Neuromuscular Diseases PDF

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Neuromuscular Diseases: From Basic Mechanisms to Clinical Management
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Monographs in
Clinical Neuroscience
Vol. 18
Series Editor M. Fisher, Worcester, Mass.

Advisory Board W.G. Bradley, Miami, Fla.
H.P. Hartung, Wurzburg
M.A. Moskowitz, Charlestown, Mass.
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Neuromuscular Diseases:
From Basic Mechanisms to
Clinical Management
Volume Editor F. Deymeer, Istanbul
16 figures and 21 tables, 2000

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Dr. Feza Deymeer
Professor of Neurology
Department of Neurology
University of Istanbul
C
apa, Istanbul
Turkey
Library of Congress Cataloging-in-Publication Data
Neuromuscular diseases: From basic mechanisms to clinical management / volume editor,

Feza Deymeer.
p. ; cm. (Monographs in clinical neuroscience, ISSN 1420-2441; vol. 18)
Includes bibliographical references and indexes.
ISBN 3805570562 (hardcover : alk. paper)
1. Neuromuscular diseases. I. Deymeer, Feza. II. Series.
[DNLM: 1. Neuromuscular Diseases. WE 550 N4935 2000]
RC925.5 .N473 2000
616.744dc21
00-030965
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents and
Index Medicus.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and
dosage set forth in this text are in accord with current recommendations and practice at the time of publication.
However, in view of ongoing research, changes in government regulations, and the constant flow of information
relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for
any change in indications and dosage and for added warnings and precautions. This is particularly important
when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or
utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying,
or by any information storage and retrieval system, without permission in writing from the publisher.
Copyright 2000 by S. Karger AG, P.O. Box, CH4009 Basel (Switzerland)

www.karger.com
Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel
ISSN 14202441
ISBN 3805570562
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Contents
VII Preface
1 Novel Approaches to Therapeutics of the Muscular Dystrophies

Hoffman, E.P. (Washington, D.C.); Buyse G.M. (Leuven)
12 Use of Normal and Genetically Modified Myoblasts for the Treatment
of Myopathies
Skuk, D.; Tremblay J.P. (Ste-Foy, Que.)
26 Disorders of the Sarcoglycan Complex (Sarcoglycanopathies)
Bonnemann, C.G. (Gottingen)
44 Facioscapulohumeral Muscular Dystrophy: Diagnostic and Molecular
Aspects
Lunt, P.W. (Bristol)
61 Myotonic Dystrophy
Moxley, R.T. (Rochester, N.Y.); Meola, G. (Milan)
79 Muscle Ion Channel Diseases
Rudel, R.; Jurkat-Rott, K.; Lehmann-Horn, F. (Ulm)
96 Congenital Myasthenic Syndromes
Engel, A.G.; Ohno, K.; Stans, A.A. (Rochester, Minn.)
113 Juvenile and Late-Onset Myasthenia gravis
zdemir, C. (Istanbul)
Deymeer, F.; Serdaroglu, P.; O
128 Hereditary Peripheral Neuropathies
De Jonghe, P.; Timmerman, V.; Nelis, E. (Antwerp)
147 Acute Inflammatory Demyelinating Polyradiculoneuropathy
Bella, I.R. (Worcester, Mass.)
163 Proximal Spinal Muscular Atrophy of Childhood
Hausmanowa-Petrusewicz, I.; Zaremba, J. (Warsaw)
177 Familial Amyotrophic Lateral Sclerosis: A Review
Morita, M.; Brown, R.H., Jr. (Charlestown, Mass.)
190 Author Index

191 Subject Index
Contents VI
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Preface
This volume of Monographs in Clinical Neuroscience is devoted to neuro-

muscular disorders, a field where much progress has been made in the last two
decades, particularly in genetics. Mainly genetic, but also some immunological
disorders, with a wide spectrum ranging from those affecting the muscle to
those affecting the motor neuron, are presented in detail, including present
and potential future therapeutic modalities.
The first two chapters discuss different approaches which can lead the way
to successful treatment of muscle diseases, particularly Duchenne muscular
dystrophy. One approach is screening pharmacological agents in animal
models with the same genetic defect as in human muscular dystrophy. Another
important advancement is new gene therapy vectors based upon adenovirus
and adeno-associated virus. Much research is aimed at improving a different
potential therapeutic modality, myoblast transfer. Major areas of concern
are suppressing the immune response to the myoblasts, promoting the migra-
tion of myoblasts into the target muscle and improving their survival in the
muscle.
Three major dystrophies are the subjects of the next three chapters: sarco-
glycanopathies, facioscapulohumeral muscular dystrophy and myotonic dys-
trophy. Clinical features, protein expression pattern and mutational spectrum
of a-, b-, c- and d-sarcoglycanopathies, a distinct group within recessive limb-
girdle muscular dystrophies, are elucidated together with animal models and
gene transfer. Facioscapulohumeral muscular dystrophy where the DNA muta-
tion is known but the gene affected is unknown has a unique mutation. The
intricacies of the mutational analysis and genotype-phenotype correlations
are addressed and hypotheses for possible mechanisms of action of the muta-
VII
tion are suggested. Possible therapeutic options are also touched upon. In the
chapter on myotonic dystrophy, molecular genetics of the disease is discussed
including the phenomenon of anticipation and questions are raised about the
reliability of predicting phenotype from genotype. Management issues and
therapeutic trials are reviewed.
The chapter on muscle ion channel diseases elaborates on the pathogenesis
and the molecular genetics of sodium, calcium and chloride channelopathies.
While the electrophysiological basis of some of the symptoms is explained,
attention is drawn to phenomena which remain unexplained such as the tem-
perature sensitivity of paramyotonia congenita patients as opposed to its
absence in other sodium channelopathies.
In the chapters on neuromuscular junction and peripheral nerves, both
genetic and immunological diseases are included. A detailed discussion of
the electrophysiological and molecular biological characteristics of congenital
myasthenic syndromes, classified into presynaptic, synaptic and postsynaptic
defects, is presented. Juvenile and late-onset myasthenia gravis stand out as
distinct subgroups within myasthenia gravis. The recognition of epidemiologi-
cal, clinical and immunological characteristics of the disease in these age
groups helps in diagnosis and treatment.
The chapter on hereditary peripheral nerve disorders discusses clinical
and genetic characteristics of a wide range of disorders including Charcot-
Marie-Tooth 1 and 2, distal hereditary motor neuropathies, hereditary neurop-
athy with liability to pressure palsies and hereditary neuralgic amyotrophy.
The next chapter deals with acute inflammatory demyelinating polyra-
diculoneuropathy and its axonal variants including AMAN and AMSAN.
Clinical features, pathogenesis, significance of autoantibodies to glycoconju-
gates and approach to treatment are some of the topics analyzed.
The chapter on proximal spinal muscular atrophy of childhood discusses
its classification and its electrophysiological and pathological features. The
role of the SMN1 gene and other candidate genes as well as that of the
SMN protein and epigenetic factors including gender influence and incomplete
penetrance of the mutated genes are considered. The last chapter concentrates
on the pathophysiology of familial amyotrophic lateral sclerosis linked to the
mutant SOD1 and on the neurotoxicity of the mutant SOD1 protein and gives
a review of therapeutic trials.
I hope that this monograph will contribute to efforts linking basic science
to clinical medicine and that it will provide the reader with new insights and
fresh perspectives in neuromuscular disorders. I thank all the authors who
have made this monograph possible.
Feza Deymeer
Preface VIII
Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.
Monogr Clin Neurosci. Basel, Karger, 2000, vol 18, pp 111
............................
Novel Approaches to Therapeutics of the
Muscular Dystrophies
Eric P. Hoffman a, Gunnar M. Buyse b
a
Research Center for Genetic Medicine, Childrens National Medical Center,
George Washington University, Washington, D.C., USA;
b
Department of Pediatrics, Division of Child Neurology, University Hospital
Gasthuisberg, KU, Leuven, Belgium
Recessively inherited muscular dystrophies are diseases caused by a lack

of a specific protein component of muscle tissue. The identification of the
dystrophin gene and protein initiated an impressive series of studies which
have led to the characterization of nearly a dozen different muscular dystrophy
genes over the last 12 years. The dramatically increased knowledge of the
molecular basis of the muscular dystrophies has led to improved diagnosis of
patients, accurate genetic counseling of their families, and an understanding
of the pathophysiology of many of these disorders. However, the advances in
molecular technology and understanding of the disease process have not yet
led to improvements in patient treatment.
Two recent approaches have shown particular promise in developing novel
therapeutics. First, the identification of animal models with the same molecular
genetic defect as human muscular dystrophy patients has allowed large-scale
screening of pharmacological agents capable of increasing the strength of
dystrophic muscle. Second, new developments in gene therapy vectors based
upon adenovirus and adeno-associated virus have led to functional recovery
of dystrophic muscle via delivery of the normal gene. This review focuses on
these two approaches.
Animal Models as an Experimental Platform for

Therapeutic Studies in Duchenne Muscular Dystrophy
The identification of the dystrophin gene and protein as causative of

Duchenne muscular dystrophy (DMD) made it possible to screen for dys-
trophic animals with the same primary biochemical defect [Koenig et al., 1987;
Hoffman et al., 1987]. A number of mouse, dog and cat models were quickly
identified which showed dystrophin deficiency of muscle and mutations of the
dystrophin gene [Hoffman et al., 1987; Sicinski et al., 1988; Kornegay et al.,
1988; Cooper et al., 1988; Gaschen et al., 1992; Winand et al., 1994; Carpenter
et al., 1989]. Subsequently, the cardiomyopathic hamster was found to show
primary delta-sarcoglycan deficiency [Nigro et al., 1997; Sakamoto et al.,
1997], and additional knockouts of sarcoglycan genes were accomplished
[Hack et al., 1998; Duclos et al., 1998; Coral-Vazquez et al., 1999].
These homologous animal models show clinical presentations and pro-
gressions which were discordant with their human counterpart. Dystrophin-
deficient cats (HFMD) show dramatic muscle hypertrophy, with lingual hyper-
trophy leading to an inability to lap water, and diaphragmatic hypertrophy
leading to a constriction of the esophagus [Gaschen et al., 1992, 1998, 1999].
Dystrophin-deficient dogs (CXMD) show rapid muscle weakness and wasting,
with some puppies dying as neonates, while most show substantial physical
disability by just 6 months of age [Kornegay et al., 1988; Cooper et al.,
1988]. Dystrophin-deficient mice (mdx) show asymptomatic hyperCKemia and
muscle hypertrophy, with weakness only relatively late in their normal life
span [Lefaucheur et al., 1995; Pastoret and Sebille, 1995]. Sarcoglycan-deficient
hamsters present with cardiomyopathy, while the human counterpart shows
no overt heart involvement [Nigro et al., 1997].
The clinical discordancy between the human disease and homologous
animal counterparts led to the perception that these were not good animal
models. Indeed, a series of studies were conducted to improve the mdx mouse
model, by adding additional gene defects to the mouse to increase the clinical
severity: double mouse knockouts for dystrophin and myoD [Megeney et al.,
1996], and dystrophin and utrophin [Grady et al., 1997] were done to hasten
the disease presentation and progression. In some instances, these confirmed
existing models of pathophysiology of the disease. For example, it is known
that dystrophin deficiency leads to secondary loss of neuronal nitric oxide
synthase (nNOS) localization at the myofiber membrane [Brenman et al.,
1995]. This loss of nNOS localization leads to abnormal vascularization of
muscle during exercise [Thomas et al., 1998]. A double knockout of nNOS
and dystrophin showed no effect on the mdx phenotype [Chao et al., 1998]:
this is to be expected, as genetic loss of nNOS (via knockout) is not likely to
be different from biochemical loss of nNOS (via mislocalization). In other
instances, the double knockouts clarified functional roles of proteins involved
in the pathophysiology of dystrophin deficiency. For example, utrophin had
been hypothesized to be capable of functionally replacing dystrophin [Tinsley
et al., 1996, 1998], and the creation of double knockouts for dystrophin and
Hoffman/Buyse 2
utrophin confirmed this hypothesis [Grady et al., 1997; Rafael et al., 1998;
Gilbert et al., 1999]. Finally, in yet other instances, double knockouts simply
clouded the pathophysiological picture. For example, deficiency of both
myoD and dystrophin showed a more severe phenotype, however the mecha-
nism(s) underlying this exacerbation of symptoms is not at all clear [Megeney
et al., 1999].
An alternative approach to improving the animal models is to rest assured
that they do indeed represent good genetic and biochemical homologues of
their human counterparts, and to utilize them to develop novel therapeutics.
Gene and/or protein replacement through gene therapy is one clear and rational
approach, which is discussed later in this review. A second approach is to
develop experimental systems through which pharmacological agents can be
screened for possible efficacy in human patients. This latter approach has been
utilized in a recent series of reports using the dystrophin-deficient mdx model.
Drug Screening in Animal Models
Drug screening of animal models of muscular dystrophy has been pursued

since the early 1970s. In these premolecular era studies, specific inherited
muscular dystrophies in animals were argued to be a valid model for human
disease based upon clinical findings. One such popular model, the dystrophic
chicken, was used to screen various drugs which targeted specific pathophysio-
logical processes (immune system, calcium traffic, muscle growth and homeo-
stasis) [Barnard et al., 1976; Hudecki et al., 1981]. These studies led to the
identification of the glucocorticosteroid prednisone as a compound able to
slow the dystrophic process, a drug which has since been applied to large
numbers of DMD patients with clear success [Fenichel et al., 1991; Griggs et al.,
1991]. The rationale of these studies was that many of the pathophysiological
processes (myofiber degeneration, regeneration, connective tissue proliferation,
muscle wasting) were probably shared by the different muscular dystrophies,
and that pharmacological agents able to target these processes could show
efficacy independent of the specific (unknown) primary biochemical defect.
The molecular identification of homologous animal models has greatly
facilitated their use as an experimental model for their human counterpart.
A number of investigators have shown that exercise exacerbates the mdx mouse
phenotype [Hudecki et al., 1993]. This knowledge has been further developed
into a drug screening protocol using exercised mdx mice, with a relatively
crude yet effective whole-body strength assay for drug efficacy [Granchelli
et al., 1999]. The initial screen of 18 drugs has shown promising agents which
target specific aspects of the pathophysiology of dystrophin deficiency. Specifi-
Novel Approaches to Therapeutics of the Muscular Dystrophies 3

cally, considerable gains in muscle strength have been shown using glutamine,
pentoxifylline, creatinine, IGF-I, oxatomide, and prednisone. Oxatomide is a
mast cell stabilizer, which inhibits degranulation of mast cells, thereby pro-
tecting myofibers from damage via proteases and immune mediators [Gorospe
et al., 1994a, b, 1996]. IGF-I is a muscle growth factor, and likely improves
muscle regeneration [Barton-Davis et al., 1998]. Glutamine is a conditionally
essential amino acid, which is largely derived from skeletal muscle stores.
Supplementation of this amino acid presumably replenishes depleted stores
due to diseased muscle, and consequently may prevent muscle necrosis through
decreased protein degradation and protection against oxidative stress. Indeed,
preliminary data on DMD patients appears to confirm the beneficial effect
of this compound on protein homeostasis in dystrophic muscle [Hankard
et al., 1998]. Pentoxifylline is a hemorheologic agent which is used to enhance
peripheral vascularization of tissues; in DMD it likely functions by counter-
acting the nNOS-deficiency-induced vascular insufficiency of dystrophin-defi-
cient muscle [Thomas et al., 1998]. Alternatively, by inhibiting phospho-
diesterase activity, it might be beneficial in DMD by decreasing the production
of pro-inflammatory cytokines. Creatine is a well-documented performance
enhancer, and likely functions through improvement of muscle energy metabo-
lism, stimulation of protein synthesis, and/or decreasing intracellular calcium
overloading in dystrophic muscle [Pulido et al., 1998]. Creatinine is a metabolite
of creatine that presumably functions in an analogous manner. Additional
recent studies have suggested that gentamycin, an aminoglycoside antibiotic
which may permit the reading-through of genetic stop codons, may show
benefit in mdx mice [Barton-Davis et al., 1999]. Human clinical trial networks
are being established internationally to test these drugs, and other agents able
to improve the strength of dystrophin-deficient mice.
Gene Delivery to Dystrophic Muscle
All pharmacological approaches to treatment of muscular dystrophy hinge

on altering the pathophysiology of the disease, and, with a few exceptions, do
not address the primary biochemical defect. Gene delivery is an alternative
approach which should halt disease onset and/or progression through biochem-
ical replacement of the missing protein. Two viral gene delivery systems have
achieved considerable success in gene therapy of the muscular dystrophies:
third generation adenovirus (aka gutted adenovirus), and adeno-associated
virus (AAV). It should be pointed out that these two types of viruses are
fundamentally different in their biology, despite the similar names. In a word,
AAV can be viewed as a parasite of more harmful viruses; it borrows ma-
Hoffman/Buyse 4
chinery for the production of its viral progeny from another virus which has
coinfected the same cell (such as adenovirus and herpes-type viruses). AAV
is not replication-competent; it is unable to produce viral progeny without
the assistance of these other, larger viruses, and instead relies on its helper
virus for completion of its life cycle. As most of the helper viruses of AAV
are themselves quite toxic to cells, AAV seems to be able to confer some
beneficial effect to its host; reports have suggested that AAV can confer protec-
tion against human papilloma virus-induced cervical carcinoma [Hermonat,
1994] and inflammatory muscle disease [Tezak et al., 1999]. Thus, harnessing
AAV as a gene delivery vehicle has intrinsic advantages, relative to other toxic
viruses; these will be pointed out below.
Gutted adenovirus is the only gene therapy vehicle that has been shown
to be able to deliver full-length dystrophin protein, at levels sufficient to rescue
significant amounts of dystrophin-deficient muscle [Kochanek et al., 1996;
Clemens et al., 1996]. This viral vector is based upon adenovirus, but has been
modified so as to remove all adenoviral genes. This gutting of the viral
genetic backbone has two important advantages relative to previous versions of
adenoviral vectors. First, the removal of adenoviral genes releases considerable
space in the virus, so that constructs of up to 30 kb can be used. This carrying
capacity is much, much larger than any other viral delivery system used to
date. Second, the removal of the adenoviral genes reduces the immunogenicity
of the virus, at least with regard to the cellular immune response against
infected cells. This is due to the lack of virally encoded antigens expressed by
the gutted adenoviral backbone.
Using this gutted adenovirus, a series of reports have shown that the full-
length dystrophin cDNA can be delivered, driven by a muscle-specific (creatine
kinase) gene promoter [Kochanek et al., 1996; Clemens et al., 1996; Haecker
et al., 1996]. Normal levels of dystrophin can be produced in an entire mdx
mouse muscle, providing that the injections are done in neonatal mice. The
recombinant viral genome persists as an episome in mature myofibers, and
expression has been seen for as much as 1 year from the time of injection
[Chen et al., 1997, 1999]. However, recent systematic studies have shown that
the degree of persistence of dystrophin expression from the recombinant virus
largely depends on the level of immune response against infected cells [Loch-
muller et al., 1996; Howell et al., 1998].
There are remaining issues which must be solved before the gutted adeno-
virus can be considered a therapeutic approach with likely efficacy. First and
foremost, mature myofibers lack viral attachment receptors for adenovirus,
and thus significant infection can only be achieved in neonatal muscle, or in
actively regenerating muscle [Feero et al., 1997b]. This maturation-dependent
infectivity of muscle may be overcome by altering the tropism of the virus;

recent experiments have been published where the protein coat of the virus
was altered to target the virus towards heparin proteoglycan receptors [Bouri
et al., 1999]. This increased infectivity of adult muscle, although not to the
extent needed. Further research on altering the topism of the virus for adult
muscle is needed. A second issue is that of sensitivity of the immune system
for this virus. This problem was recently underscored by the report of a number
of deaths due to acute immune response against recombinant adenovirus, in
different human clinical trials. An additional concern is the need to separate
recombinant virus from first generation adenovirus which is used as a helper
virus during production of infectious virions. A recent report has shown that
this can be circumvented using cre/lox recombination sites in the helper virus,
which are able to prevent the helper from packaging into infectious virions
[Aoki et al., 1999]. Third, adenovirus may show lingering problems with
persistence, due to its existence as an episome, which may be subject to loss
from the myofiber nucleus. Finally, adenovirus remains an immunogenic virus,
even with all viral genes eliminated. A recent publication suggests that infection
of professional antigen-presenting cells (dendritic cells) in muscle may be a
major reason for the antigenicity of adenovirus [Jooss et al., 1998].
AAV is a relatively recently characterized human parvovirus, which shows
proclivity for mature muscle [Tezak et al., 1999]. The normal life cycle of
the wild-type virus involves infection of nonmitotic cells, with subsequent
integration into human chromosome 19 using a virally encoded recombinase
protein (rep). It remains as a silent (latent) integrated virus, until the cell is
infected with a helper virus (adenovirus or herpes-type viruses). It seems to
confer some protection against the immune system; a recent study found that
muscle may be protected from inflammatory attacks when infected with AAV.
The virus is quite small in size, and appears able to pass through the myofiber
basal lamina, unlike larger viruses (such as adenovirus and HSV).
The ability of AAV to infect mature myofibers, to integrate stably into
host chromosomes, and to evade immune system recognition makes AAV a
seemingly ideal vector for muscle gene delivery. Its only apparent drawback
is its limited packaging capacity: AAV can only contain transgene constructs
of up to 5 kb, which is far too small for the 11-kb dystrophin coding sequence.
Due to the constraints on transgene size, all published data to date has been
centered on the sarcoglycan genes, which are much smaller in size and easily
accommodated into recombinant AAV vectors. A spontaneously occurring
hamster model of delta sarcoglycan deficiency has been characterized [Nigro
et al., 1997; Sakamoto et al., 1997], and most reports have utilized this hamster
to demonstrate the feasibility and efficacy of AAV-mediated gene therapy for
muscle [Fisher et al., 1997]. To date, impressive data on biochemical, histo-
logical and functional rescue of the hamster tibialis anterior muscle has been
Hoffman/Buyse 6
published [Li et al., 1999], as well as some data on large-scale delivery to the
hamster leg after permeabilization of the vasculature [Greelish et al., 1999].
These studies have shown persistence, high level expression and lack of immune
response against the delta sarcoglycan protein, as was observed earlier with
marker transgene studies [Xiao et al., 1996]. Human clinical studies have been
approved by regulatory agencies, with injection of small foot muscles in alpha-
sarcoglycan-deficient patients through a collaborative study of the University
of Ohio in Columbus and the University of Pennsylvania in Philadelphia.
Additional clinical trials on larger muscle groups are planned by our center
with the University of Florida in Gainesville. Long-term toxicity trials are
underway in primates, to ensure that there is no late adverse effect of sarcogly-
can overexpression in muscle.
It is important to note that only a few studies have been published regard-
ing AAV transgene delivery in muscle. For example, while delta sarcoglycan
overexpression in muscle does not appear to be toxic to myofibers, it is not
unlikely that overexpression of alpha sarcoglycan or other proteins is in fact
more deleterious. It will be critical to conduct a series of studies using many
different transgenes to determine the general sensitivity of the myofiber to
overexpression of proteins delivered by AAV. It is also important to determine
whether recombinant AAV integrates into myofiber nuclear DNA, or whether
it is able to persist long-term as an extrachromosomal episome.
There are remaining hurdles before AAV vectors can be expected to
achieve therapeutic levels of gene expression in muscular dystrophy patients.
First and foremost, widescale delivery must be accomplished, including gene
delivery (AAV infection) of the heart and respiratory muscles. This will almost
certainly entail systemic delivery through the vasculature. Such delivery routes
raise problems of targeting of the virus to only myogenic cells, permeabili-
zation of the capillary bed, and production of the very large amount of virus
needed to achieve genetic rescue of sufficient muscle. If the recombinant virus
is injected into the vascular system, then it will likely infect endothelial cells,
neurons, and other cell types. It is currently not known whether overexpression
of a muscle protein such as delta sarcoglycan in nonmuscle cells will result in
dysfunction of those cells. Two approaches to circumvent this (likely) problem
include use of muscle-specific gene promoters, and development of targeting
ligands which allow AAV to infect only myogenic cells [Feero et al., 1997a].
Successful permeabilization of the capillary bed has recently been reported
[Greelish et al., 1999], however no studies of toxicity of such approaches in
dystrophic muscle have been reported. With regard to large-scale production
of recombinant AAV, the three-plasmid cotransfection method developed by
Xiao et al. [1996] has proven highly successful in producing large amounts of
recombinant virus from tissue culture cells, with no contamination of helper

virus. However, most studies to date have used virus preparations with a
relatively low proportion of infectious particles, often with only 1 in 1,000 or
less of virions able to successfully infect cells. Recently developed chromato-
graphy methods have been reported which seem able to increase the proportion
of infectious virus particles in laboratory preparations [Clark et al., 1999],
and this field promises to continue to quickly evolve.
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Hoffman/Buyse 10
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Dr. Eric P. Hoffman, Research Center for Genetic Medicine,

