms of inherited neuropathies.

5. Immune system and epigenetic factors as modulators

of pathology

Angelo Schenone (Genova, Italy) reviewed data from the literature that

showed clinical and histopathological evidence for an involvement of the

immune system in the

pathogenesis of inherited neuropathies [13±15].

Christoph Schmid and Rudolf i (Wu»rzburg,

Germany) presented data from a possible impact of immune cells in

heterozygous P01/2 mice, a model for CMT1B. Cross breeding of these mice

with immune-deÆcient mouse mutants (e.g. RAG-12/2 mice) resulted in a

signiÆcant amelioration of the pathological phenotype as revealed by

electron microscopic and electrophysiological investigations.

The observation that isolated splenocytes from P01/2 mice show a

stronger proliferation rate than splenocytes from wild type mice when

exposed to myelin components,might reØect that chronic, genetically

mediated myelin degeneration in the mutants might elicit autoimmunity.

Ralf Gold (Wu»rzburg, Germany) summarised the principle mechanisms

underlying the pathogenesis in acquired, inØammatory neuropathies [16].

He particularly focused

on the action of T-lymphocytes as producers of cytokines or of cytotoxic

enzymes, such as metalloproteases, perforin and granzyme. In addition,

T-lymphocyte-mediated cytokine secretion can indirectly attract

macrophages that can be detrimental for neural cells by secreting

cytokines, free oxygen radicals, nitric oxide and toxic metabolites. As

antigen-presenting cells macrophages can augment the cytotoxic

action of T-lymphocytes. The B-lymphocytes interact with speciÆc

T-helper cells and serve as constituents of humoral cytotoxity. In

principle, such mechanisms could

also modulate pathogenesis in inherited neuropathies.

Although clear hints for an involvement of the immune system are rarely

found in the clinics and there might be also more coincidental than

causative links between inherited neuropathies and inØammatory diseases,

it appears plausible to assume that the immune system could act as a

modulating factor during the progression of the disease. The involvement

of immune mechanisms might provide one explanation for the response of

some reported patients on immunomodulatory drugs and possibly also for

the high variability in the severity of inherited neuropathies even

within one and the same family.

6. Gene therapy approaches

Bernd Rautenstrau° (Erlangen, Germany) reported about two cases with a

putative mosaicism for CMT1A duplication. Such mosaicisms might be the

result of a possible

repair mechanism during mitosis [17]. The understanding of these

mechanisms might have impact on gene therapeutic approaches in the far

future.

Eva Nelis (Antwerpen, Belgium) was focussing on possibilities to silence

malignant

mutations leading to gain-of-function or dominantnegative effects in

inherited peripheral neuropathies. As one possibility, speciÆc antisense

oligodeoxynucleotides

could be used to prevent translation of the mutant mRNA by complementary

binding to the transcript and destructive cleavage by RnaseH [18]. A

second possibility makes use of ribozymes which cleave the target RNA at

the putative cleavage site [19].

Melitta Schachner (Hamburg, Germany) presented the introduction of

wild-type P0 gene into Schwann cells of homozygous P0-deÆcient mice by

an adenoviral expression

vector [20]. P0-speciÆcmRNAas well as P0 protein could be detected in

the primarily P0-deÆcient Schwann cells. Although this is a

straightforward approach, there are some

drawbacks that have to be considered. First, the procedure needs

permanent immune suppression to avoid immune attack against the vector.

Second, it might be difÆcult to get the gene dosis right and third,

mutations leading to gain-offunction or dominant-negative effects cannot

be cured by this approach. Nevertheless, the study shows for the Ærst

time that gene therapeutic approaches are principally possible in

animal models for inherited peripheral neuropathies.

P.K. (London, UK) presented a `switch-off' transgene mouse model

for CMT1A overexpressing PMP22 [21] under the control of a

tetracycline-responding

promoter. The rationale of this important study is to investigate

whether the detrimental effects of PMP22-overexpression are principally

reversible. This has striking impact on the question of whether

treatment of already

damaged nerves of adults could lead to a clinical amelioration.

R. i et al. / Neuromuscular Disorders 10 (2000) 69±74 72

7. Conclusion

The meeting raised a couple of interesting questions and perspectives

that were summarised in a discussion session. There was general

agreement that gene therapeutic

approaches are far from being applicable in the near future in humans.

Rather, approaches to save the axonal integrity might be an important

symptomatic approach that might be more promising. In addition, the

investigation of modulatory genes, epigenetic factors and possibly

immune mechanisms might be instrumental to understand intrinsic rescue

or aggravating mechanisms and to develop new concepts to treat the

disorders.

Workshop advisors

V. Timmerman (Antwerpen, Belgium)

P. De Jonghe (Antwerpen, Belgium)

Workshop participants

J. Aycart Testa (Madrid, Spain)

J. Berciano (Santander, Spain)

S.T. Brady (Dallas, USA)

O. Combarros (Santander, Spain)

P. De Jonghe (Antwerpen, Belgium)

R. Frei (Wu»rzburg, Germany)

A. Gabree»ls-Festen (Nijmegen, The Netherlands)

R. Gold (Wu»rzburg, Germany)

A. Herczegfalvi (Budapest, Hungary)

K. n (London, UK)

A. Jordanova (SoÆa, Bulgaria)

S. Linet Perelli (Ariccia, Italy)

R. i (Wu»rzburg, Germany)

H-W. Mu»ller (Du»sseldorf, Germany)

E. Nelis (Antwerpen, Belgium)

B. Rautenstrau° (Erlangen, Germany)

M. Schachner (Hamburg, Germany)

A. Schenone (Genova, Italy)

H. Schmalbruch (Copenhagen, Denmark)

C. Schmid (Wu»rzburg, Germany)

M. Sereda (Berlin, Germany)

U. Suter (Zu»rich, Switzerland)

P.K. (London, UK)

V. Timmerman (Antwerpen, Belgium)

I. -Alema¬n (Madrid, Spain)

C. Van Broeckhoven (Antwerpen, Belgium)

P. Vinci (Ariccia, Italy)

Acknowledgements

This workshop was made possible by the Ænancial support of the European

Union BIOMED II grant: Clinical, genetical and functional analysis of

peripheral neuropathies:

an integrated approach (CT961614 and CT960055) and the European

Neuromuscular Center (ENMC) and its main sponsors and associated

members. We are grateful to Professor emeritus Dr A.E.H. Emery for his

scientiÆc help, and to Mr Rutgers and Mrs Janine de Vries for

the organisational assistance of the ENMC. Professor Dr

C. Van Broeckhoven is the coordinator of the European CMT consortium.

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