Childrens National Medical Center, George Washington University,
111 Michigan Ave. NW, Washington, DC 20010 (USA)
Tel. +1 202 884 6011, Fax +1 202 884 6014, E-Mail ehoffman@childrens-research.org

............................
Use of Normal and Genetically Modified
Myoblasts for the Treatment of
Myopathies
Daniel Skuk, Jacques P. Tremblay
Unite de Recherche en Genetique Humaine, Centre de Recherche de Pavillon Centre
Hospitalier de lUniversite Laval, CHUQ, et Faculte de Medecine de lUniversite
Laval, Ste-Foy, Que., Canada
The Rationale of Myoblast Transplantation
Genetic myopathies are characterized by different degrees of muscle weak-

ness determined by intrinsic muscular defects of genetic origin. Although the
underlying genetic defect of most genetic myopathies was unknown a decade
ago, efforts in molecular biology led to the identification of the genetic basis
of many diseases [1]. The first step in understanding the molecular basis of
genetic myopathies was the discovery of the molecular cause of Duchenne
muscular dystrophy (DMD). The severity and frequency of this disease made
DMD a stereotype for all severe genetic myopathies, and most of the efforts
in neuromuscular research were directed towards this disease.
The discovery of the molecular basis of a disease must be followed by
the development of its treatment. For many genetic diseases, the solution is
to replace the defective gene with a normal one, and this concept is the basis
of gene therapy [2]. Nevertheless, considering the muscle as a target for a
genetic treatment, its particularities provide other possibilities to incorporate
the affected protein into the cells. Skeletal muscles are composed of long
syncytial elements (myofibres) that cannot enter the mitotic cycle. During
adult life, precursor myogenic cells (called satellite cells) are quiescent in the
periphery of mature myofibres [3]. When a myofibre is damaged, satellite cells
proliferate and fuse to repair the damaged segment or replace it altogether
[4]. These satellite cells can be proliferated in vitro, where they are called
myoblasts. The existence of these cells has led to the idea that myoblast
transplantation (MT) into a diseased muscle could contribute to form new
and healthy fibres [5].
First Experiments in MT
As early as in 1978, Partridge et al. [5] suggested that the intramuscular

injection of muscle precursor cells could provide a treatment for recessive
inherited myopathies. However, some of the first experiments involving intra-
muscular injection of satellite cells were not performed as a therapeutic
approach but, rather, as an experimental tool for basic studies of muscle
biology [6, 7]. In 1979, Lipton and Schultz [6] implanted autologous cloned
myoblasts into muscles and demonstrated that these cells participated in the
regeneration of muscle fibres. The developmental role of myoblasts during
embryogenesis was also investigated through MT [7]. Watt [8] and Watt et al.
[9] showed, in the early 1980s, that muscle precursor cells obtained from
enzymatically disaggregated neonatal muscles fused and formed hybrid fibres
after their injection into regenerating mouse muscles. This group investigated
cyclosporin A as the first immunosuppressive treatment for MT in mouse [10].
The transplantation of mesenchymal cells from mouse embryos into dystrophic
mice was also studied as an experimental approach for the treatment of dys-
trophic muscles [11]. Thereafter, myoblasts injected into completely cryodam-
aged muscles were shown to repopulate almost 70% of these muscles [12].
Moreover, MT was tested with variable success in mouse models of different
myopathies, such as phosphorylase kinase deficiency [13], the mdx mice [14, 15]
and the dy/dy mice [16].
Early Clinical Trials
Inspired by these first experiments, several clinical MT trials were soon

conducted by various research teams [1724]. Unfortunately, the clinical trials
obtained very limited results. Functional tests showed, at best, a transient
strength increase in a few patients [18]. The presence of donor dystrophin
RNA was demonstrated in the transplanted muscles [17] as well as a significant
but small increase in dystrophin-positive fibres [18, 22, 23]. The presence of
10.3% of dystrophin-positive fibres formed by the donor myoblasts in the
transplanted muscle of a patient is currently the best histological result ob-
tained from these clinical trials [22]. Donor nuclei in some of the transplanted
patients were also confirmed by fluorescence in situ hybridization, using a
probe specific for a dystrophin exon, which was absent in the patient [25].
Use of Normal and Genetically Modified Myoblasts for the Treatment of Myopathies 13
The only group claiming to have had a modest success with their clinical
trials was the Cell Therapy Research Foundation [26, 27]. This clinic reported
slight functional improvement and the presence of dystrophin in the trans-
planted muscles of some patients. Nonetheless, the functional improvement was
not correctly evaluated since it failed to exclude a placebo effect or the beneficial
effect of immunosuppression [28]. On the other hand, it was recently demon-
strated that the dystrophin in one of these patients did not correspond to the
transplanted myoblasts but was, in fact, the product of a back-mutation [29].
Some criticisms over the clinical trials came as a result of insufficient
prior animal research [30]. Thereafter, certain research groups reexamined the
MT-related problems using experimental models.
Immunobiology of MT
The role of the immune system in MT was underestimated in some of

the clinical trials. An immune response was considered unlikely because it was
previously shown that major histocompatibility complex (MHC) molecules
were not present on normal mature myofibres [3133]. Nevertheless, MHC
was expressed by muscle fibres under certain conditions, such as inflammatory
reaction, muscle regeneration, and in DMD patients [3234]. Moreover, it was
later observed that myoblasts express MHC [3538].
Evidence of cellular rejection following allo- and xeno-MTs was observed
in unimmunosuppressed mice [3941]. Muscles injected with myoblasts were
infiltrated by CD4+ and CD8+ lymphocytes [39, 40, 42]. These lymphocytes
expressed granzyme B, an enzyme released to damage the target cells [43].
Moreover, transplantation of human myoblasts into severe combined immuno-
deficient (SCID) mice led to many myofibres that contained human dystrophin
[4446]. Human transplanted myoblasts could then form muscle fibres when
the specific immune response is avoided.
Myoblasts obtained from MHC-compatible mice, but from a strain differ-
ent from the host, were also rejected by immunocompetent hosts [47]. Rejection
due to minor antigens was also observed after syngeneic MT from males into
females, although slower than that observed following MHC-incompatible
MTs [47]. Moreover, syngeneic transplantation of normal myoblasts in mdx
mice led to specific immune responses against dystrophin, as explained later.
There is cumulating evidence that myoblasts themselves are antigen-pre-
senting cells. Following stimulation with c-interferon, a cytokine present during
the inflammatory reaction, myoblasts express MHC molecules class I and II
[36, 48, 49] and cell adhesion molecules involved in the activation of lympho-
cytes [5052]. Myoblasts can process and present the same antigens as antigen-
Skuk/Tremblay 14
presenting cells and do increase secretion of IL-2 by lymphocytes [53]. It is,
therefore, not surprising that they are rapidly rejected, following transplanta-
tion of myoblasts incompatible with major or minor antigens.
Immunosuppression in MT
Following the demonstration of the immune response after MT, attention

was focused on identifying an adequate immunosuppressive treatment. It was
observed that the degree of success of MT depended on the immunosuppressive
treatment [54]. The immunosuppressive agents used in clinical trials were
cyclosporin A [17, 22] and cyclophosphamide [21]. However, in some animal
experiments, cyclosporin A could not suppress the cellular immune reaction
after MT [44]. Cyclophosphamide-immunosuppressed animals did not exhibit
hybrid myofibres after MT, and it was proposed that this drug killed the
transplanted myoblasts by its antiproliferative properties [55]. Rapamycin was
shown to control the immune rejection after MT [56], but the best results were
obtained with FK506 [57]. Using FK506 following transplantation of normal
myoblasts expressing the gene of b-galactosidase, up to 95% of the muscle
fibres in mdx mouse muscles expressed both b-galactosidase and dystrophin
[57]. FK506 was also found very effective in controlling the immune response
after MT in non-human primates [42, 58, 59]. In monkeys, the immunosuppres-
sive benefits of FK506 were observed up to 1 year following MT [59a]. Success-
ful MTs were also obtained using a combination of monoclonal antibodies
directed against lymphocyte adhesion molecules [52].
Transplantation of Genetically Modified Autologous Myoblasts
Although used clinically for immunosuppression, the administration of

FK506 is limited by adverse effects such as nephrotoxicity [60], neurotoxicity
[61], pancreatic toxicity [62], and posttransplantation lymphoproliferative dis-
orders [63]. An alternative approach to avoid long-term immunosuppression
is to transplant the patients own genetically corrected myoblasts. For this
approach, the dystrophin gene must be introduced into the dystrophic myo-
blasts before their autologous transplantation. This has recently been done
with success in mdx mice using adenoviral vectors containing the human
dystrophin minigene [64] or the full-length dystrophin [65]. Adenoviral-medi-
ated dystrophin transfer was successfully done in human DMD myoblasts,
and these corrected myoblasts transplanted in SCID mice formed human-
dystrophin-positive muscle fibres [66]. However, the autotransplantation of
myoblasts infected with an adenoviral vector could trigger possible immune
reactions against the viral vector itself, as observed following direct gene
therapy with this vector [67, 68]. This immune reaction may eventually be
controlled by a transient immunosuppression with FK506 [68].
Although this autologous MT is presumed to have no specific immune
reactions, the introduction of dystrophin into DMD patients could be poten-
tially immunogenic. Antibodies reacting with dystrophin were observed in
some of the transplanted patients [18, 69]. There is also some evidence of
dystrophin immunogenicity after normal MT into mdx mice. Syngeneic trans-
plantation of normal myoblasts into mdx mice triggered the production of
antibodies against dystrophin [70]. These antibodies were detected 1 month
following MT, but dystrophin-positive fibres were present up to 9 months after
MT and CD4+ and CD8+ lymphocyte infiltration were not observed [47, 70].
In another study, however, the same kind of transplantation led to a slow
cellular rejection of the dystrophin-formed fibres, mediated by cytotoxic T
lymphocytes and restricted to H-2Kb [71]. Three epitopes were identified as
the principal antigenic targets of dystrophin in this study.
Another problem of autotransplanting myoblasts from the DMD patient
itself is the low proliferative capacity of these myoblasts [72]. The muscle fibres
in the DMD are submitted to recurrent cycles of degeneration-regeneration ex-
hausting the proliferative capacity of satellite cells. Some strategies to increase
the proliferative capacity of DMD myoblasts were proposed. One such strategy
is the immortalization of cells with the large tumor (T) antigen of simian virus
40 (SV40) [73]. The introduction of the SV40 large T antigen under the control of
the human vimentin promoter increased the proliferative capacity of myoblasts,
retaining their capacity to differentiate [74]. However, the success of trans-
planting myoblasts expressing the T antigen was significantly reduced compared
with that observed with control myoblasts [unpubl. data]. This was attributed
to an increased death of the T antigen-positive myoblasts following transplanta-
tion. An alternative approach to increase the proliferative capacity of cells is the
introduction of the telomerase gene [75]. In recent experiments of our research
group, the introduction of the telomerase gene in myoblasts led to better trans-
plantation success than with the introduction of the T antigen [unpubl. data].
Another strategy to circumvent the low proliferative capacity of DMD
myoblasts may be the transformation of other cell types into myoblasts. Intra-
muscular injection of dermal fibroblasts, genetically modified by introducing
MyoD1 (a master regulator gene for myogenesis), was tested but provided
limited results so far [76]. Recently, evidence has been presented suggesting
that bone marrow cells could contain myogenic progenitors [77]. The use of
these bone marrow-derived myogenic progenitors was suggested as a potential
treatment of muscular dystrophies.
Skuk/Tremblay 16
Early Survival of Transplanted Myoblasts
Most of the observations on myoblast survival after intramuscular injec-

tion demonstrated a rapid loss of these cells after transplantation. The use of
myoblasts labeled by introducing the b-galactosidase gene showed a loss of
5080% of the transplanted cells during the first 6 days that followed MT
[7882]. After labelling myoblasts with [14C]thymidine, instead of b-galactosi-
dase, 99% of cell loss was reported after 4 days [83]. These different results
may depend on the method used to evaluate cell death, since b-galactosidase
detection does not only account for cell death, because the transgene will
increase with the proliferating surviving cells. In spite of this, different results
were obtained even with the same method, suggesting that early cell death
may depend on the cell type used for transplantation [84]. Since the rapid
death of injected myoblasts was detected following autotransplantation and
transplantation in SCID and immunosuppressed mice, a specific immune reac-
tion as the reason for this early cell death is ruled out [81].
Up until now, three explanations regarding the early death of the trans-
planted myoblasts have been suggested: the conditions of cell storage before
transplantation [79], the specific survival of a subpopulation of myoblasts with
stem cell-like characteristics [83], and the damage induced by the invasion of
inflammatory cells [8082]. The last hypothesis proposed that the muscle dam-
age induced by the injections and the presence of some necrosed cells trigger
the influx of inflammatory cells that may produce a bystander damage on
the graft [80, 81]. This hypothesis was supported by the findings that an anti-
LFA-1 antibody decreased cellular mortality [80, 81], that myoblast loss is
reduced in hosts that are deficient in neutrophils or in macrophages [82], and
that myoblasts that are genetically engineered to express an inhibitor of IL-1
showed improved survival rates [84].
Complement was observed to be fixed spontaneously in vitro by human
myoblasts [85] and was suspected to be one of the reasons of the cell death.
Nevertheless, in vivo observations showed that complement was fixed only
intracellularly by necrosed cells, and the immediate cell death observed after
MT did not decrease in complement-depleted mice [86].
Diffusion of Transplanted Myoblasts
MT is also confronted with the limited capacity of myoblasts to migrate

into the muscle tissue where they are injected. In monkeys, injections of
myoblasts resuspended in a saline solution led to limited bands of hybrid
fibres in the transplanted tissue, showing that the myoblasts fused only with
the fibres located near the injection trajectories but not so far [42]. It was
reported that the migration of myoblasts was only 1.6 mm in mice [87] and
unique intramuscular injections of these cells showed that they spread on a
limited surface of 2.69.5105 lm2 [88]. The only current possibility for a
successful MT in large muscles, therefore, is to perform multiple injections
very close to each other [59a].
Some authors have shown that myoblasts can migrate under some experi-
mental conditions. Myoblasts were capable of migrating into fatty connective
tissue implanted into mouse muscles [89]. Myoblast injections into irradiated
muscles of mdx mice showed the presence of donor dystrophin-positive myo-
fibres in the adjacent muscle at the later stages (250 days) following MT [90].
Myoblasts from neighbouring normal muscles were also capable of invading
and repopulating freeze-killed muscles [12, 91].
Cells with the capacity to migrate into different tissues, such as leucocytes
and tumour cells, secrete metalloproteinases, a group of enzymes that degrade
the extracellular matrix [92]. The migration of myoblasts may be improved by
increasing the secretion of metalloproteinases. Incubation of myoblasts with
a high dose of basic fibroblast growth factor, a factor that can trigger the
secretion of metalloproteinases, increased by 4-fold the success of MT [93].
The addition of concanavalin A to the myoblast culture increased by more
than 3-fold the dispersion of these cells in a muscle [88]. This effect was
attributed to the secretion of metalloproteinases by fibroblasts in the primary
culture.
To avoid multiple cell injections into the muscles, intra-arterial administra-
tion of myoblasts could be used [94]. Although this technique could have the
potential advantage of reaching important muscles that are relatively inacces-
sible to injection, such as the diaphragm, few hybrid fibres were detected in
previously damaged muscles only.
MT in Large Animals
MT must prove to be effective under conditions approaching the clinical

situation and not only in the small muscle of a mouse. Therefore, studies of
MT in large animals are of crucial importance.
Muscular-dystrophic dogs represent a good model for the study of MT
due to an absence of dystrophin [95]. However, only one article exists on MT
in dystrophic dogs, where the absence of positive results can be attributed to
the lack of immunosuppression [96]. Better results of MT were obtained in
normal dogs treated with FK506 at low doses combined with cyclosporin A
and mycophenolate mofetil [97].
Skuk/Tremblay 18
Monkeys are anatomically and immunologically closer to humans, and
it can be expected that the results obtained with this model could be extrapo-
lated to patients. Experiments in monkeys demonstrated that the specific im-
mune response is developed against the transplanted myoblasts and against
the myofibres created by their fusion, and that this rejection can be controlled
by FK506 [42, 58, 59]. Recently, our group demonstrated that it is possible
to achieve successful MTs in the whole biceps of monkeys [59a]. Up to 75%
of hybrid fibres were obtained in the transplanted muscles 1 month after MT,
and the hybrid fibres were present in the transplanted muscles up to 1 year
after MT. Considering the size of a monkey biceps, and the immunological
similitude with humans, these experiments provide some parameters that could
make this technique applicable to humans.
Could MT Improve Patients?
The effectiveness of MT must not only be demonstrated by the presence of

the reporter gene in many muscle fibres, but by its capacity to stop histological
degeneration and progressive strength loss in the disease muscles as well. There
is already some evidence that supports potential beneficial effects of MT. In
mdx mice, the lack of dystrophin makes muscle fibres more vulnerable to
damage resulting from eccentric exercise [98, 99]. When MT was performed
1 month before submitting the mdx mice to eccentric exercise, myofibre damage
was only observed in dystrophin-negative fibres [100]. These results suggest
that MT protected the muscle tissue of mdx mice from mechanical stress, the
mechanism considered responsible for triggering myofibre necrosis in DMD.
It was also observed that the number of dystrophin-positive fibres remained
unchanged, 35250 days following MT, while the number of dystrophin-nega-
tive fibres progressively decreased [90]. This was attributed to the protective
role of the donor dystrophin.
Most of MT experiments demonstrated that myoblasts fuse with previ-
ously existing myofibres. Nevertheless, if the transplanted myoblasts could not
regenerate new fibres when the muscles are replaced by fibrosis and fat tissue,
MT would be a potential treatment only during the first years of DMD
patients. Can MT restore the muscular tissue and strength of patients with
advanced disease? Although few studies on this aspect of MT exist, preliminary
results are encouraging. It was demonstrated that myoblasts could invade the
fatty connective tissue to form myotubes and new muscle fibres [89]. Moreover,
myoblasts could form ectopic and functional muscles without the need of a
previous tissular support when they were implanted under the skin [101]. MT
was shown to restore both muscle mass and force following irreversible muscle
damage [102, 103], as well as increase twitch and tetanic tension in normal
regenerating muscles [104].
Some evidence suggests that some transplanted myoblasts survive as
muscle precursor cells, which can participate in subsequent muscle regeneration
[105, 106]. This is important because it means that these cells can provide a
permanent source of normal cells to replace the damaged muscle fibres in the
dystrophic patients.
MT in Other Myopathies
Although most of the attention on MT was given to the treatment of

DMD, other genetic myopathies could benefit from this technique as well.
Congenital Dystrophy. Merosin is a component of the skeletal muscle basal
lamina and its absence determines a type of usually severe congenital dystrophy
[107]. An animal model for this disease is the dy/dy mouse, which shows an
absence of merosin and a severe and progressive dystrophy [108]. Transplanta-
tion of normal myoblasts into dy/dy mice was shown to restore merosin
expression in the muscle fibres [109].
Metabolic Myopathies. Hybrid fibres formed in vitro by the fusion of
normal myoblasts with those from a glucose-6-phosphate dehydrogenase defi-
cient mouse contained high levels of this enzyme, suggesting that MT could
restore normal enzymatic activity in metabolic myopathies [110]. Similar in
vitro experiments suggested that MT could restore acid a-glucosidase in the
type II glycogen storage disease [111]. MT was also tested in phosphorylase
kinase-deficient mice, but results were limited [13].
Conclusion
MT remains the only treatment that could eventually not only introduce
the dystrophin gene into the already existing muscle fibres but increase their
size and perhaps their number as well, thereby increasing the strength of
myopathic patients. Although initial trials have not produced clinically ac-
ceptable results, ongoing research during of the last decade provided a
better understanding of the problems associated with MT. This cumula-
tive knowledge will permit a more effective MT, applicable to myopathic
patients. Research is still being pursued on many potential further im-
provements of MT, such as avoiding sustained immunosuppression and
improving the migration as well as the survival of myoblasts in the host
muscle.
Skuk/Tremblay 20
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Dr. Jacques P. Tremblay, Unite Recherche en Genetique Humaine, Rc 9300,

CHUQ-Pavillon CHUL, 2705 Boulevard Laurier, Ste-Foy, Que. G1V 4G2 (Canada)
E-Mail Jacques-P.Tremblay@crchul.ulaval.ca
............................
Disorders of the Sarcoglycan Complex
(Sarcoglycanopathies)
Carsten G. Bonnemann
Department of Neuropediatrics, University Childrens Hospital, Gottingen, Germany
Limb-Girdle Muscular Dystrophies
After years of inconsistent use, the diagnostic category of limb-girdle

muscular dystrophy (LGMD) more recently has undergone a reevaluation [1],
not least because of the great progress that has been made elucidating the
genetic basis for a number of these muscular dystrophies. The definition of
autosomal dominant and autosomal recessive types (also referred to as LGMD
type 1 and type 2, respectively, see table 1) [2, 3] demonstrated the existence
of genetically distinct entities within this group.The term LGMD thus is now
frequently used for a genetically as well as clinically heterogeneous group of
muscular dystrophies that are characterized by progressive weakness starting
in the muscles of the pelvic or shoulder girdles, largely sparing the face and
the extraocular muscles [4]. With the identification of a number of the responsi-
ble genes, clinical differences between as well as variability within the different
entities can now be worked out more clearly [3, 5]. In the genetically based
nomenclature [2], LGMD type 1 refers to autosomal dominant conditions
and LGMD type 2 to autosomal recessive entities (see table 1). The four
sarcoglycan (SG)-deficient muscular dystrophies (sarcoglycanopathies,
SGpathies, LGMD 2D, 2E, 2C, 2F) form a distinct subgroup within the
autosomal recessive LGMD and are the subject of this review.
Dystrophin-Associated Proteins
The SG are part of the larger group of dystrophin-associated proteins

(DAPs). The DAPs were defined essentially by virtue of their copurification
Table 1. Gene locus-based classification of LGMD
Designation Genetic Gene Protein Number

locus symbol product of exons
Autosomal dominant
LGMD 1A 5q31 MYOT myotilin 10
LGMD 1B 1q11-21 LMNA lamin A/C 12
LGMD 1C 3p25 CAV3 caveolin-3 2
LGMD 1D 6q23 ?
LGMD 1E 7q ?
Autosomal recessive
LGMD 2A 15q15 CAPN3 calpain-3 24
LGMD 2B 2p13 DYSF dysferlin D55
LGMD 2C 13q12 SGCG c-SG 8
LGMD 2D 17q21 SGCA a-SG 10
LGMD 2E 4q12 SGCB b-SG 6
LGMD 2F 5q33-34 SGCD d-SG 8
LGMD 2G 17q11-12 TCAP telethonin 2
LGMD 2H 9q31-33 ?
LGMD 2I 19q13.3 ?
along with dystrophin in digitonin-solublized skeletal muscle membrane prep-

arations using wheat germ agglutinin and anion exchange (DEAE) chromatog-
raphy [68]. Dystrophin is a large monomeric rod-like subsarcolemmal
molecule that binds cytoskeletal F-actin via its N-terminus and its rod domain
[reviewed in 9, 10]. The subdomains of dystrophins C-terminus are directly
or indirectly involved in binding a number of the DAPs [reviewed in 11]. It
has become clear that the DAPs can be subdivided into different subcomplexes
based on biochemical characteristics, binding and association to dystrophin
and the other DAPs, as well as orientation with respect to the sarcolemma
(fig. 1). A purely intracellular group of DAPs consists of the syntrophins and
the dystrobrevins (the dystrobrevins are DAPs that at the same time are
homologous to dystrophins C-terminus [12]). These molecules occur in diverse
isoforms and form variable complexes attached to dystrophins C-terminus.
This subcomplex may potentially be involved in organizing ion channels and
receptors within the membrane by binding cytoplasmic domains [reviewed
in 13]. Another transmembrane complex besides the SG complex is dystrogly-
can, composed of a- and b-dystroglycan, both of which are encoded by the same
gene on chromosome 3p21 and are generated by posttranslational processing of
a single precursor protein [reviewed in 14]. b-Dystroglycan binds dystrophins
Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 27

Fig. 1. Schematic representation of the DAPs. Only the cell-binding globular domain
of the laminin trimer is shown. Disorders resulting from mutations in the various components
are indicated by the arrows. AR>Autosomal recessive; AD>autosomal dominant;
CMD>congenital muscular dystrophy.
C-terminus at the cysteine-rich domain, spans the membrane and extracellu-

larly binds to a-dystroglycan. One of the extracellular binding partners of
a-dystroglycan is the a2 chain of the heterotrimer laminin-2, also known as
merosin [reviewed in 15]. A link from dystrophin via dystroglycan to the
basement membrane component laminin-2 can thus be postulated. This link
may serve purely mechanical functions of force transduction but may also be
involved in transmembrane signalling and basement membrane organization
[reviewed in 15]. A 25-kD component that was known to copurify with the
DAPs has only recently been characterized [16]. It is predicted that the protein
includes four putative transmembrane domains and was found to be identical
to the human KRAG gene (Kirsten ras-associated gene) on chromosome
12p11.2 [16]. Because of its structural relation to the tetraspanin family of
molecules it was renamed sarcospan. Sarcospan is transcribed in many tissues,
including muscle and heart. It is particularly closely associated with the SG
complex [17] so that the protein is lost from the membrane together with the
SG complex in patients and animal models of SGpathies.
Bonnemann 28
Sarcoglycan Complex
SG was established as an independent transmembrane complex amongst
the DAPs by demonstrating its separation from dystrophin and the other
DAPs using the detergent n-octyl-glucoside [18]. Its currently known members
in skeletal muscle include a-, b-, c- and d-SG. The genes for a-, b- and c-SG
were identified on the basis of partial peptide sequences [1922], whereas the
d-SG cDNA was identified as an EST on the basis of homology to c-SG [23].
It was then formally shown to be a member of the SG complex in skeletal
muscle by biochemical methods [24, 25]. Another SG protein identified elec-
tronically by homology this time to a-SG is e-SG, encoded on chromosome
7q21 [26, 27]. Whereas a- and c-SG are largely restricted in their expression
to striated muscle [19, 22, 28], d-SG is found in addition in smooth muscle
[23]. b-SG is transcribed at highest levels in skeletal muscle, but also in smooth
muscle and in a number of other tissues [20, 21]. e-SG is found rather more
ubiquitously [26, 27]. Although e-SG is not highly expressed in striated muscle
and does not appear to be a regular member of the SG complex in skeletal
or cardiac muscle, in smooth muscle there appears to be a version of the SG
complex that contains b-, d- and e-SG [29].
The four SG proteins in striated muscle are single membrane spanning
molecules that are glycosylated on the extracellular side [30, 31]. a-SG, encoded
on chromosome 17q21, is a type I transmembrane molecule (the protein is
preceded by a signal peptide so that its C-terminus is within the cell) with a
molecular weight of 50 kD [19, 32]. The other three molecules are type II
transmembrane molecules (there is no preceding signal peptide, so that the
N-terminus is within the cell). b-SG, encoded on 4q12, has a molecular weight
of 43 kD [20, 21], c-SG (13q12) of 35 kD [22], and d-SG (5q33) also of 35
kD [23]. The extracellular domain of all SG is rich in b-pleated sheets inter-
rupted by a-helices [30, 31]. Although there is no direct sequence homology
between b-SG on the one hand and c- and d-SG on the other, in all three
molecules there is a similar arrangement of four spaced cysteine residues close
to the (extracellular) C-terminus resembling a partial epidermal growth factor
(EGF) module [33].
The biochemical structure of the complex is beginning to be elucidated
and cross-linking as well as coimmunoprecipitation experiments suggest that
b- and d-SG in particular are closely associated, along with c-SG. a-SG on
the other hand may be situated more separately within the complex [18, 25, 34].
However, there is close interdependency of the SGs, since the entire complex
is usually severely diminished in case of mutations of only one of its members
(see below). In cell culture systems, assembly of the SG complex in the sarco-
lemma is also dependent on the simultaneous presence of all of its members
[35]. Mutations introduced in one of the SGs do not disturb translation and

glycosylation of the remaining SG molecules in the ER, but the complex fails
to assemble in its proper position in the membrane [35].
It is still unclear how exactly the SG complex is associated with dystrophin
or dystroglycan and what its function may be. There is evidence that a-dystro-
glycan in particular is reduced in cases of SG deficiency in animal models
[3638] as well as in patients [39], suggesting perhaps a direct or indirect link
(e.g. mediated by an additional protein) between SG and dystroglycan. Other
evidence indicates reciprocal influence between the SG complex and the inte-
grin system [40]. There appears to be close physical proximity of the SG-
dystrophin complex with integrin a5b1 and its associated proteins [40]. Differ-
ent work suggested that a-SG may function as an ectoATPase, based on the
fact that it binds ATP and has ATPase activity, thus potentially regulating
the activity of purinergic receptors [41]. However, a coherent picture of SG
function has yet to emerge. Obviously, any theory of SG function has to
explain the severe muscular dystrophy resulting from its absence as well as
the substantial loss of membrane integrity that is evident from analysis of
animal models of sarcoglycanopathies [37, 38, 42, 43]. In these animals there
is considerable in vivo uptake of the albumin-bound tracer Evans blue into
affected muscle with an age-dependent onset [38]. Recent evidence from the
c-SG-deficient mouse model suggests that muscle degeneration occurs indepen-
dently from mechanical stress [44], thus directing attention away from a purely
mechanical function of the complex towards a a role in signalling, possibly
promoting cell survival.
Sarcoglycanopathies: Clinical Features
Deficiency of SG proteins was demonstrated in dystrophin-positive

autosomal recessive muscular dystrophy even before the identification of the
first SG mutations [45, 46]. Primary a-SGpathy was first confirmed in a
large family from France with evidence of genetic linkage to the a-SG gene
locus on chromosome 17 [47]. b-SGpathy was independently identified in a
genetic isolate of the Indiana Amish where it coexists with LGMD 2A
(calpainopathy) [21] as well as by screening sporadic cases of muscular
dystrophy for b-SG mutations [20]. c-SG was first found to be mutated in
consanguineous families with autosomal recessive muscular dystrophy from
northern Africa [22, 48] with evidence for genetic linkage to the c-SG gene
locus on chromosome 13 [49], but the disease is now recognized worldwide.
d-SGpathy was discovered by independently mapping the d-SG gene and a
form of autosomal recessive muscular dystrophy to the same locus on chromo-
some 5q33-34 [23, 50, 51].
Bonnemann 30
Population studies of the SGpathies are difficult to compare and will not
be listed in detail, however, certain common trends seem to emerge. The
proportion of SGpathies as a group within a population of LGMD patients
clearly depends on the age group analyzed as well as the severity of the
phenotype. In one large series, the proportion of SGpathies among autosomal
recessive and sporadic LGMD patients of all ages was 25% [52]. In the well-
studied population in Brazil the proportion of SGpathies in the entire group
of LGMD patients was 20%; however, calculated for just the severe cases it
was as high as 68%, whereas for the milder cases the proportion was only
8.5% [53]. Comparable numbers were found in one region in Italy, where the
proportion of SGpathy among severe childhood LGMD presentations was
54.5% as opposed to 17.5% for the milder group [54]. Similar relationships in
principle were also found in the Turkish childhood LGMD population [55]
and in an American series [56]. Comparing the relative proportion of the
different SG gene mutations in various populations in which there is no strong
founder effect for one single mutation, a-SG seems to be the most common
SG gene mutated (4050% of SG mutations), followed by both b- and c-SG
[52, 54, 56]. Although d-SGpathy seems to be more common in Brazil [53],
it is probably the least common form in other populations [57].
From the analysis of patients with SGpathies identified so far, no discrim-
inating clinical differences between the four disorders have emerged as of yet.
Therefore, their clinical features will be discussed jointly, emphasizing possible
differences. Manifestation in childhood is the most common presentation with
an age at onset of around 68 years on the average, but weakness may start
earlier or considerably later, including adulthood [3]. Early motor milestones
tend to be normal in the majority of cases but may be delayed with abnormal
ambulation or toe walking from the beginning. The presenting complaints/
features in addition include waddling gait, difficulties with running or getting
up from the floor, but also excercise intolerance, potentially muscle cramps
or pain (pseudometabolic presentation [58]). The initial muscle weakness
usually presents in the pelvic girdle followed by the muscles of the shoulder
girdle. The clinical pattern of muscle involvement is reminiscent of Duchenne
and Becker muscular dystrophies (DMD/BMD) with early involvement of
glutei and hip adductors [58, 59], although involvement of the scapular fixators
and the deltoid muscle may be more significant in the SGpathies compared
to the dystrophinopathies, leading to early scapular winging and loss of arm
elevation. Quadriceps and hamstrings may be equally involved. There may
also be early Achilles tendon contractures and lordosis. Calf hypertrophy
usually is present at some point, hypertrophy of other muscles as well as
sometimes macroglossia can occur. The weakness is progressive and often
rapidly so. In childhood onset cases there may be loss of ambulation at around

1216 years of age, although wheelchair confinement before the age of 10 has
been seen [51, 58, 60]. While later onset and milder progression are also
possible in b- [21, 39, 52] and c-SGpathy [61, 62], the highest proportion of
milder cases is found in of a-SGpathy [56, 58, 59, 63], including a case of an
almost asymptomatic adult with high serum creatine kinase (CK) levels and
some lordosis only [64]. There have been very few cases of genetically confirmed
d-SGpathy so far; the ones reported have been severe [51, 65]. However, for
a-SGpathy [58, 64], for b-SGpathy [21] as well as in particular for c-SGpathy
[48, 61], there can be significant intra- and interfamilial variability that makes
predictions of prognosis in an individual case difficult, even if there had been
an affected sib in the same family. CK levels are significantly elevated, especially
early in the course. A significant difference to the dystrophinopathy spectrum
is the lack of intellectual involvement in the SGpathies. Although in the
majority of cases there is no overt cardiomyopathy, a smaller number of patients
may present with clinically significant dilative cardiomyopathy, sometimes
even requiring cardiac transplantation [66]. Evidence for subclinical cardiac
involvement by ECG and ECHO criteria may be seen more often [6769]
but less prominently so than in DMD/BMD. Significant cardiomyopathy can
definitely occur in b-SGpathy [70]. Although reported patients with confirmed
d-SGpathy have not had cardiomyopathy, there are patients with very probable
d-SGpathy and significant cardiomyopathy [71; Voit, pers. commun.]. There
have been no reported cases of a- or c-SGpathy with clinically overt cardiomy-
opathy so far, but the number may still be too small to reach definite conclu-
sions (see discussion under Animal Models). In the later course of SGpathy
there is also the possibility of progressive respiratory compromise concomitant
with the development of progressive scoliosis.
Diagnosis, Protein Expression Patterns and Mutational Spectrum
The diagnosis of a SGpathy rests on history and clinical examination,

analysis of protein expression patterns in the muscle biopsy and ultimately on
demonstrating a causative gene mutation in one of the SG genes. In informative
families, genetic linkage analysis may help to include, and perhaps more impor-
tantly, to exclude known genetic loci from diagnostic considerations. More
frequently encountered though is the sporadic patient with a proximal muscu-
lar dystrophy of unknown cause. Analysis of the muscle biopsy will frequently
be the next step in the evaluation of such a patient, in particular when there
is no genetic evidence for a dystrophinopathy based on the PCR deletion
screening [72]. A characteristic feature of the disorders of the SG complex is
the disintegration of the entire complex when mutations in one of the four
Bonnemann 32
proteins occur [30, 31]. Thus, the primary mutated gene may not be obvious
from analyzing the biopsy by immunohistochemistry or by Western blot.
However, on utilizing all four SG antibodies certain helpful protein deficiency
patterns may become evident. In a-SGpathy and c-SGpathy the protein most
reduced or absent is the mutated one, while the other components may show
significant preservation at the membrane, in particular d-SG in cases of
c-SGpathy [53, 55, 73, 74]. On the other hand, in b- and also d-SGpathy in most
cases there is complete absence of the entire complex or at least considerable
reduction of all SG proteins [20, 21, 39, 53, 60, 73], although in d-SGpathy
there may be some partial preservation of c-SG [75]. Only in a-SGpathy is
there some correlation between the residual immunoreactivity for the mutated
protein and the clinical severity [58, 59]; the same correlation does not hold
true for the other SG. In c-SGpathy for instance, complete absence of c-SG
immunoreactivity does not automatically imply a severe phenotype as this
can be seen in severe as well as in milder cases [61].
In some patients there may be some decrease in dystrophin immunoreactiv-
ity as well, sometimes presenting with an immunohistochemical picture remin-
iscent of BMD [67, 73, 75]. Nonetheless, according to Western blot analysis
the dystrophin molecule is of normal molecular weight, and the residual
amount of dystrophin in a SGpathy is likely to be more than 2030%, although
it may be as low as 10% of normal [75]. Vice versa, there can also be a
substantial reduction of the SG complex along with dystroglycan and the other
DAPs and sarcospan in the dystrophinopathies [76]. It has been speculated that
this loss of DAPs is in fact responsible for the development of muscular
dystrophy [77, 78].
Mutations: a-SG
Mutational analysis in the a-SG gene has revealed a predominance of
missense mutations over truncating mutations [54, 79, 80]. Genotype/pheno-
type correlations are quite difficult to establish, in particular because of the
high number of compound heterozygotes [80]. However, null mutations in
a-SG consistently appear to lead to a severe phenotype, whereas there is much
more clincical variability associated with the missense mutations [58, 80]. The
misssense mutations described so far are located exclusively in the extracellular
domain. One third of mutated chromosomes in the largest series from Europe
carried the single most prevalent missense mutation, Arg77Cys [80]. This
mutation in a CpG dinucleotide occurs on different genetic backgrounds and
must, therefore, have arisen indepedently a number of times. The phenotype
associated with this mutation again is quite variable, ranging from intermediate
to mild in one study, the severity being correlated with the residual a-SG
staining in the biopsy [58, 59]. Another relatively frequent mutation in the

large European series was Arg284Cys (12% of mutated chromosomes), which
in the homozygous state was always associated with a mild clinical presenta-
tion [59, 80]. This mild phenotype probably is the only consistent genotype/
phenotype correlation for a-SG missense mutations. In this largest study so
far, there was only one clinically severe homozygous a-SG missense mutation,
Arg34Cys [80].
Mutations: b-SG
In b-SG again there is a mixture of missense as well as truncating
mutations. All the truncating mutations were associated with a severe pheno-
type [56, 59, 60]. In contrast to a-SG, in b-SG there is a relatively high
proportion of missense mutations associated with a phenotype as severe as
found for the truncating mutations [60]. Thus, overall the proportion of severe
cases associated with missense mutations is considerably higher in b-SGpathy
than in a-SGpathy. Most b-SG mutations are in the extracellular domain
with the notable exception of Gln11Glu, located at the very N-terminus [56].
There is a certain cluster of b-SG mutations in a region immediately adjacent
to the transmembrane domain, encoded on exon 3, most, but not all of
which [39] are associated with a severe phenotype [60, 81]. The missense
mutations that are associated with comparatively milder phenotypes and
much more variability of presentation and progression tend to be located
further away from that region [21, 52]. This data may imply, but does not
prove, that the region immediately adjacent to the transmembrane domain
in b-SG could be crucial for b-SG function within the complex, maybe
mediating some of the interactions within the complex. Thr151Arg is the
mutation that was identified in the original Indiana Amish pedgrees [21],
although there appears to be a second mild b-SG mutation segregating within
that population (Arg91Cys) [39].
Mutations: c-SG
In c-SG most of the mutations identified so far have been truncating
mutations, frequently caused by deletions [22, 33, 55, 56, 59, 61]. The deletions
may also be larger, potentially encompassing most of the gene locus [97,
Bonnemann and Kunkel, unpubl. obs.]. Two recurrent mutations in the c-SG
gene are important: A deletion of a single T residue within exon 6 (del521T),
causing a frameshift and truncation of the protein, was originally described
in the North African population [22] and subsequently in a number of patients
originating from the Mediterranean region [61]. The mutation is associated
with a rare 122-bp allele of the intragenic dinucleotide repeat marker (D13S232)
suggesting a founder effect of the mutation [82]. Although most patients with
this mutation are rather severly affected, some show much milder disease [61].
Bonnemann 34
Since this is a truncating mutation, for c-SG the dictum that null mutations
always are associated with a severe phenotype does not appear to be true.
Thus, there probably are modifiers of severity that still remain to be identified.
Another recurrent and relevant mutation is the missense mutation Cys283Tyr,
found almost exclusively among Romano Gypsy patients, based on a founder
effect [83, 84]. The missense mutation alters one of the terminal cysteine
residues that form a partial EGF module. The consistently severe phenotype
associated with this mutation [83] probably reflects the functional importance
of the motif that could be involved in protein-protein interactions, although
a binding partner has not been identified yet. Other missense mutations in
the c-SG gene have been rare, and only one was in a homozygous state that
would allow for genotype/phenotype correlations (Leu193Ser), in this case
causing a milder phenotype [62].
Mutations: d-SG
In d-SG only very few mutations have been identified so far, all of them
causing a severe phenotype of early childhood onset and rapid progression
to wheelchair confinement [51, 57]. This includes the only missense mutation
known in the gene (Glu262Lys) [65]. In this tendency towards a more severe
phenotype, d-SGpathy may be similar to b-SGpathy, even though the protein
structure as well as the gene structure resemble more c-SG.
Animal Models
The first known animal model of an SGpathy is the cardiomyopathic

Syrian hamster which also has muscular dystrophy [85]. Originating from the
original strain BIO 1.50, animals with predominantly hypertrophic (BIO 14.6
and UMX 7.1) as well as those with predominantly dilated cardiomyopathy
(TO-2) have been bred [86]. The animals were found to lack expression of the
entire SG complex [36, 87]. Subsequently a large 29.8-kb deletion in the 5
region upstream of exon 2 in the d-SG gene was found to cause the disease
in all the different strains [8890]. Since the deletion was identical in the
different strains with dilated versus hypertrophic cardiomyopathy, the genetic
background plays a role in determining the exact phenotype of the cardiomyo-
pathy.
In addition to this spontaneous animal model, mice strains with null
mutations in each of four SGs have now been generated by gene targeting
techniques [37, 42, 43, 91]. All mice develop significant muscular dystrophy
that becomes evident histologically at an early age. Whereas the a-SG mice
were clinically quite normal [37], the c-SG-deficient mice had clinically evident

muscular dystrophy with abnormal gait, motor difficulties and stunted growth
[42]. From the reports published it is not clear how severe the muscular
dystrophy was clinically in the b- and d-SG-deficient animals [43, 91]. All mice
showed active necrosis and regeneration of skeletal muscle, as well as uptake
of the dye Evans blue into muscle, indicating sarcolemmal disruption [37, 38,
42, 43, 91]. In addition, apoptotic cell death was observed in the c-SG-deficient
animals [42]. In each of the animals there was evidence of disruption of the
SG complex with severe reduction of the other three SGs. In the null mutants
in which sarcospan expression was analyzed [a-, b-, d-SG-deficient mice;
37, 43, 91], there was concomitant severe reduction of this molecule as well,
confirming its close association with the SG complex. a-Dystroglycan was
analyzed in the a-SG- and the b-SG-deficient mice and appeared to be more
loosely associated with the sarcolemma in membrane preparations from muscle
lysates [37, 43]. These results indicate that the close membrane association of
a-dystroglycan may at least in part be dependent on the SG complex.
Similar to the d-SG-deficient hamster, the d-SG-deficient mice also develop
significant cardiomyopathy [91]. Loss of d-SG-disrupts the smooth muscle SG
complex composed of at least b-SG, d-SG and e-SG [29]. In the d-SG deficient
mice, the lumen of the coronary arteries was irregular with a cardiac pathology
that suggested ischemic lesions. The authors in that study thus propose that
the cardiomyopathy develops on the basis of vascular dysfunction caused by
the loss of the smooth muscle SG complex [91]. Part of the resulting cardiac
pathology in mice after exercise could be reversed by pretreating the mice with
the vasodilator nicorandil, opening important therapeutic considerations in
these muscular dystrophies. In view of the vascular hypothesis of cardiomyo-
pathy it is interesting to note that a reduced coronary reserve was reported
in patients with SGpathy [69] and that the likelihood of significant cardiomyo-
pathy is probably highest in patients with either b- or d-SGpathy. The b-SG-
deficient mice should also have lost the smooth muscle SG complex although
this was not directly shown. With increasing age these mice show larger fibrotic
patches in the heart, even though there does not appear to be a clinical correlate
such as increased mortality [43]. The a-SG-deficient mice, on the other hand, do
not develop cardiomyopathy, even though the entire SG complex is significantly
reduced in cardiac muscle [37]. Since a-SG is not part of the smooth muscle
SG complex this finding would be consistent with a vascular pathogenesis of
SG-deficient cardiomyopathy. However, one strain of c-SG-deficient mouse
also develops significant hypertrophic cardiomyopathy of the left as well as
the right ventricle contributing to significant early mortality in these animals
[42], even though c-SG is also not expressed in smooth muscle [29]. Thus, the
pathophysiology of cardiomyopathy in SGpathies may be multifactorial and
heterogeneous.
Bonnemann 36
Treatment
There are a few observations in the literature suggesting that in the

SGpathies there may be a response to corticosteroid treatment that is reminis-
cent of the response seen in DMD with a sustained benefit over a number of
years [64, 92, 93]. However, there are no controlled studies yet and no data
on long-term outcome. In any case, the similarity of the response is intriguing
also from a physiological point of view, since dystrophinopathies and SGpa-
thies may have certain aspects of pathophysiology in common [78].
Gene transfer has been accomplished in the SG-deficient cardiomyopathic
hamster [9496] as well as in the a-SG-deficient mouse [37], facilitated by the
small size of SG cDNAs. In all attempts, the introduction of the cDNA for
the mutated SG resulted in a restoration of the entire SG complex and led to
a restoration of sarcolemmal integrity, as assayed by Evans blue [94] or Procion
orange dye assays [95]. Even marked overexpression and intracellular accumu-
lation of the excess-induced protein did not influence this result at the mem-
brane [95, 96]. The transfer was performed by intramuscular recombinant
adenovirus injection [37, 94], or using recombinant adenovirus-associated virus
[96], in one study as an intra-arterial injection after pretreatment with papaver-
ine and histamine to open the endothelial barrier of the vessels [95].
From these studies on SG gene transfer, there is good reason to surmise
that successful gene transfer will result in the functional recovery of the
treated muscle. However, while these results are encouraging with respect to
human gene therapy in the SGpathies, the formidable challenge of safe delivery
to all skeletal muscles (and potentially the heart including coronary arteries),
long-term expression of the introduced gene, the degree of fibrosis, and
immunotolerance remain to be addressed as prerequisites for such a treatment
to become a success. Meanwhile, we currently have the means for a proper
and precise genetic diagnosis of these disorders, certainly a sine qua non
for any gene-based therapeutic approach to the patient with a SG-deficient
muscular dystrophy.
Note Added in Proof
A muscle-specific protein, filamin 2, has recently been identified as an

intracellular interactor of the SG complex [98]. Another b-SG knock-out
mouse model notably showed similar vascular anomalies as described for the
d-SG knock-out mice before [99]. This and another paper also show that there
appears to be a separate SG complex in striated muscle in which e-SG replaces
a-SG [100].

Acknowledgements
I would like to express my gratitude to Dr. Eijiro Ozawa, Tokyo, for his encouragement
to write this review and to Dr. Volker Straub, Essen, for critically reviewing the text and for
helpful discussions. The support of the Deutsche Gesellschaft fur Muskelkranke e.V. (DGM)
is gratefully acknowledged.
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Carsten G. Bonnemann, Department of Neuropediatrics, University Childrens Hospital,

Robert-Koch-Strasse 40, D37075 Gottingen (Germany)
Fax +49 551 39 6252, E-Mail cbonne@med.uni-goettingen.de

............................
Facioscapulohumeral
Muscular Dystrophy:
Diagnostic and Molecular Aspects
Peter W. Lunt
Clinical Genetics Unit, Bristol Royal Hospital for Sick Children, Bristol, UK
Facioscapulohumeral muscular dystrophy (FSHD) has a prevalence esti-

mated at between 2.55.0/100,000, and a birth incidence of heterozygote gene
carriers around 1/20,000 [1, 2]. The genetic defect follows autosomal dominant
inheritance, but with new mutation accounting for around 10% of recognised
cases [3]. A typical symptomatic presentation is in teenage (the 2nd decade
of life), with shoulder girdle weakness and associated muscle wasting, with
examination at that time revealing characteristic peri-oral and/or peri-orbital
facial weakness, which in retrospect has usually preceded the shoulder girdle
symptoms. The condition is progressive, with proximal lower limb involvement
sufficient to require a wheelchair developing in 1020% of cases [2]. However,
the range of age and severity of presentation between families, and also to a
considerable degree within families, is extremely wide, varying from minimal
signs of asymptomatic scapular girdle or facial weakness in the 7th decade,
to cases with severe progressive proximal and distal upper and lower limb
involvement together with Mobius-like expressionless facies by early child-
hood [4]. The molecular basis underlying this variability is now beginning to
be understood, and facilitating some degree of prediction within a family.
FSHD is caused by a so far unique mutation mechanism, and is perhaps
also unique amongst genetic conditions in being one where the DNA mutation
is known, but where there is yet no knowledge of the gene (or genes) affected
by this. Thus, the genetic defect causing FSHD is deletion of an integral
number of 3.3-kb repeat units located at subtelomeric chromosome region
4q35, reducing the normal 1096 tandem repeat copies (40- to 320-kb frag-
ment) down to a residual 110 copies (10- to 40-kb fragment) [5, 6]. There is
a closely homologous 3.3-kb repeat array located at the subtelomeric chromo-
some region 10q26, and some evidence for chromosomal translocation (or
sequence conversion) between these two regions, possibly predisposing to the
DNA rearrangement causing FSHD [7]. Diagnostic molecular testing can be
offered through this uniform mutation mechanism, both within families for
prediction, and for single representative cases where confirmation of diagnosis
is sought. This is made possible with a high specificity where there is a positive
molecular result [3]. The sensitivity of testing has still to be improved, and
exclusion of diagnosis in a single representative case with a neuromuscular
presentation is still not possible from a molecular genetic laboratory result
alone.
It is found that the age at onset and severity of clinical presentation
correlates broadly and inversely with the size of the residual DNA fragment
at 4q35, and, by inference, therefore correlates directly with the number of
repeat units deleted [810]. Thus, the smallest residual fragment lengths at
1017 kb (13 repeat copies) are usually associated with a severe infantile or
childhood presentation [4, 11], medium lengths (1830 kb, or 47 repeat copies)
are often found in the largest recognised dominant families, while the largest
lengths (3138 kb, or 810 repeat copies) have been associated with a milder
predominantly scapulohumeral presentation, and may well have reduced
penetrance, particularly in females [8, 12, 13]. New mutation cases are seen
predominantly with the smallest residual fragment lengths, giving matching
clinical severity, and may originate predominantly on the maternal copy of
chromosome 4 [6, 13]. Study of parental DNA suggests that around 2030%
of new mutations occur as somatic and germline events in one of the parents,
this usually also being the mother [3, 13, 14, 43].
The favoured hypothesis for the molecular pathogenesis is for the deletion
of 3.3-kb repeats to alter the chromosomal configuration or folding at terminal
4q region, and thereby exert a positional effect on a gene or genes located
proximally to this, possibly through proximal spread of telomeric heterochro-
matin [15]. The 3.3-kb repeat units appear not to be transcribed themselves,
although the presence of a paired homeodomain sequence within each repeat,
with sequence similarity to other transcription regulators, induces other plau-
sible hypotheses for a more direct role in regulation of expression of other
genes [16, 17]. To date, no gene has been identified whose expression is influ-
enced directly by the 3.3-kb repeat lengths. Clinically, it is now recognised
that many patients with FSHD have evidence for a subclinical hearing loss,
most marked at 6,000 Hz [18]. Others may have evidence of a retinal vasculopa-
thy, manifesting at its most severe (as Coats disease-like changes) in the
youngest onset FSHD cases [19, 20]. Similarly, it is now being reported that
epilepsy and mental retardation may be associated with some of the most
Facioscapulohumeral Muscular Dystrophy: Diagnostic and Molecular Aspects 45

severe FSHD presentations, suggesting the possibility of a CNS effect with
the largest deletions [11].
The concept of the clinical presentation in an FSHD case being determined
by the probability of significant disturbance of site-specific gene expression
dependent on the total length of the 3.3-kb repeats is emerging. With such a
model, there would be no strict threshold at the mildest end of the spectrum,
but rather an overlap between minimally affected cases and unaffected subjects
from the normal population, with overlap between repeat copy numbers at
the lowest end of the normal range with those which could manifest as a
mildest presentation of FSHD or scapulohumeral muscular dystrophy. Delin-
eation of factors controlling this probability, particularly demonstrated by
wide clinical variability within families, could potentially lead to methods of
therapeutic intervention, which currently are restricted to ameliorative surgery,
such as scapular fixation, tendon transfers, and blepharorrhaphy [20, 21].
Meantime empirical treatments with corticosteroids or b2-agonists are being
evaluated on a trial basis [22], while increasing interest in FSHD patient groups
is providing a focus for raising and providing support.
Diagnosis of Index Case
The diagnosis of FSHD in an apparently isolated case, or in the index case

in a family context, now depends on a combination of the clinical presentation,
family history, and molecular genetic analysis. Published clinical diagnostic
criteria comprise inclusion and exclusion criteria with additional comments
[23]. The inclusion criteria broadly require shoulder girdle involvement prior
to any pelvic girdle involvement, facial weakness (peri-oral, peri-orbital or
both) in an index case, or in at least 50% of the affected cases in a family,
and proximal prior to distal involvement in the upper limbs, but with early
distal (peroneal) weakness often a characteristic feature in the lower limbs.
Initial asymmetry of shoulder girdle involvement is usual, with the right side
affected first in over 75% of cases, and possibly with a relationship to handed-
ness [2]. Suggested exclusion criteria (or at least cause for considerable caution)
are a sustained improvement of symptoms, presence of ptosis or extra-ocular
muscle involvement, and onset of weakness in the pelvic girdle prior to the
shoulder girdle, without typical facial weakness [23]. Muscle hypertrophy,
particularly in forearm and calf muscles, can be seen, and muscular pain can
be a prominent symptom [20]. Muscle biopsy may show few or non-specific
changes, but can show typical dystrophic features [1]. Inflammatory change
and small angular fibres are often seen. Specific biopsy features of other
neuromuscular conditions should not be present. However, in a clinically
Lunt 46
suspected case, DNA testing rather than muscle biopsy could now be the first
line investigation.
Clinical presentation in a typical case is usually as a teenager who first be-
comes aware of developing symptoms of shoulder girdle weakness, or of signs
of muscle wasting in this region. Data from large families with many affected
individuals suggests a median onset age of 11 years for clinical signs (usually as
facial weakness) to be first recognisable by an examiner, preceding by a few years
the median age for the patient to first complain of symptoms [2, 24]. Typically
the teenager notices a dropped shoulder contour or thinning of the upper arms,
possibly with increased winging of the scapulae, and admits to difficulty raising
one arm (usually the right one). Facial examination will usually show weakness
of eye closure minimal involvement being an inability to bury the eyelashes on
attempted tight eye closure. Weakness of peri-oral muscles is often evident to
the attuned eye as a slight asymmetry of the lips, but is best demonstrated by air
escape when the examiner tries to force air out of the patients puffed cheeks. As
well as confirmatory findings of shoulder girdle weakness, an exaggeration of
the normal lumbar lordosis with a positive Beevors sign [25], and a degree of
peroneal weakness may already be present at this stage.
In more severe infantile-onset cases, facial weakness is the earliest and
most prominent sign. Thus, the infant may show little or no facial expression,
appearing unable to smile, and may be initially misdiagnosed as having Mobius
syndrome [4]. Pelvic girdle weakness in the most severe cases can be prominent
by age 10 years, leading to consideration of Xp21 or limb girdle types of
muscular dystrophy, but unlike these conditions, FSHD is still characterised
by an even greater degree of shoulder girdle weakness rather than pelvic
weakness. FSHD is inevitably progressive, and an overall 20% of patients
require a wheelchair by the 5th decade, although this can be required before
age 20 years in many of the most severe new mutation cases [2]. Harder to
recognise is the milder, later onset presentation, which tends to be associated
with larger (3238 kb) DNA fragment sizes at 4q35 with fewer copies deleted
of the 3.3-kb repeats. Many of these gene carriers may remain unaware of
symptoms, or not attribute symptoms to the family condition, but can be
recognised in a family context by signs of peri-oral or peri-orbital weakness,
by a minor degree of scapular winging or scapular weakness, or most often
by an asymmetrically dropped shoulder contour. However, in this group,
signs of facial weakness may be minimal or absent, and some families classified
with a dominant scapulohumeral form of muscular dystrophy represent the
mild end of the spectrum of FSHD-associated 3.3-kb repeat deletion with
fragment size of 3538 kb [12].
Penetrance of clinical signs, previously estimated at 95% by age 20 years
[24], now appears to be significantly lower in females compared to males,

particularly with the larger DNA fragment sizes [13]. The family history is
therefore often particularly helpful in establishing diagnosis. Stories of relatives
who have had difficulty raising their arms, have had a rounded shoulder
contour, or have apparently thrown their leg when walking are particularly
helpful. Since it is only in the more severely affected cases that reproductive
fitness may be reduced, one can expect a high proportion of FSHD cases in
the milder half of the spectrum (i.e. with fragment size q25 kb) to have other
affected relatives. Examination of parents and sibs of all cases can therefore
be very helpful diagnostically, particularly in relation to facial weakness, in
providing more than one clinical presentation for evaluating against the stand-
ard clinical criteria, but this has to be balanced against the problems inherent
in uncalled-for genetic diagnosis of relatives who might have preferred to
remain unaware of the presence of clinical signs and diagnosis in themselves.
Information on relatives identified in family histories should also be verified
where possible.
Anticipation
It is uncertain whether there may be evidence of clinical anticipation in
FSHD, whereby the severity tends to increase with successive generations
[810]. At present it is not clear how this could come about with a fixed
mutation in each family, but the same has been proposed in at least one other
neurological condition with a fixed mutation familial amyloid polyneurop-
athy [26]. One could hypothesise that the fixed deletional mutation, by affecting
chromosome folding or telomeric pairing at meiosis, might be inducing a
dynamic expansion (say) in a different type of tandem repeat located more
proximally on chromosome 4q, leading to further expansion at subsequent
meioses, but this must remain as pure conjecture in the absence of any known
dynamic repeat sequence in the 4q35 region. Furthermore, the finding of DNA
evidence for somatic and germline mosaicism for a severe mutation [13, 14],
as an explanation for minimal symptoms in one parent in some new mutation
cases, and the knowledge that females may generally have a milder presentation
than males [2, 14], might provide more plausible explanations for at least some
cases of apparent anticipation [43].
Molecular Testing: Confirmation of Diagnosis
In 9095% of cases of FSHD, as defined by meeting the diagnostic criteria,

the diagnosis can effectively be confirmed by showing the presence of a
shortened (=35 kb) DNA fragment at 4q35 (recognised by probe p13E-11),
which arises from deletion of an integral number of copies of the 3.3-kb repeats
Lunt 48
from that region [3, 5]. The DNA probe used (p13E-11) also detects the closely
homologous 3.3-kb repeat array from 10q26 [7]. However, each chromosome
10-type repeat has an additional BlnI restriction site [27]. For the specific
diagnostic test, a double digest with EcoRI/BlnI is employed on genomic DNA
(obtained from peripheral blood), which removes chromosome 10-type repeats,
but leaves chromosome 4-type repeats intact (albeit reduced by 3 kb in size
compared to EcoRI single digest) [27]. Test specificity for BlnI-resistant frag-
ments smaller than 35 kb (approximately 83.3-kb repeats) in someone with
a neuromuscular presentation is very high (98%), since BlnI-resistant fragments
of p35 kb are found in =2% of the normal population [3]. However, the
sensitivity of the test is lower. In around 6% of clinically affected cases which
satisfy the diagnostic criteria but appear as false-negatives, and in the 2% or
so of controls who would be false-positives, a more complicated situation
exists with respect to the arrangement of 3.3-kb repeat sequencies at 4q35 and
10q26, requiring characterisation of all four repeat arrays for interpretation
[7, 28].
This is achieved by using pulse field gel electrophoresis (PFGE) or field
inversion type (FIGE), with EcoRI and EcoRI/BlnI digests, to identify DNA
fragments up to 200 kb in size, hence the length and Bln sensitivity type of
the four separate repeat arrays [6]. Only 80% of the normal control population
have two repeat arrays of each type; 20% show polymorphism for either a
translocation or gene sequence conversion between the repeats at the 4q35
and the 10q26 regions, recognised on PFGE as having three fragments of one
type and one of the other, or rarely by four fragments of the same type [7].
FSHD arises when the repeat array on chromosome 4 is reduced in size (copy
number), irrespective of whether the repeat units are of 4-type or 10-type.
Hence there are 5% of patients with FSHD who have a =38-kb fragment,
but which is Bln-sensitive, owing to this being a shortened 10-type repeat array
on one chromosome 4, and usually associated with a 1:3 ratio of 4-type:10-
type repeats on PFGE. Variation in length of the repeat arrays attached to
chromosome 10 seems to be of no consequence, and nearly 60% of controls
have a fragment which appears in the same size range as FSHD-associated
fragments (=38 kb, if families with scapulohumeral presentation are included)
[6], although in =4% is this Bln-resistant and hence might confuse diagnostic
testing [3].
Table 1 shows the distribution of fragment size and type in FSHD patients
and controls. The presence of a shortened Bln-resistant fragment on conven-
tional gel electrophoresis as a diagnostic test for FSHD would for fragments
=32 kb give test sensitivity of 85% and specificity of 98.5%, and for fragments
=38 kb give test sensitivity of 94% and specificity of 96.5%. For milder pre-
sentations with fragment sizes 3238 kb, or for Bln-sensitive fragments, or for

Table 1. Distribution of fragment size and type (Bln-resistant or Bln-sensitive) detected
on standard gel electrophoresis in FSHD patients and in controls
Conventional gel fragment seen
Bln-resistant no Bln-resistant, but Bln-sensitive total

=32 kb 3238 kb =32 kb 3238 kb no frag.=38 kb
FSHD, %
Overall 85 9 4.7 0.9 0.4 100
With 2EB:2E 73 8 0.5 0.05 Not FSHD 81.5
With 1EB:23E 4 0.4 4 0.8 0.34 9.5
With 0EB:34E 0.2 0.05 0.02 0.3
With 23EB:1E 8 0.9 0.015 0.02 0.04 9
With 34EB:0E 0.2 0.02 0.001 0.2
Controls, %
Overall 1.5 2 19 39 39 100
With 2EB:2E 0.6 0.2 16 33 31 81
With 1EB:23E 0.01 0.005 2 3.7 3.8 9.5
With 0EB:34E 0.05 0.1 0.1 0.25
With 23EB:1E 0.85 1.7 1 1.8 3.8 9
With 34EB:0E 0.04 0.1 0.01 0.15
Likelihood ratios of FSHD:control
With 2EB:2E 120:1 40:1 1:30 1:600 Not FSHD
With 1EB:23E 400:1 80:1 2:1 1:5 1:10
With 0EB:34E 4:1 1:2 1:5
With 23EB:1E 10:1 1:2 1:60 1:100 1:100
With 34EB:0E 5:1 1:5 1:10
This is given as the overall percentage distribution seen in FSHD or in controls, and also
as the distribution according to the possible ratios of 4-type (EB):10-type (E) repeat arrays
seen on PFGE. From this a likelihood ratio of FSHD: control has been calculated for each
fragment size/type and possible 4-type:10-type fragment ratio.
testing to exclude a diagnosis of FSHD, it is essential to add in PFGE.

Therefore, for each category of shortened fragment, table 1 also gives a calcu-
lated likelihood ratio of FSHD:control according to the overall ratio of
4-type:10-type repeat arrays seen on PFGE.
Two other rare situations have been observed which can add further
complication: (1) deletion extending into the p13E-11 hybridisation site, and
(2) hybrid fragments, comprising repeats of both chromosome 10 and chromo-
some 4 type [28]. Recognition of either of these situations requires use of a
Lunt 50
second probe 9B6A which is complimentary to the repeat unit (D4Z4) itself.
Subjects deleted for the p13E-11 site will appear to have no small fragment
on 4q35 when hybridised with p13E-11, which may lead to an initial interpreta-
tion as exclusion of FSHD. However, they show only 3 bands on PFGE/FIGE
using this pobe, but a hidden fourth band (which is =38 kb in FSHD) if
9B6A is used instead [28]. This has been observed with a normal length repeat
array in an unaffected father and son, ascertained through further extension
of the deletion resulting in a shorter repeat array and de novo FSHD in an
affected son [28]. It is not known whether p13E-11 site deletion may be
prevalent in the general population, or whether it would have a high propensity
to further extension and inevitably be ascertained only as a rare association
with de novo FSHD. If we are correct that a more proximal gene locus is
being influenced by the repeat deletion, since 4q35 haploinsufficient states
(with cytogenetically detectable terminal deletions) are not known to show
any features of FSHD [29], the proximal limit of molecular deletions causing
FSHD defines a distal limit for the location of an influenced gene locus [28].
Hybrid repeat arrays, consisting of a run of 10-type repeats attached to
the distal end of a 4-type repeat array, could lead to a false-positive diagnosis
of FSHD since EcoRI/BlnI double digest may show a small Bln-resistant
fragment segregating with 4q35, but corresponding to the length of the 4-type
repeats only. Using PFGE /FIGE the apparent disappearence of a larger EcoRI
fragment (from the hybrid array) may be possible to recognise [28], but also
requires use of probe 9B6A to avoid confusion where a 10-type repeat array
at 10q26 happens to be of similar size to the Bln-resistant residual fragment.
Development of a new technique, using a BlnI/BglII double digest, is antici-
pated to provide a dosage test which on standard gel electrophoresis using
p13E-11 will be able to assess the ratio of 4-type to 10-type repeats in the
most proximal repeat unit for all four chromosomes concerned, and hence
help identify translocated (or sequence-converted) repeats and p13E-11 site
deletions [30].
The clinical importance of these laboratory findings is that in sending a
sample to the laboratory for diagnostic testing, given that although test speci-
ficity may be very high, because the sensitivity is lower the clinician must inform
the lab whether he is expecting a diagnosis of FSHD to be confirmed, or whether
he is trying to exclude the diagnosis. An estimated prior probability of the diagno-
sis being truly FSHD can be combined by Bayes calculation with a likelihood
ratio for a fragment of given size and Bln-type to be associated with FSHD or
not (FSHD:control ratio in table 1). Note that owing principally to 0.5% of
FSHD subjects who have a double exchange with a 4-type array at 10q26 and a
shortened 10-type array at 4q35, and 6% of controls who have three 10-type
fragments with a shortened one at 10q26, the sensitivity and specificity of testing

cannot yet reach higher overall than 99.5 and 94%, respectively. Also, DNA
samples suitable for PFGE must be extracted in a specific way and from a fresh
blood sample, imposing some practical limitations on the testing. Our own
experience of running a diagnostic molecular service in Bristol, UK based on
conventional gel electrophoresis only has been that 21% of 270 referrals have no
fragments =40 kb and are probably not FSHD; 14% of those with fragments
=35 kb have this as a Bln-sensitive fragment. This data suggests that if 25% of
controls have a Bln-sensitive fragment =35 kb, then it will be 5% of true FSHD
who have a shortened 10-type fragment on 4, as predicted.
In the above calculations for sensitivity and specificity of testing, inde-
pendence of deletional mutation and background chromosome translocation
pattern has been assumed. However, if parents of new mutation cases might
have an excess of shorter or translocated repeat types, perhaps predisposing to
de novo deletion, the sensitivity and specificity of testing may be less good
[31, 43]. This would also imply that some people (or perhaps the fertilised eggs
of some couples) could have an increased susceptibilty to deletional reduction
of the 4q35 repeat array, and therefore to having a child with de novo FSHD.
Scapulohumeral Presentation and Fragment Lengths of 3440 kb

An as yet undefined proportion of families with FSHD have a milder scapu-
lohumeral presentation with a Bln-resistant fragment on 4q35 in the size range
3440 kb (810 repeats) [8]. In one family from Somerset, UK, in which affected
subjects demonstrated a scapulohumeral syndrome without facial weakness, a
4q35-cosegregating 38-kb fragment is found in all definitely affected members,
and in 2 at-risk young adults who are presumed to be presymptomatic [12]. In
this and similar families, penetrance is likely to be reduced, particularly in fe-
males [13]. Bln-resistant fragments of this size can also be seen in some normal
controls thereby reducing test specificity in this size range to a level which may
be impracticable for diagnostic use. Hence there is probably no definite threshold
between the normal unaffected situation and the most mild degree of shoulder
girdle weakness. Rather, one can envisage with increasing repeat lengths (frag-
ment size) a gradually decreasing probability that a shorter than usual repeat
array will in any particular cell in the body affect the expression of some other
gene (or genes) adversely, and hence an overall decreasing probability that symp-
toms will develop in any one individual.
Addition of 4q35 and 10q26 Linkage Analysis

In families where there remains doubt about the diagnosis from molecular
testing, the addition of linked polymorphism analysis may be very helpful in
determining whether particular fragments cosegregate with 4q35 or 10q26
markers. The author has experience of 2 teenage brothers who presented with
Lunt 52
scapular winging (in both) and spondylolisthesis (in one), but normal muscle
biopsy. The finding of Bln-sensitive fragments of size 28 kb in one boy and
33 kb and 37 kb in the other was resolved by 4q35 and 10q26 marker analysis
in both boys, their sister and their parents. FSHD was excluded by showing
that none of these fragments appeared to segregate with 4q35 markers, but
all 3 were compatable with 10q26 linkage of the fragments.
Questions of Patients and Parents Once Diagnosis

Has Been Confirmed
Prognosis
FSHD inevitably involves a progressive weakness, although can plateau for
long periods. Loss of ambulation, requiring wheelchair use, is found in 20% of
patients aged between 40 and 83 years, but in this group, all patients with dis-
abling proximal lower limb weakness had in retrospect already been aware of
at least some lower limb weakness by age 20 years [2]. Several studies of the
retrospectively reported age at onset of first clinical signs have now each shown
a broad correlation between average age at onset and FSHD-associated DNA
fragment size [810], this being 1018 kb in most infantile-onset cases; 1834 kb
in most typical teenage-onset cases, and 30 kb or above in the eldest-onset cases.
There may be an additional generational (anticipation) effect, with the mildest
clinical cases within a family (after exclusion of the direct family line) still tending
to be those in the top generation [810], but further study of additional pedigrees
is required. Males may have a slightly younger average onset age than females,
and adult females may be more likely than males of the same age to show non-
penetrance [13]. This may be a hormonal effect rather than a genetic one, with
severity more equal above age 5060 years [32]. Currently, parents can be advised
that the presentation of FSHD in any affected offspring is likely to be at least as
severe as in a same-sex affected parent and quite possibly greater where a mother
is passing the condition to a son. Prediction of the likely severity in a daughter
receiving the condition from her father is less certain, although anecdotally it is
hard to think of any definite examples where it has been significantly later onset
or milder.
Therapeutic Strategies and Other Support

There are no specific treatments identified for FSHD. The observation of a
tendency to earlier and greater periscapular and upper arm weakness on the side
of the dominant hand (usually right side) [2], together with the often irreversible
muscle wasting of a limb following enforced immobility (e.g. secondary to a
fracture) [1] suggest that the primarily impaired process might be muscle re-

generation following damage. If so, strenuous body-building exercise would cer-
tainly be contraindicated, while investigation of factors controlling the process
of muscle regeneration could lead to potential treatments. A trial of steroids has
not shown any sustained benefit [33], but a trial of a b2-agonist (albuterol) is
currently being evaluated [22]. For certain patients, surgery to fixate the scapula
to the chest wall has proved successful, and enabled greater use of the arms,
albeit at the restriction of becoming unable to raise them above shoulder level
[20, 21]. A proper understanding of factors controlling variation of severity
within a family, and even within a sibship, might potentially lead to therapeutic
intervention to influence a genetic modifier mechanism. General support can be
very helpful, including physiotherapy and occupational therapy, but equally,
contact with specific or more general neuromuscular patient support groups will
become increasingly helpful, particularly if contact between patients in different
families can be facilitated through the Internet.
Genetic and Clinical Testing Wider in a Family

De novo cases of FSHD can only be proven as such by recognition of
de novo appearence of a typical Bln-resistant short fragment, where DNA
from both parents is available and paternity is proven. In all other cases there
remains a possibility of genetic risk wider in the family, through unrecognised
or asymptomatic gene carriers, and a wider family study is indicated [31].
Proven de novo cases have almost all involved a DNA fragment smaller than
the average size for familial affected cases (i.e.=25 kb) [8]. This may reflect
an ascertainment bias for paediatric onset cases, or a higher genetic fitness
and smaller proportion of new mutations amongst cases with larger fragments,
but could also possibly indicate a higher overall mutation rate for smaller
fragments. New mutations, whether occurring in meiosis or mitosis (the latter
giving rise to somatic and germline mosaicism), have predominantly been
maternal (80% of recognised parental mosaics being in the mother) [6, 3,
13, 14], but whether this might indicate a parental sex difference for the size
of de novo deletion, analagous to the parental sex difference between de novo
deletion and point mutation in Xp21 dystrophy, has not yet been tested.
Alternatively, more male mosaics may show symptoms [43].
A family study involves both clinical and molecular evaluation, which is
only possible if the index case has a proven and readily recognisable repeat
array deletion at 4q35, giving a characteristic DNA fragment. Quite often,
the attuned clinical examiner will detect subtle but typical clinical signs in a
relative, which had previously gone unnoticed, particularly with respect to
minor peri-oral or peri-orbital weakness. The issue of informed consent be-
comes paramount, and if a relative believes themselves to be clear, perhaps
clinical examination as well as DNA testing of that person should follow a
Lunt 54
similar protocol to predictive DNA testing in other late-onset conditions.
Supportive linkage analysis at 4q35 and 10q26 should also be performed to
minimise any chance of a spurious positive prediction from a 10q26 fragment.
This would usually require DNA samples from unaffected spouses as well as
blood relatives. In apparently clinically isolated cases, the family study should
commence with both parents of the affected subject, to recognise in particular
the 20% of cases where one parent is mosaic for the mutation, which shows
as a faint band on the gel corresponding to the affected band in their offspring.
In a further 12% of cases, one parent (equal sex ratio) has demonstrated the
fragment in full dose, but remains asymptomatic [3], including at least 2 cases
where a very small (13 kb) fragment has been associated with a very severe
presentation in their offspring [31], although in one of these, further PFGE
study identifying 5 bands in the father suggests that he may be a mosaic. The
presence in two other families of a 25-kb 4q35-cosegregating fragment in
both a parent and grandparent who each remain asymptomatic would seem
inadequately explained by the possibility of a reduced penetrance in females,
and has led to the suggestion of a 2-step mutation process [31].
Penetrance and Risk to Offspring

Original data suggesting that a family member, born at 50% risk, has only
a 4.8% chance after age 20 years of still carrying the FSHD mutation if they
show no detectable clinical sign of the condition (95% penetrance) was based
on data from families demonstrating dominant inheritance and of sufficient size
to be able to exclude the proband, his sibs, parents and grandparents from analy-
sis [24]. Most such families have fragments from the middle size range (1830 kb),
since ascertainment of families with larger fragments is likely to be less complete,
while with the shortest fragment lengths the greater severity limits family size.
With smaller fragments (1018 kb) the age-dependent penetrance will approach
100% by age 20 years, whereas with larger fragments (3038 kb) it may well be
much lower, particularly in women, but the presentation is also likely to remain
relatively mild. Hence, a relative of someone with a severe FSHD presentation
(apart from the parents, one of whom may be a mosaic) can generally be reas-
sured that they are very unlikely to be carrying the same gene defect if they
remain clear above age 20 years. However, details of the fragment size in the
index case should be checked first, and with a potential possibility of a 2-step
mutation process, reassurance can only really be given to a relative following a
negative DNA test. If, as seems likely, there is a gradually reducing penetrance
with increasing fragment size, there will be a significant region of overlap between
normality and a mild FSHD presentation, such that any proactive widening of
a family study in families with the larger fragment sizes (3240 kb) would seem
unwise. However, this is an area requiring further research.

Testing of Children
It is usually inappropriate to test a child for a potentially later-onset
genetic condition for which there is no treatment, if they are not showing any
sign of the condition. For FSHD, only if an affected sibling has had a severe
early childhood-onset presentation with matching small fragment size, such
that a younger sib inheriting the same 3.3-kb repeat deletion would be expected
inevitably to develop significant symptoms also in their early childhood, might
testing be considered. Linked DNA marker analysis should be performed
simultaneously. However, a single report of deletional expansion occurring
between (an albeit asymptomatic) parent and one offspring [28] behoves cau-
tion even in the situation above, to avoid predicting for a milder later-onset
presentation.
Prenatal Testing
This is possible from chorion villus biopsy (CVB) from 11 weeks gestation
onwards, but similarly requires certainty about the FSHD-associated fragment
in the family, must be run together with DNA from both parents, and must
be backed up by linked markers. Analysis is by Southern blot, which requires
a larger CVB sample and a longer time for analysis than in other conditions
which can employ PCR techniques. The possibility of developing a PCR assay
for the fragments is being investigated, but so far has only been able to detect
fragments up to 25 kb in length [3].
Basic Science
FSHD is a condition where the mutation is known, but not the gene or
genes on which it acts. If there were unequivocal genetic heterogeneity, the
localisation and possibility of cloning of a gene involved in a second and non-
4q35 locus could well add additional clues to the molecular and cellular
pathogenesis. To date, there are only two large FSHD families published which
appear to be unlinked to 4q35 (both from one US genetic centre) [34], but in
neither has any alternative chromosomal localisation, including 10q26, been
identified [35]. Hypotheses for possible mechanisms of action of the deletion
of 3.3 kb repeats include the following.
Position Effects: (1) If the repeats are normally important for subtelomeric
chromosome folding, the disturbance of this may well alter the expression of
a gene or genes sited more proximally along chromosome 4. (2) The telomeric
region of all chromosomes is folded/methylated as heterochromatin. Deletion
of part of the area involved in this might cause a region more proximally to
be folded/methylated as heterochromatin, and therefore genes from that area
Lunt 56
could be inactivated by spreading heterochromatisation. (3) The 3.3-kb re-
peats might help align the 4q and 10q telomeres on the spindle during cell
division, perhaps facilitating some expressional interaction between genes on
these chromosomes, which would be disturbed by 3.3-kb repeat deletion.
More Direct Regulator Role of the 3.3-kb Repeats: (4) Each repeat contains
a paired homeodomain suggesting some role in temporal, regional or tissue-
specific regulation of gene expression [16]. STOP codons in the DNA sequence
of the repeat preclude an open reading frame, and the repeats appear not to
be transcribed. However, sequence homology with genes for a DUX family
of DNA-binding regulatory proteins seems more than a coincidence [17].
Perhaps an RNA transcript itself might have a regulatory role.
In all these possible models, it is suggested that the effect of the mutation
occurs at a distance from the mutation itself. Attempts to find a gene more
proximally on 4q35, whose protein product is expressed in muscle and is
regulated by the number of 3.3-kb repeats, have to date been unsuccessful. At
least three possibilities for candidate genes have been identified (FRG1, ALP,
TUB4q), but none of these show altered expression in FSHD muscle tissue
[3638]. Some evidence suggests that the expression of multiple genes may be
affected by the repeat deletion, and variously up- or downregulated, but it is
not yet clear whether this might be a secondary phenomenon consequent on
muscle damage. Cases of karyotypic deletion of 4q35 have not exhibited any
muscular dystrophy [29]. This suggests that FSHD cannot be consequent on
haploinsufficiency, but is probably a gain of function effect. The correlation
of deletion size with severity, the absence of facial weakness with the smallest
deletions, and the singular distribution of muscle involvement in FSHD suggest
that a small deletion may leave intact the regulatory mechanism for expression
of some gene in facial muscles but not in the shoulder girdle. Similarly the
tendency for the most severe cases to be the ones with the most marked retinal
changes and hearing loss, and possibly also to have associated seizures and
mental retardation [11], suggests that the largest deletions may tend to affect
gene expression at sites additional to muscle. Several parallels with myotonic
dystrophy could be more than a coincidence; in both conditions facial and
peroneal muscle weakness can be a prominent feature, and in both there is a
paired homeodomain sequence involved in, or in the vicinity of, the mutation
[39]. That the only limb girdle muscular dystrophy in which the upper limb
girdle is the one predominantly affected (type 2A) is the only one where the
gene codes for a proteolytic enzyme (calpain 3) rather than a structural protein
[40] is also noteworthy.
Comparative studies with the genomes of other organisms have not yet
provided useful leads. The 3.3-kb repeats seem to be limited to higher primate
species [41]. The 4q35 region shares homologies in the mouse with sequencies

and genes on murine chromosome 8, but the only neuromuscular phenotype
(myodystrophy) determined by a gene (myd) in this murine chromosome region
is inherited as a recessive condition, and has closest flanking markers which
show homology much more proximally on human 4q [42].
Future (Speculation)
Further research is expected eventually to identify the nature of the gene

(or genes) which are influenced by the 3.3-kb repeat deletions, the normal role
of the repeats themselves, and the mechanism of mutation. Clues may come
potentially from genetic linkage studies for polymorphisms which may influ-
ence variation of severity between siblings. Identification of any such modifier
gene could potentially lead to initial forms of therapy, at least to help minimise
the severity of presentation. Knowledge of the mutation mechanism may have
wider implications for understanding the control of chromosome folding, and
the possible role of this in regulation of gene expression.
References
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facioscapulohumeral muscular dystrophy (FSHD). J Med Genet 1996;33:361365.
28 Lemmers RJLF, van der Maarel S, van Deutekom JCT, van der Wielen MJR, Deidda G, Dauwerse
HG, Hewitt J, Hofker M, Bakker E, Padberg G, Frants RR: Inter- and intrachromosomal sub-
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29 Tupler R, Berardinelli A, Barbierato L, Frants R, Hewitt JE, Lanzi G, Maraschio P, Tiepolo L:
Monosomy of distal 4q does not cause facioscapulohumeral muscular dystrophy. J Med Genet
1996;33:366370.
30 van der Maarel SM, Deidda G, Lemmers RJLF, Bakker E, van der Wielen MJR, Sandkuijl L,
Hewitt JE, Padberg GW, Frants RR: A new dosage test for subtelomeric 4;10 translocations improves
conventional facioscapulohumeral muscular dystrophy (FSHD) diagnosis. J Med Genet 1999;36:
823828.
31 Lunt PW, Jardine PE, Stevenson A, Tyfield L: Genetic counselling in facioscapulohumeral muscular
dystrophy (FSHD): Lessons from clinically unaffected mutation carriers. Muscle Nerve 1998
(suppl 7):S25.
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(suppl 7):S25.
33 Tawil R, McDermott MP, Pandya S, King W, Kissel J, Mendell JR, Griggs RC, FSH-DY group:
A pilot trial of prednisone in facioscapulohumeral muscular dystrophy. Neurology 1997;48:4649.
34 Gilbert JR, Stajich JM, Wall S, Carter SC, Qiu H, Vance JM, Stewart CS, Speer MC, Pufky J,
Yamaoka LH, Rozear M, Samson F, Fardeau M, Roses AD, Pericak-Vance MA: Evidence for
heterogeneity in facioscapulohumeral muscular dystrophy (FSHD). Am J Hum Genet 1993;53:
401408.
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which cross hybridise to FSHD1A associated markers in FSHD1B. J Med Genet 1995;32:770773.
36 van Deutekom JCT, Lemmers RJLF, Grewal PK, van Geel M, Romberg S, Dauwerse HG, Wright
T, Padberg GW, Hofker MH, Hewitt JE, Frants RR: Identification of the first gene (FRG1) from
the FSHD region on human chromosome 4q35. Hum Mol Genet 1996;5:581590.
37 Bouju S, Pietu G, Le Cunff M, Cros N, Malzac P, Pellissier J-F, Pons F, Leger J-J, Auffray C,
Dechesne CA: Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35
facioscapulohumeral muscular dystrophy (FSHD) candidate genes. Neuromuscul Disord 1999;9:
310.
38 van Geel M, Beck AF, Eichler EE, Frants RR, de Jong PJ: Possible activation of a b-tubulin
pseudogene (TUB4q) could explain the FSHD molecular defect. Am J Hum Genet 1999;65(suppl 4):
A93.
39 Harris S, Moncrieff C, Johnson K: Myotonic dystrophy: Will the real gene please step forward!
Hum Mol Genet 1996;5:14171423.
40 Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud
C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno M-R, Zatz M, Tischfield JA, Fardeau M,
Jackson CE, Cohen D, Beckmann JS: Mutations in the proteolytic enzyme calpain 3 cause limb-
girdle muscular dystrophy type 2A. Cell 1995;81:2740.
41 Winokur ST, Bengtsson U, Vargas JC, Wasmuth JJ, Altherr MR: The evolutionary distribution
and structural organization of the homeobox-containing repeat D4Z4 indicates a functional role
for the ancestral copy in the FSHD region. Hum Mol Genet 1996;5:15671575 & corrigendum:
Hum Mol Genet 1997;6:502.
42 Grewal PK, van Deutekom JC, Mills KA, Lemmers RJ, Mathews KD, Frants RR, Hewitt JE: The
mouse homolog of FRG1, a candidate gene for FSHD, maps proximal to the myodystrophy mutation
on chromosome 8. Mamm Genome 1997;8:394398.
43 van der Maarel SM, Deidda G, Lemmers RJLF, van Overveld PGM, van der Wielen M, Hewitt
JE, Sandkuijl L, Bakker B, van Ommen GJB, Padberg GW, Frants RR: De novo facioscapulohumeral
muscular dystrophy: Frequent somatic mosaicism, sex-dependent phenotype, and the role of mitotic
transchromosomal repeat interaction between chromosomes 4 and 10. Am J Hum Genet 2000;66:
2635.
Dr. Peter W. Lunt, Clinical Genetics Unit, Bristol Royal Hospital for Sick Children,
St. Michael Hill, Bristol BS2 8BJ (UK)
Fax +44 117 9285167
Lunt 60
............................
Myotonic Dystrophy
Richard T. Moxley a, b, Giovanni Meola c, d
a
University of Rochester School of Medicine and
b
University of Rochester Medical Center, Rochester, N.Y., USA;
c
University of Milan and
d
San Donato Hospital, Milan, Italy
The Trinucleotide Expansion [CTG]n in Myotonic Dystrophy:

Impact of This Discovery on Diagnosis and Genetic Counseling
Myotonic dystrophy (DM) is an autosomal dominant disorder with

highly variable clinical manifestations [1, 2]. It affects specific tissues, such
as distal limb and facial muscles, smooth muscles (gastrointestinal tract,
uterus), the heart (primarily, the conducting system), the eye (primarily, the
lens), the brain (especially, anterior temporal and frontal lobes), and endo-
crine function (testosterone deficiency, abnormal growth hormone regulation,
insulin resistance) [1, 2]. DM results from an unstable trinucleotide repeat
expansion containing cytosine, thymine and guanine [CTG]n, located in the
3 untranslated region of chromosome 19q13.3 [35]. The DM gene codes
for a serine-threonine kinase (DMPK) [35]. This type of enzyme often
regulates various intracellular processes by phosphorylation-dephosphoryl-
ation reactions [36]. The function of DMPK in normals and in DM is
unknown. A deficiency of DMPK probably plays a pathophysiologic role in
DM [6, 7], but other factors, such as an altered function of flanking genes
[813] and toxic effects of the abnormally enlarged DMPK mRNA [1219],
are likely to contribute in a critical way to the variable multisystem manifesta-
tions of DM.
Diagnosis
The gold standard for the diagnosis of DM is the identification of an
abnormally enlarged [CTG]n repeat expansion in the DM gene. Normal alleles
for the DM gene range in size from [CTG]5 to [CTG]37 [35, 20]. DM patients
Table 1. Overview of genetics of DM
Frequency 1/8,000 (likely to be a significant underestimate;

new calculations in the future will use data obtained by
using specific gene probes)
Inheritance Autosomal dominant with anticipation
Sex bias for transmission of the Female (almost all cases of congenital DM result from
severe form female transmission of the DM allele)
Protein/expression Serine/threonine kinase
Disease-causing mechanism Unknown speculation (see text)
Repeat/location CTG/3 untranslated region
Size of repeat in normal alleles (CTG)537
Predisposing normal alleles Predisposed normal alleles have repeat size of (CTG)1930
(frequency) (approximately 0.10)
Linkage disequilibrium Yes (absolute)
Table 2. Correlations of clinical manifestations with the age of onset
Mildest [CTG]n =150 repeats

Onset: middle to old age
Major findings: cataracts, minimal or no muscle weakness
Classical [CTG]n 3001,000 repeats
Onset: adolescence and early adult life
Major findings: myotonia, muscle weakness (face, distal muscles in arms and legs)
Congenital [CTG]n ?1,500 repeats with some cases occurring with sizes as small as 800 repeats
Onset: at birth, frequent history of hydramnios and reduced fetal movements
Major findings: neonatal respiratory distress, hypotonia, bilateral facial weakness, feeding
difficulty, talipes, mental retardation
have repeat expansions ranging from 50 to over 2,000 [2047]. Table 1 provides
an overview of the genetics in DM.
The specific alteration that leads to the unstable expansion of [CTG]n
repeat expansion in DM remains a mystery. There is a general correlation
between the degree of repeat expansion and the severity of the manifestations
of DM [1747]. However, a recent report, evaluating the relationship between
leukocyte [CTG]n repeat length and clinical onset, has found that a significant
correlation for age of onset only occurs in patients with relatively small repeat
expansions [29]. These observations point out that the proposed classification
of DM using age of onset, clinical severity and the length of the [CTG]n repeat
expansion given in table 2 below is an approximation and not a rigorously
proven classification.
Moxley/Meola 62
Diagnostic tests of primary value in DM include (1) DNA testing for the
abnormal enlargement of the [CTG]n repeat, (2) a focused clinical examination
to look for the skeletal muscle and nonmuscular manifestations, (3) electromy-
ography to identify subclinical myotonia, and (4) slit lamp examination to
detect characteristic cataracts [1, 2]. Tests of secondary value include (1) serum
creatine kinase, which is often mildly elevated, and (2) muscle biopsy, which
frequently shows an increase in central nuclei, type I fiber atrophy, and ringed
fibers [1, 2].
The major challenge in diagnosis is to consider the possibility of DM.
DM is so variable. It disguises itself in many ways. It may present as a floppy
infant to a neonatalogist, a patient with failed labor or placenta previa to the
obstetrician; a case of postoperative apnea to the anesthesiologist or surgeon,
a patient with sudden heart block or tachyarrhythmias to the cardiologist, a
patient with pseudoobstruction of the intestine to the gastroenterologist, a
patient with recurrent atelectatic pneumonitis or sleep apnea to the pulmo-
nologist, a baby with talipes to the orthopedist, a child with behavior disorder
to the psychologist, and a young or old adult with cataracts to the ophthalmol-
ogist [1, 2]. Other associated clinical manifestations of DM may elude detection
by each of these specialists because they have focused on their own area.
Genetic Counseling
Analysis of DNA isolated from amniocytes or chorionic villus samples
is able to predict the delivery of infants with DM [17, 18, 2125]. However,
there are certain limitations. The [CTG]n repeat enlargement varies in the DM
alleles in different tissues [12, 13, 23, 26, 30, 3336, 3847]. On occasion the
[CTG]n repeat expansion in the DM gene isolated from amniocytes is smaller
than the repeat isolated from maternal or fetal leukocyte DNA and yet the
child ultimately turns out to have the severe form of disease, congenital DM
[17]. Prenatal diagnosis of congenital DM, as opposed to the less severe
types of DM, needs to rely on a combination of factors, including maternal
pregnancy history, clinical findings, and cautious interpretation of maternal
and fetal DNA analysis [17]. Clearly DNA analysis permits identification of
whether the fetus has the abnormally expanded DM allele and proves that
the disease allele is present. It is not, however, a reliable means to predict the
severity of DM after birth.
At the time of prenatal evaluation of at risk mothers and infants it is
useful to discuss the different obstetric complications so that optimal use of
preventive therapy is available [1, 2]. Counseling of women with DM is impor-
tant so that they have an understanding of the increased risk of complications
during pregnancy and delivery and are aware of the fact that the degree of
[CTG]n repeat expansion may not reliably predict problems [19].
Myotonic Dystrophy 63
Fig. 1. Serial photographs of a father and son affected with DM are shown. To the
right are two photographs in the upper panel of the father (age 77 years) and two in the
lower panel of the son (age 41 years) obtained in 1991. The two photographs furthest on
the left were obtained when the father was 45 years of age and the son was 18 years old.
Those photographs immediately adjacent to the ones obtained in 1991 show the father at
age 62 years and the son at age 26 years. Both patients have had cataract surgery, right eye
lens implant for the father and left eye for the son. Ptosis, bifacial weakness, wasting of the
sternocleidomastoid, temporalis, and masseter muscles as well as frontal balding are apparent
in the son and less obvious in the father.
The DM Gene and the Phenomenon of Anticipation
The phenomenon of anticipation, the earlier onset of more severe symp-

toms in individuals in successive generations within a family, occurs in DM
[1]. The discovery of the unstable CTG repeat expansion in the DM gene
provides a genetic explanation for this clinical phenomenon. Figure 1 illus-
trates anticipation between a father and son. The father has mild DM and
his son has earlier onset classical type of DM (table 2).
Moxley/Meola 64
Fig. 2. Mother with mild to moderate DM with her two children, both of whom have
the congenital form of DM.
Figure 2 presents a mother with mild DM and her two daughters who
have congenital DM. Figure 2 is also a reminder that almost all cases of
congenital DM occur in offspring of mothers with DM [1, 17, 2125, 27, 31].
There is a genetic explanation for the relatively exclusive predilection of females
with myotonic dystrophy to have offspring with the congenital form of DM.
Oocytes remain viable in DM mothers despite having CTG repeat expansions
up to several thousand repeats in length. Males have some mechanism that
provides an upper limit in repeat size for sperm, such that the male gametes with
hugely enlarged CTG repeats do not survive or are not capable of producing a
viable pregnancy [31].
In contrast to the female preponderance that accounts for the congenital
form of DM, there is an excess of male grandparental transmissions in DM
[27, 31]. In particular, there is an excess of males among those first documented
in a pedigree to have transmitted DM to individuals who have clear clinical
signs of disease [27, 31]. A similar male predilection to transmit unstable
repeat expansions occurs in Huntingtons disease and suggests a possible
similarity between DM and Huntingtons related to the influence of male
gender on repeat instability [31].
Anticipation occurs because of the predilection of the CTG repeat in the
DM gene to enlarge. However, there are examples of the opposite phenomenon,
that is the contraction in CTG repeat length in successive generations
[20, 28, 33]. The mechanism responsible for the maintenance and regulation
of CTG repeat size in the DM gene is unknown. As our knowledge of this
process increases, a new, perhaps curative, strategy for treatment may emerge.
[CTG]n Repeat Mosaicism in DM: Its Impact on Clinical Features and
Treatment
Marked variability in the clinical presentation and in the severity of the

different manifestations are hallmarks of DM [1, 2]. Certain organs have an
increased incidence of symptoms in DM [1, 2]. DNA analysis has demonstrated
somatic mosaicism affecting these organs. There is an increased length of the
CTG repeat expansion (greater than that observed in leukocyte DNA) in
skeletal muscle [30, 3342], brain [34, 4042], and heart [34, 4042]. These
findings and other related observations have led to the hypothesis that the
severity of alterations in the genotype (the degree of abnormal CTG repeat
expansion in the DM gene) predicts the severity of clinical manifestations in
the specific tissue under study. There are several reports that support this
hypothesis and describe an increased severity of skeletal muscle manifestations
(phenotype) that directly correlates with an increase in CTG repeat length
measured in leukocyte DNA [35, 21, 4447]. These observations offer encour-
agement to clinicians and researchers that there may be, at least in some
patients, a straightforward, direct relationship between genotype and pheno-
type.
However, the results of recent investigations raise important questions
about the reliability of predicting phenotype from genotype. These reports
indicate a need for caution in individual patients before concluding that they
show a direct relationship between the CTG repeat expansion and severity. For
example, there is often a much greater enlargement of CTG repeat expansion in
skeletal muscle DNA compared to that in circulating leukocytes [30, 3342].
This agrees with the clinical observation that skeletal muscle dysfunction is a
dominant feature in DM and that abnormal function of leukocytes is uncom-
mon. However, specific muscle groups waste to a greater degree in DM patients.
The muscles of the face and of the distal limbs waste much more than the
proximal limb muscles [1, 2]. Recently, investigators have examined muscle
biopsy specimens obtained from different muscles in the same patient to
determine if the extent of CTG repeat expansion in specific muscles predicts
their degree of weakness [36]. The investigators have found that the degree of
abnormal CTG repeat expansion in the vastus lateralis (proximal) and tibialis
anterior (distal) muscles is similar, but the degree of muscle weakness is much
greater in the tibialis anterior compared to the quadriceps femoris muscles
[36]. More study is necessary to identify those factors, other than CTG repeat
expansion, that account for the selective pattern of muscle wasting and weak-
ness.
Cognitive and behavioral abnormalities, as well as alterations on imaging
of the brain (ventricular enlargement, white matter changes in temporal lobes)
Moxley/Meola 66
occur in DM [1, 2]. Recent investigations indicate that there is a lack of direct
correlation between the degree of CTG repeat enlargement in leukocyte DNA
and (1) the severity of cognitive/behavioral impairment in adults with DM
[48], (2) the alterations that occur in brain structure [4952], and (3) the mental
retardation that is characteristic in congenital DM [50]. Brain manifestations
attributable to DM can sometimes occur years before the development of
skeletal muscle weakness [50]. These examples of alterations in brain function
and structure provide further evidence of the restricted manifestation of symp-
toms that can occur in DM and point out the tenuous nature of the hypothesis
that there is a straight forward, direct correlation between CTG repeat expan-
sion in leukocyte DNA and the various manifestations of DM in different
tissues.
The pathomechanism responsible for the brain manifestations of DM
may differ from that in other target tissues. There is a decrease in cerebral
blood flow in the anterior temporal and frontal lobes of patients with DM
[48]. Metabolic factors and alterations in the control of cerebral blood flow
may play a role in the development of brain manifestations in adult DM [48].
Cardiac conduction defects are common in DM and are a major cause for
sudden death in patients [1, 2, 5358]. However, there is no direct relationship
between the degree of CTG repeat enlargement in leukocyte DNA and atrial
or ventricular arrhythmias [5355], between cardiac conduction disturbance
and skeletal muscle involvement [53], or between cardiac conduction dis-
turbance and the extent of fibrolipomatous infiltration and cardiac dysfunction
[58]. The lack of a straightforward correlation between CTG repeat length in
leukocyte DNA and cardiac symptoms may relate to the somatic mosaicism
of repeat expansion and indicate greater expansion of the CTG repeat in fibers
of the Purkinje system than in circulating leukocytes. Further investigations
of postmortem cardiac tissue and of cardiac function are necessary to clarify
the relationship between the severity of cardiac manifestations of DM and
the gene defect.
Studies of human fetal development in DM demonstrate that the CTG
repeat instability develops only after 13 weeks gestational age and before 16
weeks [40]. This observation suggests that CTG repeat size in different tissues
is significantly influenced by developmental genes at specific times in fetal
growth. It further suggests that repeat expansion and somatic mosaicism are
largely complete by a gestational age of 16 weeks. However, CTG repeat
expansion occurs in leucocyte DNA [38, 52] and sperm [38] during adult life.
These observations raise the possibility that the DM gene may expand further
during adult life in tissues, such as skeletal muscle. This ongoing additional
expansion of the DM gene may influence flanking genes and/or increase the
accumulation of DMPK mRNA in a way that triggers cellular dysfunction
or cell death. These ideas and other theories about the genetic pathomechanism
in DM are under investigation.
Clinical Features and Management

Leukocyte DNA analysis provides an earlier opportunity to diagnose [20]
and to anticipate clinical problems in patients with DM. It is now possible to
pursue a more aggressive monitoring program to detect early cardiac conduc-
tion disturbance, cataract formation and respiratory difficulties [1, 2]. Table 3
presents a summary of the major problems and their treatment in patients
with congenital and childhood onset DM. Table 4 outlines the problems and
management of complaints related to skeletal muscle dysfunction in DM.
Females with DM can encounter significant complications during pregnancy
and have an increased frequency of problems at the time of delivery. These
are summarized in table 5. Patients with DM are at risk for developing intraop-
erative and postoperative complications of anesthesia, especially the develop-
ment of delayed onset apnea [1, 2]. Table 6 outlines strategies to identify and/
or to avoid complications from anesthesia.
Pathophysiology and Therapeutic Trials
Natural History: Opportunity to Establish New Measures

DNA diagnostic testing can establish the diagnosis of DM in individuals
with only minimal as well as those with advanced symptoms of DM. This
capability provides us with a new opportunity to establish the natural history
of all of the various clinical manifestations of DM. The development of
standardized, reliable, reproducible testing to assess the various clinical mani-
festations of DM is in progress [38]. Table 7 provides examples of possible
clinical tests that may be useful in documenting the natural history of these
various manifestations.
Trials of Testosterone, Growth Hormone, Insulin-Like Growth Factor-1,

Troglitazone and Dehydroepiandrosterone Sulfate
The pathomechanism responsible for the muscle wasting in DM remains
unclear. Possible causes include toxic DMPK mRNA-mediated muscle fiber
death [12, 1316, 38], DMPK deficiency [6, 10, 12, 13], abnormal function of
genes that flank the DM gene [813], failed muscle fiber growth [59], insulin
resistance [2, 60, 61], and deficient protein anabolism [2, 60, 62, 63].
Gonadal failure and a deficiency of testosterone are common in men with
DM [1, 2]. To determine if maintaining a high physiologic level of testosterone
increases muscle mass and function in DM, investigators have given either
Moxley/Meola 68
Table 3. Management of DM in infancy and childhood [adapted from 1]
Problem Management
Respiratory insufficiency or failure Neonatal intensive care, ventilator support, chest

x-ray to check on elevation of diaphragm, poor
prognosis for survival if on ventilator longer than 4
weeks; standard care as necessary for pulmonary
immaturity
Feeding problems and aspiration Frequent monitoring of respiration and check chest
x-ray; poor swallowing monitor for recurrent
episodes of aspiration; neonatal intensive care or
highly skilled nursing care needed; evaluate
esophageal function if problems persist
Risk of anoxia and cerebral Neonatal intensive care; serial cerebral ultrasound
hemorrhage in infants measurements are helpful
Talipes Early diagnosis and active correction; splinting;

surgery, if indicated
General delay in development Accurate evaluation of contributing factors:

especially, muscular and cerebral components
Speech problems Anticipate early: distinguish palatal weakness and

other muscular defects from mental retardation
Abdominal pain A frequent and difficult problem; treat as for spastic

colon; role of smooth muscle disease and myotonia
in this complaint is unknown; response to
medications is variable and not established
Surgery and anesthesia Be aware of risks; avoid general anesthesia if possible
Recurrent otitis, hearing difficulty Active management of otitis, awareness of later

neural hearing loss
School decisions May need special classroom setting and vocational
education in later school years; requires full
assessment of functional capacities, awareness of
facial immobility and hearing and speech defects
which exaggerate mental retardation; physical
disability rarely severe and usually is static during
childhood
Should physical activity be restricted Not feasible or justified; obtain yearly ECG to search
for signs of cardiac conduction disturbance
Table 4. Treatment of skeletal muscle problems in DM
Problem Management
Foot drop Light-weight molded ankle foot orthoses
Knee extensor weakness Light-weight long leg knee-ankle-foot orthoses may allow prolonged
weight bearing and prevent the pain and chronic effusion in the knee joint
Low back strain/pain Often occurs when patients have to arise from sitting with their legs
abducted their thorax flexed forward, and their back in exaggerated
lordosis; PT to advise about elevated seats, self-help devices to assist in
arising and reducing low back strain; use NSAIDs and low-dose tricyclic
antidepressants for several weeks or as maintenance therapy; use
cyclobenzaprine for several days during acute severe pain; mexiletine
treatment to reduce myotonia in paraspinous muscles may be necessary on
a chronic basis to decrease the susceptibility of these muscles to recurrent
strain/pain
Anterior shoulder pain Develops, as scapular fixator weakness becomes apparent; rotator cuff
strain, tendonitis, and overuse spasm in the anterior deltoid and upper
trapezius muscles contribute individually or in combination; need to avoid
lifting with arms outstretched and abducted; PT instruction is helpful; for
acute pain use rest, NSAIDs, low dose tricyclic antidepressants, and
cyclobenzaprine; long-term mexiletine therapy may also be necessary for
chronic or recurrent shoulder muscle pain; obtain orthopedic consultation
if rotator cuff injury is suspected
Myotonia Mexiletine is often more effective than phenytoin

Generalized weakness Wheelchair; use intermittently with braces in earlier stages of generalized
weakness (e.g. for traveling long distances within the home or trips
outside)
Respiratory muscle weakness Consider nasal ventilation, initially at night and subsequently during
with ventilatory failure daytime; in selected cases, consider positive pressure ventilation via
tracheostomy
Neck weakness Fitted collar; head supports in car seats and chairs
Ptosis Lid elevating crutches or props on eyeglasses; lid surgery is necessary only
infrequently
Recurrent dislocation of the Mandible surgery is infrequently required; guidelines for preoperative and
mandible due to weakness postoperative care need to be followed carefully; often there is muscle
of temporalis and spasm in the masseter muscles during attacks of pain with dislocation;
masseter muscles local heat packs and mexiletine therapy on a long-term basis can lessen the
severity of the episodes; occasionally an attack of mouth closure with
dislocation of the mandible creates a medical emergency for mouth
breathers; patients on critically timed oral medication
Moxley/Meola 70
Table 5. Maternal-obstetric complications in DM
Maternal complications
Increased muscle weakness, especially respiratory
Increased rate of spontaneous abortion
Reduced fetal movements and hydramnios
Prolonged and often ineffective labor
Sensitivity to general anesthesia (arrhythmias, apnea
following cesarean section)
Obstetric complications
Retained placenta
Placenta previa
Neonatal deaths
Table 6. Preoperative and postoperative care and complications of anesthesia in DM
Preoperative evaluation
ECG, pulmonary function testing (including supine and upright forced vital capacity)
and arterial blood gas measurements
Intraoperative monitoring
Monitor ECG; measure arterial blood pressure; use a peripheral nerve stimulator to
monitor blockade of peripheral muscle; monitor temperature; warm mattress; warm
intravenous fluids; maintain humidification of anesthetic gases
Postoperative care
Retain endotracheal tube in place and ventilate if necessary on an intensive care unit;
monitor respiratory efficiency with checks of oxygen saturation and pCO2 for at least
24 h postoperatively to avoid overlooking delayed onset apnea; use controlled flow
oxygen therapy with close monitoring of ventilation in patients relying upon hypoxic
drive due to chronic respiratory insufficiency; provide early physiotherapy; monitor ECG;
keep patient warm; monitor swallowing closely to check for signs of aspiration; treat all
infections vigorously
Anesthetic agents
When possible use local or regional anesthesia, such as, an epidural block; avoid
suxamethonium and other depolarizing muscle relaxants; avoid or use only minimal
doses of thiopental; for muscle relaxation use short-acting agents, such as atracurium
or vecuronium; avoid or use only minimal doses of opiates to avoid respiratory depres-
sion; when possible, avoid general anesthesia; if necessary, use combination of nitrous
oxide/oxygen mixture with an agent, such as 0.8% enflurane or 1.0% isoflurane; use
anticholinesterases, such as neostigmine, with care; may be preferable to ventilate the
patient until residual curarization wears off
Table 7. Different measures to define the natural history of DM
Skeletal muscle weakness (manual muscle testing, quantitative isometric muscle contraction
force testing) and wasting (dual energy x-ray absorptiometry, total body potassium, magnetic
resonance imaging, urinary creatinine)
Myotonia (electromechanical measures, in vitro techniques)
CNS (neuropsychological tests, imaging, position emission tomography)
Respiratory (forced vital capacity, sleep apnea testing, electromyography, ventilatory drive)
Cardiac (electrocardiogram, 24 h monitor, echocardiography, magnetic resonance imaging,
position emission tomography)
G1 (cinevideoswallowing, gastric emptying time)
Endocrine (gonadal, pituitary axis, thyroid, pancreas, hormone levels, hormone challenges,
oral glucose tolerance testing, intravenous glucose tolerance testing)
testosterone or placebo in a randomized controlled fashion to 40 men with

DM [64]. Active treatment involved testosterone enanthate (3 mg/kg per week)
for 12 months. After 1 year those patients who received testosterone had a
significant increase in muscle mass. However, they had no significant improve-
ment in strength or function [64]. There is a definite role for testosterone in
providing replacement therapy in DM for testosterone deficiency. The use of
testosterone as a treatment for muscle wasting requires further study.
In addition to the occurrence of testosterone deficiency, many DM patients
have an abnormal release and regulation of growth hormone [1, 2]. A thera-
peutic trial of growth hormone isolated from pooled human brains [65] raised
hope that it might be a useful treatment. Daily intramuscular injections for
2 weeks led to an improvement in nitrogen balance and a shortening of the
abnormally prolonged PR interval that was present at baseline [65]. However,
the patients had no increase in muscle strength or function. A more recent
trial using recombinant human growth hormone given daily for 16 weeks to
7 men with classical DM produced a 10% increase in lean body mass and a
28% increase in muscle protein synthesis [66]. Despite this improvement in
protein anabolism there was no significant increase in strength or function.
These studies of growth hormone and testosterone treatment suggest that
future longer-term therapeutic trials may prove beneficial since both growth
hormone and testosterone produced an increase in muscle mass. However, it
is unclear whether the increase in muscle tissue actually represented an increase
in the contractile proteins. Further studies are needed.
Moxley/Meola 72
In recent years human recombinant DNA-derived insulin-like growth
factor-1 (IGF-1) has become available. To determine if IGF-1 is more effective
than its parent, growth hormone, investigators have recently completed a
therapeutic trial of IGF-1 in DM [67]. Seven men and 2 women received a
4-month trial of 5 mg of human recombinant IGF-1 subcutaneously at 12-
hour intervals. Four patients, whose dosage of IGF-1 was greater than 0.070 mg/
kg per dose, showed an improvement both in strength and in function. Patients
also showed improvements in the rate of protein synthesis and in insulin
sensitivity. The results of this trial of IGF-1 are promising. Future trials merit
consideration. However, there are restraining circumstances. The cost of
IGF-1 (as well as growth hormone) is very high. There is also a risk of
accelerating the growth of a coexisting neoplasm or of worsening prostatic
hypertrophy with IGF-1 therapy.
Insulin is an essential anabolic hormone and skeletal muscle resistance
to insulin occurs in DM [2, 60]. A loss of the usual anabolic actions of insulin
may contribute to the muscle wasting and weakness in DM [60]. Therapeutic
trials to reverse skeletal muscle insulin resistance are in progress. One trial is
using the newly marketed thiazolidinedione derivative, troglitazone [38, 68].
A recent case report indicates that troglitazone has reduced insulin resistance
and decreased myotonia in a patient with DM [69]. This forecasts a beneficial
result of the trial of troglitazone in DM. The results of the troglitazone
study that is in progress will help to establish if insulin-enhancing agents, like
thiazolidinediones, are useful in DM.
A previous study has identified a decrease in circulating levels of dehydro-
epiandrosterone (DHEAS) in patients with DM [70]. This finding has raised
the possibility that a deficiency of this adrenal hormone may contribute to
the muscle wasting and weakness. To investigate this possibility researchers
have initiated therapeutic trials using daily intravenous infusions of 200 mg
of DHEAS. They have found very encouraging results in their initial open
trials [71, 72]. Patients have shown increased muscle function and decreased
myotonia. Further controlled studies are in progress.
Trials of Antimyotonia Therapy

The pathomechanism that underlies the myotonia in DM is unclear. Some
evidence suggests that it involves apamin-sensitive potassium channels
[38, 73, 74]. However, previous reports have also described alterations in
sodium conductance [38, 75]. No explanation is available to account for the
absence of myotonia during the first several years of life in patients with
congenital DM, and no explanation is available to account for the gradual
appearance of clinical and electrical myotonia that occurs later in childhood
[1, 2].
Only a few small scale controlled studies that evaluate antimyotonia therapy
are available in DM [7579]. In one investigation 7 men and 3 women participated
in a randomized, blinded, crossover study [78]. Each received a 2-week treatment
of procainamide (250 mg every 6 h for 7 days and then 500 mg every 6 h for the
2nd week) or disopyramide (100 mg every 8 h for the 1st week then 200 mg every
8 h for the 2nd week). Both medications decreased myotonia but did not produce
a change in grip strength. Five patients developed side effects with procainamide
and 6 patients had side effects with disopyramide.
Another trial of antimyotonia treatment involved 6 patients in a compli-
cated double-blind study design [77]. The study involved 8 separate 15-day treat-
ment periods: 2 periods of phenytoin therapy (1 of 200 mg per day, the other of
300 mg per day), and 2 periods of treatment with carbamazepine (1 using 600 mg
per day, the other using 800 mg per day). Each of the above 15-day trial periods
had a preceding 15-day trial of placebo treatment. Both phenytoin and carbama-
zepine reduced myotonic stiffness. Patients tolerated both medications well.
Another trial involved 6 men and 4 women treated in an open fashion
with tocainide [76]. Each patient received 400 mg a day followed by increases
in increments of 400 mg a day to a total dose of 1,200 mg daily. After 2 weeks
of treatment the patients had a 3680% reduction in myotonic stiffness. No
significant change in their electrocardiograms occurred. Three of the 10 pa-
tients experienced transient dizziness and nausea which disappeared following
a reduction in the dosage of tocainide to 800 mg a day.
The most encouraging trial of antimyotonia therapy has demonstrated a
superior efficacy of mexiletine and tocainide compared to phenytoin and
disopyramide in 9 patients with DM [75]. Daily doses of 400600 mg of
mexiletine produced a maximum decrease in myotonic stiffness equal to that
of 1,200 mg of tocainide and produced a significantly better reduction of
myotonia than that achieved with 300 mg of phenytoin daily. The investigators
have recommended mexiletine treatment over tocainide in view of the increased
risk of bone marrow suppression with tocainide. Future large scale trials of
mexiletine in DM deserve consideration. Such trials will help to determine if
mexiletine is safe and effective in reducing not only myotonic stiffness of
skeletal muscle, but whether it can ameliorate the myotonia that interferes
with swallowing and gastrointestinal function [1, 2].
Opportunities for Future Treatment: Studies Using Animal Models
The development of useful animal models for DM remains a challenge.

Mouse knockout models of the DM gene and overexpression models have
not produced animals with disease manifestations that closely resemble human
Moxley/Meola 74
DM [12, 13, 38, 80]. However, more recent studies have raised hopes that
abnormal CTG repeat expansions in the small and moderate range can be
successfully transmitted in successive litters of mice and that the clinical and
histopathologic alterations observed are similar to human DM [13, 38, 81,
82]. There are also hopes that knockout models in mice of genes that flank
the DM locus will provide clues about the molecular pathomechanism of DM
and about the role that flanking genes may have in some of the manifestations
of the disease [8, 12, 13, 38]. There is optimism that in the near future new
ideas will be forthcoming from animal studies, and that ultimately the investi-
gations using animal models will lead to effective treatments for the various
manifestations of DM in humans.
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Dr. Richard T. Moxley, Department of Neurology, University of Rochester,

School of Medicine and Dentistry, New York, NY 14642 (USA)
E-Mail tforrester@mail.neurology.rochester.edu
Moxley/Meola 78
............................
Muscle Ion Channel Diseases
Reinhardt Rudel, Karin Jurkat-Rott, Frank Lehmann-Horn
Department of Physiology, University of Ulm, Germany
The combination of electrophysiological and molecular genetic investi-

gations has led to the identification of a growing family of ion channel diseases
that are caused by mutations in various genes encoding voltage-gated or ligand-
gated ion channels. Since ion channels provide the basis for the regulation of
excitability in nerve and muscle cells, it is not surprising that such mutations
may lead to dysfunction of these highly specific, membrane-spanning proteins
that results in hyper- or hypoexcitability of the respective cells. Channelopathies
are known both with skeletal and cardiac muscle. These are the myotonias,
periodic paralyses, and the inherited cardiac arrhythmias (the long QT syn-
dromes), respectively. Malignant hyperthermia susceptibility (MHS) and cen-
tral core disease are two skeletal muscle disorders that are not associated with
excitation proper, but with excitation-contraction coupling. This chapter deals
with hereditary skeletal muscle diseases caused by mutations in genes encoding
voltage-gated ion channels. Defects in the sodium, calcium and chloride chan-
nel, but not in the potassium channels of skeletal muscle are known (see
table 1).
Sodium Channelopathies
Hyperkalemic Periodic Paralysis, Paramyotonia congenita and

Potassium-Aggravated Myotonia
Clinical and Electrophysiological Characterizations. These three domi-
nantly inherited diseases are all linked to SCN4A, the gene encoding the a
subunit of the skeletal muscle sodium channel. Hyperkalemic periodic paralysis
(HyperPP) is characterized by episodes of muscle weakness associated with
hyperkalemia, often with signs of myotonia in the interval between attacks.
Table 1. List of hereditary muscle diseases caused by pathologic function of voltage-dependent ion
channels (voltage dependence of RYR1 is given via DHPR)
Channel Disease Locus Protein Mutation Pathology

number
Na+ HyperPP 4 Incomplete channel inactivation

PC 17q23 hSkm-1 10 causes sustained small or large
PAM 7 membrane depolarization
Ca+
H q31-32
HypoPP DHPR 3 Large membrane depolarization
H
MHS-5 DHPR 2 Altered muscle metabolism caused
?20
H 19q12-13.2
MHS-1 RYR1 by faulty excitation-contraction
CCD RYR1 ?5 coupling
Cl DMC 7q35 ClC1 ?5 Reduced Cl conductance causing

RMC ClC1 ?30 after-depolarization
MHS-1 and MHS-5>Malignant hyperthermia susceptibility type 1 and type 5; CCD>central core
disease; DMC>dominant myotonia congenita; RMC>recessive myotonia congenita.
Paramyotonia congenita (PC) is characterized by a stiffening of the muscles

during exercise or when exposed to the cold. In PC the stiffness can give way
to weakness, which then may persist for several hours even when the muscles
are rapidly rewarmed. Potassium-aggravated myotonia (PAM) is characterized
by severe permanent myotonia or fluctuating muscle stiffness that is most
prominent about 20 min after exercise [delayed onset myotonia; 1, 2]; the
mild form is symptomatically very similar to dominant myotonia congenita
Thomsen (which is a chloride channel disease [3]).
To date, 21 missense mutations have been discovered leading to the differ-
ent symptoms described above (fig. 1).
The three allelic diseases do not always appear in their pure forms, e.g. PC
patients often suffer from spontaneous episodes of weakness which may go along
with an elevated serum potassium level. As a clear distinction, HyperPP patients
never show substantial stiffness when cold, and muscle weakness never occurs
in PAM. Although intermediate forms are frequent, it seems reasonable to retain
the classification of three separate nosological entities because, in the pure forms,
not only the symptoms but also the recommended treatments differ.
The clinical symptoms of the three diseases, muscle stiffness and in
HyperPP and PC muscle weakness, are not present all the time. Rather, they
are elicited by various stimuli. A typical trigger for an episode of weakness
in HyperPP is rest after a heavy workload; stiffness and weakness in PC are
Rudel/Jurkat-Rott/Lehmann-Horn 80
Fig. 1. Subunits of the voltage-gated sodium channel of skeletal muscle. The a subunit
consists of four highly homologous domains (repeats IIV) containing six transmembrane
segments each (S1S6). The S5S6 loops form the ion selective pore, and the S4 segments
contain positively charged residues conferring voltage dependence to the protein. The repeats
are connected by intracellular loops; one of them, the IIIIV linker, contains the supposed
inactivation particle of the channel. b is an auxiliary subunit. When inserted in the membrane,
the four repeats of the protein fold to generate a central pore as schematically indicated at
the bottom on the right. Conventional 1-letter abbreviations are used for the replaced amino
acids. The different symbols used for the point mutations indicate the resulting diseases as
explained at the bottom of the left-hand side.
triggered by muscle exercise and/or exposure of the muscles to the cold; inges-
tion of potassium-rich food may induce muscle stiffness in PAM patients. In
each case, the symptoms taper off spontaneously within a few hours. The
episodes reduce the patients quality of life considerably, although they may
be prevented to a certain extent by appropriate behavior and symptomatic
treatment with drugs [for more details, see 1].
Electrophysiological Basis of the Symptoms of the Three Sodium Channel-
opathies. Stiffness and weakness are caused by the same pathogenetic mecha-
nism, namely a long-lasting depolarization of the muscle fiber membranes. For
the explanation of the pathogenesis of the diseases it is important that patients
possess two populations of sodium channels, i.e. mutant and wild type.
Muscle Ion Channel Diseases 81

When after an action potential the membrane depolarization caused by
the mutant channels is mild (510 mV), the wild-type sodium channels that
physiologically recover from inactivation can be reactivated by this low resting
potential [2]. This activation may lead to one or more successive action
potentials, which is the basis for the involuntary muscle activity that the
patients experience as muscle stiffness. The repetitive firing ends either with
an increase of the depolarization, which would leave the wild-type channels
in the state of inactivation, or a decrease of the depolarization, which would
prevent the wild-type channels from getting reactivated. Such a hyperexcitable
state can be computer-simulated and mimicked with anemone toxin [4].
When the depolarization is strong (2030 mV), the majority of the wild-
type channels remain in the state of inactivation, which gradually renders
more and more muscle fibers inexcitable. This is the basis of the process of
muscle weakness turning into complete flaccid paralysis. This state is also
temporary, as the excitability of the muscle fibers returns when, by action of
the sodium/potassium pump, the membrane resting potential slowly assumes
the physiological value of about 80 mV [2].
Knowledge Obtained from Electrophysiological Experiments on Mutant
Channels Expressed in Cultured Cells. A detailed specification of the altered
channel properties produced by the various disease-causing mutations was
possible by heterologous expression of the respective mutant cDNA in cultured
cells and subsequent studies of the sodium currents conducted by the mem-
branes of such cells upon depolarizing steps. Whole-cell and patch-clamp
recordings showed that all mutations affect the channel inactivation in one
way or another (fig. 2). The alterations that were observed with such mutant
channels were a reduced speed of current decay following a depolarization
step, a more or less incomplete decay of the current, an increased speed of
the recovery from inactivation, a shifted position of the steady-state inactiva-
tion curve (Hodgkin and Huxleys h= curve) and an altered degree of uncoup-
ling of inactivation from activation [2].
The alterations found with the different disease-causing mutants are sum-
marized in table 2. They can be generalized as follows. (1) Sodium currents con-
ducted by HyperPP mutants show a large fraction of persistent current and an
incomplete slow inactivation. These changes may cause strong and long-lasting
depolarization, which is the basis of weakness in HyperPP [4]. (2) Sodium cur-
rents conducted by PC mutants are characterized by a slowing of fast inactiva-
tion. This change explains paradoxical myotonia. Another typical change is
acceleration of recovery from inactivation and a left shift of the steady-state
inactivation curve. In combination with an increased persistent current it could
explain the cold-induced weakness. (3) Sodium currents conducted by PAM
mutants are characterized by an increased persistent fraction and/or slowing of
Fig. 2. Hinged-lid model of fast inactivation of sodium channels and the effects of
mutations at various locations on the current decay. a Birds eye view on the channel consisting
of four similar repeats (IIV). The channel is cut and spread open between repeats II and
III to allow the view on the intracellular loop between repeats III and IV. The loop acts as
the inactivation gate whose hinge GG (>a pair of glycines) allows it to swing between
two positions, i.e. the noninactivated channel state (pore open, left panel) and the inactivated
state (pore blocked by the plug IFM representing amino acid sequence isoleucine, phenylal-
anine, methionine, right panel). b The substitution of E (Glu) for Gly-1306 slows channel
inactivation (left two panels, compare fast current decay in wild-type channel on far left)
and leads to a life-threatening form of PAM. The designed substitution of QQQ (Gln-Gln-
Gln) for IFM (Ile-Phe-Met) completely abolishes channel inactivation (two right hand panels)
proving that the loop between repeats III and IV is indeed the inactivation gate [adapted
from 15, 16].
fast inactivation. These alterations explain the slight depolarization which

causes the myotonia. An also existing right shift of the steady-state inactivation
curve might be the reason why PAM patients do not experience weakness.
Discussion of the Various Disease-Causing Mutations. Most of the amino
acid substitutions caused by disease-causing mutations are in the inactivating
linker between repeats III and IV or adjacent in the voltage-sensing segment
S4 of repeat IV. Almost all remaining mutations are situated at the inner side
of the membrane where they could impair the docking site for the inactivation
particle (fig. 2).

Table 2. Summary of the electrophysiological properties of PAM, PC and HyperPP
sodium channel mutations
Disease Slowing of fast Persistent Steady-state fast Recovery from Impaired slow
inactivation current inactivation fast inactivation inactivation
PC ++ + k ++
PAM + +/++ K /+
HyperPP +++ +
+ to +++ indicate the severity of the alteration; means no change. Arrows show
direction of shift of the steady-state fast inactivation curve (to the left>to more negative
potentials).
Three of the mutations in the III/IV linker (Gly-1306-Ala,Val,Glu) produce

different amino acid substitutions for one of a pair of glycines proposed to act
as the hinge for the inactivation gate. They all cause PAM and, interestingly,
the more the structure of the substituting amino acid differs from the physio-
logical Gly-1306 (longer side chains and/or greater charge) the more intensive
is the hyperexcitability of the muscles and the more severe the myotonia. Alanine,
with a short side chain, results in a benign, often subclinical form of myotonia.
Valine, having a side chain of intermediate size, causes moderate exercise-in-
duced myotonia. Glutamic acid, an amino acid with a long side chain, causes
permanent myotonia, the most severe form of the disease.
Thus the natural mutations affecting Gly-1306 provided evidence for an
increased rigidity of the amino acid chain at the position of the highly con-
served pair of glycines increasingly hampering channel inactivation [5]. Muta-
tions at this hinge site also altered channel activation and deactivation.
A similar correlation between the structural differences between wild-type
and mutant amino acids on one hand, and the severity of clinical symptoms
on the other was found for four PC-causing mutants at another identical site
(Arg-1448-Cys,His,Pro,Ser) near the extracellular face of IVS4. This finding
led to a systematic application of site-directed mutagenesis in this supposed
channel activation domain. All tested mutants primarily affected channel inac-
tivation. Therefore, it was hypothesized that depolarization-induced move-
ments in IVS4 concern both the inactivation gate and the docking site for the
inactivation particle [6].
Pathogenesis of Symptoms. Although the symptoms in PC are very much
aggravated in the cold, the sodium currents conducted by PC-causing mutants
expressed in heterologous cells did not show a corresponding dependence on
temperature. The mechanism by which the cold enhances muscle stiffness in
PC patients is not completely clear. Both the time constant of fast inactivation
and the persistent current increase with cooling, and mutant and normal
channels show the same temperature dependence; however, the absolute figures
are larger for mutant channels at any temperature. Therefore, it was proposed
that a certain threshold has to be exceeded in the cold environment to induce
myotonic and/or paralytic symptoms. In contrast to the cold-induced symp-
toms, the pathogenesis of the potassium-induced stiffness and paralysis is well
understood. The physiological depolarization, which follows an elevation of
serum potassium according to Nernst increases the open probability of the
sodium channels and unmasks their inactivation defect. Thus, potassium exerts
its effect via depolarization.
Open Questions. It is not entirely clear why patients with PC are temper-
ature-sensitive whereas those with PAM and HyperPP are not, since a specific
temperature dependence could not be found with any of the PC-causing mu-
tants in vitro. On the other hand, the cold-induced weakness is clearly linked
to membrane depolarization due to the increased sodium inward current, so
that a mechanism other than via the sodium channel seems unlikely. As to
the aggravation of the clinical symptoms upon potassium intake with PAM
and HyperPP patients, studies on PAM- or PC-causing mutants also showed
sensitivity to extracellular potassium. Therefore, the effect of potassium is
most likely explained by a membrane depolarization. Further not yet explained
problems with sodium channelopathies are the occurrence of a myopathy with
the HyperPP-causing mutation Thr-704-Met and the pathology of normoka-
lemic periodic paralysis.
Calcium Channelopathies
Two Important Calcium Channels Are Expressed in Skeletal Muscle

These are the dihydropyridine receptor, DHPR, an L-type voltage-depend-
ent calcium channel, and the ryanodine receptor, RYR1, coupled to it (fig. 3, 4).
They are situated in the triadic junctions of the transverse tubular system and
the sarcoplasmic reticulum (SR), respectively.
In skeletal muscle, the DHPR appears to be physiologically unimportant
as ion-conducting channel; it rather functions as a voltage sensor for the
ryanodine receptor (RYR1) which releases calcium from the SR, thus initiating
contraction [7]. The a1S subunit of the a1S-a2/d-b1-c pentameric DHPR com-
plex interacts with the RYR1 via the IIIII interlinker. Disease-causing muta-
tions are known in the genes for either channel. Certain point mutations in
CACNAS, the gene encoding a1S, cause familial hypokalemic periodic paralysis
(HypoPP), a disease characterized by episodes of muscle weakness. Other

point mutations in this gene, as well as mutations in the RYR1 gene, cause
malignant hyperthermia, a potentially lethal condition triggered by certain
anesthetics.
Hypokalemic Periodic Paralysis

Characterization of the Disease. The major symptom of this dominantly
inherited disease, episodes of generalized paralysis, may occur less frequently
and be on average of longer duration than in HyperPP. Decisive for the
classification is the level of serum potassium, [K]e, during a paralytic attack,
which may fall below 2 mM in HypoPP, whereas in HyperPP it may rise
beyond 4.5 mM. The hypokalemia is assumed to be caused by stimulation of
the Na/K pump by insulin which is the physiological mechanism by which
potassium ions are transported from the extracellular to the intracellular space.
Very low [K]e causes instability of the membrane potential because then the
potassium conductance approaches zero. Even in normal muscle, a [K]e
01.0 mM causes membrane depolarization, not hyperpolarization. An in-
crease in [K]e is then the easiest therapy for normalization and stabilization
of the resting potential [for a review, see 1].
The Genetic Defect in HypoPP. The disease is linked to chromosome 1q31-
32 and cosegregates with the gene encoding the DHPR a1S subunit which is
located in this region. Sequencing of cDNA derived from muscle biopsies of
patients revealed three mutations [6, 8]. Two of them are analogous predicting
arginine to histidine substitutions within the highly conserved S4 regions of
repeats II and IV (Arg-528-His and Arg-1239-His, respectively), the rare third
mutation predicts an arginine to glycine substitution in IVS4 (Arg-1239-Gly)
(fig. 3).
Pathogenesis. Electrophysiological investigation of biopsied muscle speci-
mens from HypoPP patients in vitro showed that the paralysis induced by
hypokalemia is due to a sustained depolarization of the sarcolemma to about
50 mV. The crucial role of the depolarization for the pathogenesis of paralysis
was demonstrated by experiments in which openers of ATP-sensitive potassium
channels were given in vitro. Their hyperpolarizing action was able to prevent
muscle weakness or even restore normal force.
The pathogenesis of the membrane depolarization is still unclear. L-type
calcium currents conducted by Arg-528-His and Arg-1239-His mutant chan-
nels were studied in various native and heterologous expression systems. In
summary, they revealed neither a significant alteration of gating nor a change
of the resulting calcium transients that could explain the membrane depolariza-
tion following a decrease in serum potassium. It seems likely that additional
structures are involved that interact somehow with the DHPR and/or may
respond to hypokalemia. Recently, a reduced potassium current conducted
Fig. 3. Subunits of the voltage-gated calcium channel of skeletal muscle. The a1 subunit
resembles that of the sodium channel, however the function of the various parts, e.g. the
IIIIV linker, may not be the same. a2/d, b1b4, and c are auxiliary subunits. Mutations in
the a1 subunit have been described for HypoPP and MHS. Conventional 1-letter abbreviations
are used for the replaced amino acids. The symbols used for the point mutations indicate
the resulting diseases as explained at the bottom of the left-hand side.
by ATP-dependent channels was reported in biopsied muscle specimens from

three HypoPP patients carrying the Arg-528-His mutation [9]. The authors
proposed a contribution of this finding to the pathogenesis of HypoPP.
Malignant Hyperthermia Susceptibility

Not a Disease, But a Genetically Determined Disposition. Susceptibility to
malignant hyperthermia (MH), a potentially lethal event in carriers of voltage-
or ligand-gated calcium channel mutations, may cause clinically inconspicuous
individuals to respond abnormally when exposed to volatile anesthetics or
depolarizing muscle relaxants. MH is generated by a pathologically high in-
crease of the myoplasmic calcium concentration during exposure to the trig-
gering agents. This leads to increased muscle metabolism and heat production
resulting in symptoms of muscle rigidity, hyperthermia associated with meta-
bolic acidosis, hyperkalemia and hypoxia [10, 11] The metabolic alterations
usually progress rapidly. Early administration of dantrolene, an inhibitor of

Fig. 4. Cartoon of the homotetrameric ryanodine receptor (RYR1), the calcium release
channel situated in the membrane of the SR. The cytosolic part of the protein complex, the
so-called foot, bridges the gap between the transverse tubular system and the SR. The
mutations shown cause susceptibility to MH and central core disease. Conventional 1-letter
abbreviations are used for the replaced amino acids whose positions are given by the ascribed
amino acid numbers of RYR1 [modified after 7].
calcium release from the SR has successfully aborted numerous fulminant

crises and has reduced the mortality rate to less than 10%.
MHS is genetically heterogeneous. In many families, mutations in the
gene encoding RYR1 (fig. 4) have been found. Also two mutations in the
DHPR a1 subunit have been described (see below), which underlines the
functional link between the two protein complexes. Another possible locus
contains the gene encoding the a2/d subunit of the DHPR [6].
MHS Due to RYR1 Mutations. More than 20 disease-causing point muta-
tions in RYR1 have been identified, all situated in the long N terminus of the
protein, the so-called foot of the channel complex (see fig. 4) which contains the
binding sites for various activating ligands like calcium (lM), ATP, calmodulin
(which binds in the absence of calcium), caffeine and ryanodine (nM), and
inactivating ligands like calcium (110 lM) and magnesium in mM concentra-
tions. The diagnostically important increased sensitivity of MH-susceptible
muscle to caffeine is considered to be caused by an altered RYR1 function.
Functional tests, so far only performed with mutant porcine muscle showing
an MHS equivalent in isolated SR vesicles, have shown that calcium regulation
is disturbed. Lower calcium concentrations activate the channel to a higher
than normal level, and higher than normal calcium concentrations are required
to inhibit the channel. Investigations of reconstituted RYR1 in lipid bilayers,
designed to find the reason for the increased caffeine sensitivity, led to contro-
versial results. Single-channel measurements on RYR1 did not show increased
caffeine sensitivity whereas pharmacological studies showed increased sensitiv-
ity [11]. Functional characterization of the various mutations in the N-terminus
and the central part of the foot gave similar results, i.e. increased sensitivity
of the mutant RYR1 to activating concentrations of calcium and exogenous
and diagnostically used ligands such as caffeine, halothane, and 4-chloro-m-
cresol.
Overexpression of mutant ryanodine receptors in normal human primary
muscle cells also led to an increased calcium response during exposure to a
triggering agent [11]. A reduced inhibition of calcium release by magnesium has
been reported for MHS muscle and proposed as the major pathomechanism of
MHS.
MHS Caused by L-Type Channel a1 Subunit Mutations. Cosegregation of
MHS with markers on chromosome 1q32 was shown for two families. Screen-
ing for causative mutations in the candidate gene, CACNA1S, revealed arginine-
1086 to histidine and cysteine substitutions in the intracellular interlinker
connecting domains III and IV of the protein (fig. 3), a region that hitherto
was not known to be important for excitation-contraction coupling [6, 11].
Central Core Disease

This congenital proximal myopathy with structural alterations mainly of
type 1 fibers is allelic to MH [10]. It is transmitted as an autosomal dominant
trait. Name giving are central areas along the whole fiber length that contain
structured or unstructured myofibrils and lack mitochondria. Affected indi-
viduals show hypotonia at birth (floppy infant syndrome). Later in life muscle
strength usually improves except for rare cases showing progressive muscle
weakness. Exercise-induced muscle cramps are often reported. Anesthesia-

induced events of MH have been reported, and central core disease patients
usually give positive results in the diagnostic in vitro contracture test. These
observations induced genetic linkage studies on chromosome 19q12-13.2, the
first MH locus. Linkage was indeed found and mutations in RYR1 were
detected (see fig. 4).
Even though events similar to MH may occur in numerous muscle dis-
orders during general anesthesia, a genetic relation to MH exists with certainty
only in central core disease and possibly in the King-Denborough syndrome,
a disease characterized by dwarfism, scoliosis, ptosis and further skeletal or
muscular symptoms [11]. Data on the molecular genetics of the latter disease
is still missing. Lethal events during anesthesia have been reported for both
of these rare diseases.
Chloride Channelopathies
Myotonia may not only be due to sodium channel mutations as in PAM,

but also to changes in the chloride channel CLC-1 as in myotonia congenita
(Thomsen). The clinical symptoms of these two diseases are almost indistin-
guishable although their pathogenetic mechanisms are rather different [12].
Myotonia congenita
Characterization of the Two Types. This disease is transmitted with either
a dominant or recessive mode of inheritance; both types are caused by muta-
tions in CLCN1, the gene encoding the major skeletal muscle chloride channel
[13]. Muscle stiffness is temporary and can affect every skeletal muscle of the
body. Myotonic stiffness is most pronounced when a forceful movement is
abruptly initiated after the muscles were rested for 5 min or more. For instance,
after making a hard fist, the patient may not be able to extend the fingers
fully for several seconds. Myotonia decreases or completely subsides when the
same movement is repeated several times (warm-up phenomenon), but it always
recurs after a few minutes of rest. On rare occasions, a sudden, frightening
noise may cause instantaneous generalized stiffness. The patient may then fall
to the ground and remain rigid and helpless for some seconds or even minutes.
Even more disabling may be a transient weakness. Typically myotonic muscles
generate a characteristic pattern in the acoustic electromyogram, i.e. bursts of
action potentials with amplitude and frequency modulation, so-called dive
bombers [3].
The dominant form is very rare, as less than 10 different families were
identified at the molecular level. The recessive form is much more frequent,
between 1:23,000 and 1:50,000. Males seem to be affected more often than
Fig. 5. Membrane topology model of the skeletal muscle chloride channel monomer,
CLC-1, originally based on hydropathy analysis. The functional channel is a homodimer.
The different symbols used for the known mutations leading to dominant Thomsen-type
myotonia and recessive Becker-type myotonia are explained at the bottom on the left.
Conventional 1-letter abbreviations are used for replaced amino acids [modified after 13].
females with a ratio of 3:1 when only the typical clinical features are taken
into account. However, family studies disclosed that women are affected at
the same frequency though to a much lesser degree.
Molecular Pathology. The muscle stiffness is caused by the fact that,
following voluntary excitation, the membranes of individual muscle fibers may
continue for some seconds to generate runs of action potentials. This activity
prevents immediate muscle relaxation from occurring. The overexcitability is
caused by a permanent reduction of the resting chloride conductance of the
muscle fiber membranes. The high chloride conductance is necessary for a
fast repolarization of the transverse tubular membranes, when these tend to
stay depolarized by potassium accumulated in the tubules during tetanic muscle
excitation [3].
Both dominant and recessive myotonia congenita are linked to chromo-
some 7q35 and CLCN1, the gene encoding the chloride channel. It spans at
least 40 kb and contains 23 exons whose boundaries have been located [13].
Knowledge Obtained from Electrophysiological Experiments on Mutant
Channels Expressed in Cultured Cells. Functional expression of CLCN1 has

6
been accomplished in Xenopus oocytes, human embryonic kidney (HEK-293)
cells and the insect cell line Sf-9 [13]. The resulting currents were similar to
those found in native muscle fibers. Electrophysiological studies of wild-type
and mutant channel proteins have provided first insight into the pharmacology
and structure-function relationships of CLC-1, and led to the identification
of regions involved in gating and permeation [13]. Inferences from experiments
with the chloride channel CLC-0 and studies of CLC-1 constructs strongly
suggest that functional channels are formed as homodimers [6].
More than 30 point mutations and three deletions have been found in
the channel gene, and they cause either dominant or recessive myotonia con-
genita by producing change or loss of function of the gene product (fig. 5).
Experiments with myotonia-generating drugs showed that blockade of 50%
of the physiological chloride current is not sufficient to produce myotonic
activity. This could be the reason why heterozygous carriers of recessive muta-
tions that completely destroy the channel function are without clinical myo-
tonia. Dominant inheritance is explained by a mutant channel monomer that
can form dimers and, in doing so, produces a dominant negative effect. The
most common feature of the thereby resulting chloride currents is a shift of
the activation curve towards more positive membrane potentials reducing the
total chloride conductance (fig. 6). Surprisingly, the degree of the shift and
the clinical severity sometimes disagree, e.g. Gln-552-Arg causes an unusually
large potential shift, however a very mild clinical phenotype, myotonia levior
[6].
Open Questions. No modern experiments have been reported designed to
test the explanation for the warm-up phenomenon that stems unchallenged
from pre-molecular biology days. Several mutations were found that lead to
myotonia congenita which under certain circumstances is dominantly and
under other circumstances recessively transmitted. What the decisive circum-
stance is has so far not been elucidated. Perhaps it is connected to one or the
other polymorphisms in CLCN1 that have no functional consequences within
the wild type.
Fig. 6. Recordings from human skeletal muscle CLC-1 channels expressed in a mamma-
lian cell line. Currents from normal (WT) and dominant myotonia-causing mutant (Gly-
200-Arg) channels are compared. a, b Macroscopic currents, recorded in the whole-cell mode,
were activated from a holding potential of 0 mV by voltage steps to potentials of 145 to
+95 mV, and deactivated after 400 ms by polarization to 105 mV. c Voltage dependence of
the relative open probability that is much reduced for the mutant channel in the physiological
potential range. All mutations that cause such a voltage shift have dominant effects [modified
after 14].

All dominant mutations shift the activation curve of CLC-1 towards more
positive membrane potentials. There is, however, no simple relation between
the amount of shift and the severity of symptoms seen in patients carrying
the various mutations. Apparently other, not yet detected factors act in an
ancillary fashion. Such factors may also play a role in cases of recessive
myotonia where the symptoms fluctuate [14]. Even more enigmatic is the
finding that some recessive mutations do not lead to the loss of a gene product
but to channels that in the usual expression systems conduct chloride currents
with normal amplitude and gating behavior.
Relatively little is known about the structure/function relation of CLC-1.
Several groups of investigators are involved in finding the pore region of the
channel and the mechanism of gating. Molecular biological methods, such as
site-directed mutagenesis or use of channel chimeras, will hopefully help to
overcome this unsatisfactory situation.
Acknowledgments
The support by the Deutsche Forschungsgemeinschaft (Ru 138-20), the Interdisziplin-

ares Zentrum fur Klinische Forschung (IZKF) of the University of Ulm, the Muscular
Dystrophy Association (MDA), and the European Community, TMR Programme on Excita-
tion-Contraction Coupling, is gratefully acknowledged.
References
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Dr. Reinhardt Rudel, Department of Physiology, University of Ulm,

D89069 Ulm (Germany)
E-Mail reinhardt.ruedel@medizin.uni.ulm.de

............................
Congenital Myasthenic Syndromes
Andrew G. Engel a, Kinji Ohno a, Anthony A. Stans b
a
Department of Neurology and
b
Orthopedic Surgery, Mayo Clinic, Rochester, Minn., USA
Congenital myasthenic syndromes (CMS) are inherited diseases in which

the safety margin of neuromuscular transmission is compromised by one
or more distinct mechanisms [1, 2]. Although congenital myasthenia was
recognized as early as 1937 [3], the CMS were not investigated until after the
discovery of the autoimmune origin of myasthenia gravis (MG) in the 1970s. In
the 1970s and 1980s, ultrastructural, cytochemical, and in vitro microelectrode
studies of CMS patients revealed a heterogeneous group of disorders: endplate
(EP) acetylcholinesterase (AChE) deficiency [4], the slow-channel syndrome
[5], a defect in the synthesis or vesicular packaging of acetylcholine (ACh)
[6], decreased quantal release due to paucity of synaptic vesicles [7], and EP
acetylcholine receptor (AChR) deficiency [8,9]. In 1990s, following the discov-
ery of the cDNA sequences of all human AChR subunits and of the catalytic
and tail subunits of AChE, molecular genetic studies took center stage. In
addition, patch clamping of human intercostal muscle EPs allowed resolution
and analysis of single channel currents through EP AChRs [10], and the advent
of mammalian expression systems facilitated detailed analysis of the properties
of engineered mutant AChRs and AChEs.
Classification of CMS
During the past 12 years, my coworkers and I investigated 120 CMS

kinships at the Mayo Clinic. On the basis of our studies, we classify the CMS
as presynaptic (7%), synaptic (12.5%), postsynaptic (78%), and of unknown
origin (2.5%) (see table 1).
Table 1. Classification of CMS based on 120 index patients
Defect site Index cases
Presynaptic defects
Paucity of synaptic vesicles 1
Defect in ACh synthesis/packaging 6
Congenital Lambert-Eaton-like syndrome 1
Synaptic defect
EP AChE deficiency 15
Postsynaptic defects
Primary kinetic abnormality with or without AChR deficiency 30
Primary AChR deficiency with or without minor kinetic abnormality 64
No identified defecta 3
Total 120
a
Includes 2 cases of limb-girdle myasthenia.
Diagnosis of a CMS
A typical clinical history for CMS is one of ocular, bulbar or respiratory

muscle symptoms worsened by crying or activity in the neonatal period, fluc-
tuating ocular palsies and abnormal fatigability on exertion during infancy
and childhood, normal or delayed motor milestones, sometimes progression
of symptoms during adolescence or adult life, and negative tests for anti-
AChR antibodies. Some syndromes (e.g. the slow-channel syndrome and some
CMS caused by mutations in the AChR e-subunit) may not present until the
2nd or 3rd decade of life, and in the CMS with episodic apnea the patients
have only mild symptoms or are asymptomatic between attacks of respiratory
and bulbar paralysis precipitated by fever, infection, excitement or overexertion
[6]. The intravenous edrophonium test is positive except in EP AChE deficiency
and in the CMS with episodic apnea between attacks, and is inconsistently
positive in the slow-channel syndrome.
On examination, the most important clue to a defect of neuromuscular
transmission is increasing weakness on sustained exertion, as shown by increas-
ing ptosis during upward gaze, an arm elevation time less than 30 s, or increas-
ing difficulty in performing even a few deep-knee bends. Patients with severe
involvement of the truncal muscles rapidly develop postural scoliosis and shift
their weight from one foot to another on standing. Selectively severe weakness
of cervical and of wrist and finger extensor muscles is found in older patients
with EP AChE deficiency [11] and in the slow-channel syndrome [5]. The
pupillary light reflexes are delayed in patients with EP AChE deficiency [11].
Congenital Myasthenic Syndromes 97

Ocular muscle involvement can be absent or mild in some cases of EP AChE
deficiency [11], the slow-channel syndrome [5], and in the CMS with episodic
apnea [12]. The tendon reflexes are preserved but can be hypoactive. Despite
the above diagnostic clues, in most instances the clinical examination cannot
reliably distinguish between different types of CMS.
The generic diagnosis of a CMS must be supported by a decremental
electromyographic (EMG) response at low-frequency (2 Hz) stimulation in at
least one muscle, or by abnormal jitter and blocking during single-fiber EMG.
The decremental response may be absent during the interictal periods in the
CMS with episodic apnea. Here a decremental response can be elicited by 10
Hz stimulation for 510 min, or by exercise for several minutes before 2 Hz
stimulation [6]. In patients taking high doses of AChE inhibitors, in patients
with EP AChE deficiency [4, 11], and in the slow-channel CMS (SCCMS)
[5], single-nerve stimuli evoke a primary compound muscle action potential
(CMAP) followed by one or more repetitive CMAPs, each separated by an
interval of 510 ms. The repetitive potentials are smaller and decrement faster
than the primary response at all frequencies of stimulation. Therefore, the test
must be done in patients not exposed to AChE inhibitors, after a period of rest,
and initially with single nerve stimuli. Observations in the EMG laboratory can
provide an objective estimate of responsiveness to AChE inhibitors or other
cholinergic agents. For example, one can compare the decrement observed in
a given muscle before and a few minutes after an intravenous dose of edrophon-
ium, or 6090 min after an oral dose of 3,4-diaminopyridine (3,4-DAP).
The differential diagnosis of CMS in the neonatal period, infancy and
childhood includes spinal muscular atrophy, morphologically distinct congen-
ital myopathies, congenital muscular dystrophies, infantile myotonic dystrophy,
mitochondrial myopathy, brainstem anomaly, Mobius syndrome, congenital
fibrosis of the extraocular muscles, infantile botulism and seropositive- and
seronegative-autoimmune MG. In older patients, the differential diagnosis
includes motor neuron disease, limb girdle or facioscapulohumeral dystrophy,
mitochondrial myopathy, chronic fatigue syndrome, and seropositive- and
seronegative-autoimmune MG. Radial nerve palsy, peripheral neuropathy and
syringomyelia have been incorrectly diagnosed in some cases of the SC CMS.
Most entities can be excluded by careful physical examination, serologic tests
and EMG studies.
Investigation of the CMS
A deeper understanding of disease mechanisms and a precise classification

of the CMS requires estimation of the number of AChRs per EP, light-
Engel/Ohno/Stans 98
and electron-microscopic analysis of EP morphology, and electrophysiologic
assessment of EP function in vitro. Conventional microelectrode studies of
EP potentials and currents readily reveal whether the transmission defect is
presynaptic or postsynaptic. Patch-clamp recordings of currents flowing
through single AChR channels provide precise information on the conductance
and kinetic properties of the channels. All the above studies can be performed
only on small bundles of muscle intact from origin to insertion. These are
dissected from a larger strip of intercostal or anconeus muscle, also intact
from origin to insertion, removed from the patient with minimal trauma.
If the foregoing studies point to a defect in candidate gene or protein,
then molecular genetic analysis becomes feasible. If a mutation is discovered
in the candidate gene, then expression studies with the genetically engineered
mutant molecule can be used to confirm pathogenicity and to analyze the
kinetic or structural consequences of the mutation. To date, the candidate
gene approach has resulted in the discovery of 18 mutations in the gene
encoding the collagenic tail subunit of AChE, and close to 70 mutations in
the genes coding for AChR subunits.
Presynaptic Syndromes
The presynaptic origin of these syndromes has been established by electro-

physiologic and morphologic methods, but the underlying defects at the protein
and gene level are still not known.
Paucity of Synaptic Vesicles and Reduced Quantal Release

The clinical features of this rare CMS closely mimic those of autoimmune
MG and the symptoms respond to anticholinesterase drugs. The paucity
of synaptic vesicles in the nerve terminal reduces the number of releasable
transmitter quanta, which reduces the safety margin of neuromuscular trans-
mission [7]. The decreased synaptic vesicle density could arise from (1) a defect
in the formation of synaptic vesicle precursors in the anterior horn cell, (2) a
defect in the axonal transport of one or more species of precursor vesicles to
the nerve terminal, (3) impaired assembly of mature synaptic vesicles from
their precursors in the nerve terminal, or (4) impaired recycling of the synaptic
vesicles in the nerve terminal.
CMS with Episodic Apnea

This rare syndrome usually presents at birth or in the neonatal period
with hypotonia, variable ptosis but normal ocular ductions, severe bulbar
weakness causing dysphagia, and respiratory insufficiency with cyanosis and

apnea. If the infant survives, the symptoms improve but the crises recur with
infections, fever, excitement, vomiting or overexertion. During crises, episodes
of apnea can cause sudden death or anoxic brain injury. Between crises, patients
may have only mild or no myasthenic symptoms. When weakness is absent,
it can be readily induced by exercise. With increasing age, the exacerbations
become less frequent [6, 1219]. The clinical features of the disease were
described by Greer and Schotland [13] in 1960. In 1975, Conomy et al. [12]
referred to the disease under the rubric of familial infantile myasthenia, a
term that subsequently became entrenched in the medical literature. Because
all CMS can be familial and because most CMS present in infancy, the term
familial infantile myasthenia has become a source of confusion [20]. We
therefore refer to the disease as CMS with episodic apnea.
Electron microscopy studies show no postsynaptic abnormality [6]. Stimu-
lation of small muscle bundles at 10 Hz in vitro results in an abnormal decrease
of the amplitude of the CMAP, EP potential, and miniature EP potential,
pointing to impaired resynthesis or vesicular packaging of ACh [6, 21]. Treat-
ment consists of anticholinesterase drugs given orally between crises, and
parenterally, together with respiratory support, during crises.
CMS Resembling the Lambert-Eaton Myasthenic Syndrome

In one patient reported with this syndrome, the amplitude of the CMAP
was reduced but facilitated severalfold on tetanic stimulation and the symptoms
were improved by guanidine [22]. In a second patient, observed at the Mayo
Clinic, quantal release by nerve impulse was very low at 1 Hz stimulation but
increased markedly with stimulation rates ?10 Hz. The pre- and postsynaptic
regions were structurally intact by electron microscopy and the nerve terminals
harbored abundant synaptic vesicles. The patient responded only partially to
combined treatment with pyridostigmine and 3,4-DAP. The defect likely resides
in the presynaptic voltage-gated calcium channel or in a component of the
synaptic vesicle release complex.
Synaptic AChE Deficiency
The synaptic type of CMS is caused by absence or marked decrease of

the asymmetric species of AChE in the synaptic basal lamina [4]. Owing to this,
the synaptic currents are prolonged and evoke repetitive CMAPs. Prolonged
exposure of AChR to ACh causes desensitization of AChR [23], a depolariza-
tion block at physiologic rates of stimulation [24], and an EP myopathy stem-
ming from cationic overloading of the postsynaptic region [25]. The presynaptic
terminals are abnormally small and often encased by Schwann cells, reducing
Engel/Ohno/Stans 100
a b
Fig. 1. Schematic diagram showing domains of a ColQ strand with 18 identified ColQ
mutations (a) and components of the A12 species of asymmetric AChE (b). The N-terminal
region of each ColQ strand contains a PRAD that binds a homotetramer of the catalytic
AChET subunit. The triple helical collagenic domain contains two positively charged heparan
sulfate proteoglycan-binding domains (HSPBD) that participate in anchoring the tail subunit
in the synaptic basal lamina. The C-terminal region of ColQ is essential for assembly of the
triple helix of the collagen domain in a C- to N-terminal direction and may also participate
in anchoring the tail subunit. Reprinted with permission [2].
the number of quanta released by nerve impulse [4, 11]. The safety margin of
neuromuscular transmission is compromised by reduced quantal release, loss
of AChR due to degeneration of junctional folds, and by desensitization and
a depolarization block during activity.
There are two major types of AChE in skeletal muscle [26, 27]: (1) globular
forms consisting of monomers (G1), dimers (G2), or tetramers (G4) of the T
isoform of the catalytic subunit (ACHET), and (2) asymmetric forms consisting
of ACHET subunits linked to a tail subunit formed by the association of three
collagen-like strands, ColQ (fig. 1). The conserved domains of the tail subunit
include a proline-rich attachment domain (PRAD) in the N-terminal region
of ColQ that binds an ACHET tetramer producing A4, A8 and A12 moieties
[28, 29], a central collagen domain where three ColQ strands composed of
GXY triplets form a triple helix, and a C-terminal region where the ColQ
strands are enriched in charged residues and cysteines. The collagen domain

also harbors two positively charged heparan sulfate proteoglycan-binding do-
mains implicated in binding the triple helical collagen domain to negatively
charged molecules in the basal lamina [30, 31]. The function of the tail subunit
is to insert asymmetric AChE into synaptic basal lamina, and asymmetric
AChE is the predominant species of AChE at the EP [32, 33].
In 1998, human COLQ cDNA was cloned [34, 35] and the genomic
structure of COLQ determined [34]. This, in turn, led to the discovery of 6
truncation [34] and 1 missense [35] mutations of COLQ in 7 AChE-deficient
patients. One truncation mutation was upstream of PRAD, 4 were in the
collagen domain, and 1 truncation and the missense mutation were in the
C-terminal region. Coexpression of each COLQ mutant with wild-type ACHET
in COS cells revealed that a mutation proximal to PRAD prevented the associ-
ation of ColQ with ACHET; mutations distal to PRAD generated a mutant
species of AChE composed of one ACHET tetramer and a truncated single
and insertion-incompetent ColQ strand. Subsequent studies in our laboratory
led to the discovery of 11 more ColQ mutations in 8 additional kinships
[3639]. Expression studies indicate that 4 of the new mutations prevent the
formation of asymmetric AChE, but 7 others, all in the C terminal region,
produce some asymmetric AChE. However, the mutant asymmetric AChEs
are likely insertion incompetent because patients harboring these mutations
have EP AChE deficiency.
Postsynaptic Syndromes
Increased Response to ACh: The SCCMS

The phenotypic consequences of the syndrome stem from prolonged open-
ing episodes of the AChR channel (see fig. 2b) and spontaneous channel
openings even in the absence of ACh. These cause (1) cationic overloading of
the junctional sarcoplasm and an EP myopathy with loss of AChR from
degenerating junctional folds, and (2) a depolarization block due to staircase
summation of the markedly prolonged EP potentials during physiologic
activity [4042].
Eleven SCCMS mutations have been reported to date [4048]. The differ-
ent mutations occur in different AChR subunits and in different functional
domains of the subunits (see table 2, fig. 2a). Each is dominant, causing a
pathologic gain of function. Patch clamp studies at the EP, mutation analysis,
and expression studies in human embryonic kidney fibroblast (HEK) cells
identify three types of mutations. Those residing in the second transmembrane
domain (M2], which lines the channel pore, decrease the free energy required
for channel opening, as indicated by an increased channel opening rate even
a
Fig. 2. a Schematic diagram of slow-channel (solid circles) and fast-channel (shaded

circles) mutations reported to date. The drawing on the left shows a section through the
AChR lodged in the lipid bilayer with two slow-channel mutations, aG153S and aV156M,
in the extracellular domain near the ACh-binding site of the a subunit, and 3 fast channel
mutations: eP121L near the ACh-binding site of the e subunit, aV156M in the M3 domain
of the a subunit, and e1254ins18 in the long-cytoplasmic loop of the e subunit. The drawing
on the right shows slow-channel mutations detected between the M2 and M3 domains of
the a subunit, in the M2 domains of the a, b and e subunits, and in the M1 domain of the
a subunit. b Examples of single-channel currents from wild-type, slow-channel and fast-
channel AChRs expressed in HEK cells. Reprinted with permission [2].

Table 2. Distribution of 70 mutations in AChR subunit genes
Mutation Subunit genes Total

a b d e
Point mutations
Slow-channel mutation 8 2 3 13
Low affinity fast-channel mutation 2 2
Fast-channel mutation with abnormal gating 1 1 2
Null mutation 3a 3
Reduced expression 6 2 10 18
Inframe rearrangement
Reduced expression 1 1 2
Fast-channel mutation with mode-switching kinetics 1 1
Premature chain termination (null mutations)
Frameshifting rearrangement 1 17a 18
Splice-site mutation 7a 7
Nonsense mutation 4a 4
Total 15 4 2 49 70
Includes 33 published and 32 unpublished CMS mutations identified in our laboratory

and 5 mutations reported from other laboratories. Numbers in italics indicate recessive
mutations that reduce AChR expression.
a
Null mutations.
in the absence of ACh, and increase the free energy required for channel
closure, as indicated by a delay in channel closure [40, 42, 45]. Mutations near
the ACh-binding site on the a subunit increases affinity for ACh, causing
repeated channel reopenings during the prolonged ACh occupancy [41, 47].
A third type of SCCMS has features of the two preceding types and the
mutations reside in the M1 or M2 domain [42, 44, 45].
Following the lead that quinidine is a long-lived open-channel blocker of
AChR [49], Fukudome et al. [50] showed that clinically attainable levels of
quinidine normalized the prolonged opening episodes of mutant slow-channels
expressed in HEK cells. On the basis of these findings, Harper and Engel [51]
treated SCCMS patients with quinidine-producing serum levels of 0.72.5
lg/ml (2.17.7 lM/l) and found that the patients were improved by clinical
as well as by EMG criteria.
Decreased Response to ACh: The Fast-Channel Syndromes
A reduced synaptic response to ACh occurs with recessive, loss-of-function
mutations of AChR that reduce the affinity for ACh, or primarily affect channel
gating, or cause mode-switching kinetics (Table 2, fig. 2). Each type of mutation
results in brief activation episodes (see fig. 2b) and reduces the probability of
channel opening. In all three disorders, the mutated allele causing the kinetic
abnormality is accompanied by a null mutation in the second allele so that
the kinetic mutation dominates the clinical phenotype. Fast-channel syndrome
patients respond well to combined therapy with 3,4-DAP (1 mg/kg/day given
in 35 divided doses), which increases the number of quanta released by nerve
impulse, and cholinesterase inhibitors, which increase the number of AChRs
activated by each quantum.
Low-Affinity Fast-Channel Syndrome. This disorder was observed in 2
patients. Both had very small MEPCs but normally abundant EP AChR and
normal EP ultrastructure. Patch-clamp studies revealed infrequent and briefer
than normal channel opening events, and resistance to desensitization by ACh.
Each patient had two heteroallelic AChR e subunit gene mutations: a common
eP121L mutation that affects ACh binding, and a signal peptide mutation
(eG-8R; patient 1), and a glycosylation consensus site mutation (eS143L;
patient 2). AChR expression in HEK cells was normal with eP121L but was
markedly reduced with the other mutations. Therefore eP121L defines the
clinical phenotype. Studies of engineered eP121L AChR revealed a markedly
decreased rate of channel opening, and a reduced affinity for ACh in the open
channel and desensitized states [52].
Fast-Channel Syndrome Due to a Gating Abnormality. This syndrome is
caused by a kinetic and relatively low-expressor mutation in the M3 domain
of the AChR a subunit, aV285I, together with a null mutation, aF233V, that
unmasks the kinetic consequences of aV285I. In this CMS, as in the low-
affinity fast-channel syndrome, the duration of channel open intervals and
bursts is markedly shortened, but the primary abnormality resides in the
channel gating mechanism and not in affinity for ACh. Studies of genetically
engineered aV285I-AChR in HEK cells revealed brief channel opening events
(see fig. 2b), due to a slow opening rate constant, b, and fast closing rate
constant, a, and a reduced probability of channel opening [53].
Fast-Channel Syndrome Due to Mode-Switching Kinetics. In this disorder,
the kinetic abnormality is caused by an inframe duplication in the long cyto-
plasmic loop of e, e1254ins18, which also reduces AChR expression, plus
a cysteine-loop null mutation, eC128S [54]. When expressed in HEK cells,
e1254ins18 causes mode switching in the kinetics of receptor activation in
which the normal high efficiency of gating is accompanied by two new modes
that gate inefficiently, opening more slowly and closing more rapidly than

Fig. 3. Schematic diagram of 28 low-
expressor and null mutations in the e sub-
unit. >Low-expressor promoter muta-
tions; >low-expressor missense muta-
tions; T>truncating null mutations.
normal. At the EP, e1254ins18-AChR shows very brief activation episodes

during steady-state application of ACh and appears electrically silent during
the synaptic response to ACh. The phenotypic consequences are EP AChR
deficiency and compensatory expression of fetal AChR harboring the c instead
of the e subunit (c-AChR), which restores electrical activity at EP and rescues
the phenotype [54].
Mutations Causing AChR Deficiency with or without Minor

Kinetic Abnormalities
CMS with severe EP AChR deficiency result from different types of
homozygous or, more frequently, heterozygous recessive mutations in AChR
subunit genes. The mutations are concentrated in the e subunit (see tables 2, 3,
fig. 3). There are two possible reasons for this. (1) Expression of the fetal type
c subunit, although at a low level, may compensate for absence of the e subunit
[5456], whereas patients harboring null mutations in subunits other than e
might not survive for lack of a substituting subunit. (2) The gene encoding
the e subunit, and especially the exons coding for the long cytoplasmic loop,
have a high GC content that likely predisposes to DNA rearrangements [57].
Morphologic studies show an increased number of EP regions distributed
over an increased span of the muscle fiber. The integrity of the junctional
folds is preserved but AChR expression on the folds is patchy and faint. Some
EP regions are simplified and small. The amplitude of miniature EP potentials
and currents is reduced but quantal release by nerve impulse is often higher
than normal. With null or low-expressor mutations in the e subunit, single
channel recordings at the EP [56, 58] or immunocytochemical studies [55]
reveal the presence of c-AChR at the EP that likely rescues the phenotype.
Table 3. Twenty-nine low-expressor or null mutations in the AChR e
subunit
Mutations Domains Ref. No.
e-156CKT Ets binding site 65

e-155GKA Ets binding site 64
eG-8R Signal peptide 52
eV-13D Signal peptide 61
eT51P Extracellular domain 61
T eR64X Extracellular domain 56
T e59ins5 Extracellular domain 59
T e70insG Extracellular domain 59
T e1275 Extracellular domain 56
eC128S Disulfide loop 54
eS143L N-glycosylation site 52
eR147L Extracellular domain 56
T e553del7 Extracellular domain 56, 58, 63
T e723delC Ml domain 61
* eP245L M1 domain 56
T eIVS7+2TKC Link between M1 and M2 59
T e760ins8 M2 domain 61
* eR311W Long cytoplasmic loop 56
T eIVS9-1GKC Long cytoplasmic loop 63
T e1012del20 Long cytoplasmic loop 60
T e1033delG Long cytoplasmic loop 67
T e1101insT Long cytoplasmic loop 55
T eIVS10+2TKG Long cytoplasmic loop 61
T e1206ins19 Long cytoplasmic loop 59
* e1254ins18 Long cytoplasmic loop 54
T e1260del23 Long cytoplasmic loop 67
T e1267delG Long cytoplasmic loop 61, 62
T e1276delG Long cytoplasmic loop 59
T e1293insG Long cytoplasmic loop 55
>Promoter mutations, reduced expression; >missense mutations, re-

duced expression; T>premature chain termination, null mutations; *>muta-
tion also has significant kinetic effects.
Most patients respond to anticholinesterase drugs and some derive additional

benefit from 3,4-DAP.
Different types of recessive mutations causing severe EP AChR deficiency
have been identified (see table 2). (1) Mutations causing premature termination
of the translational chain, these mutations are frameshifting [55, 56, 5962],

occur at a splice site [59, 61, 63], or produce a stop codon directly [56]. (2) Point
mutations in the promoter region of a subunit gene (e-155GKA [64] and
e-156CKT [65]). (3) Missense mutation in a signal peptide region (eG-8R)
[52]. (4) Mutations involving residues essential for assembly of the pentameric
receptor. Mutations of this type were observed in the e subunit at an N-
glycosylation site (eS143L) [52], in cysteine 128 (eC128S), a residue that is an
essential part of the C128-C142 disulfide loop in the extracellular domain [54],
and in arginine 147 (eR147L) in the extracellular domain, which lies between
isoleucine 145 and threonine 150, residues that contribute to subunit assembly
[56], and with a 3-codon deletion in the long cytoplasmic loop of the b subunit
[66]. (5) Missense mutations affecting both AChR expression and kinetics. For
example, eR311W in the long cytoplasmic loop between M3 and M4 decreases
[56], whereas eP245L in the M1 domain increases [56] the open duration of
channel events. In the case of eR311W and eP245L, the kinetic consequences
are modest and are likely overshadowed by the reduced expression of the
mutant gene.
Conclusion
CMS are heterogeneous disorders caused by presynaptic, synaptic or

postsynaptic defects. Full characterization of a given syndrome requires cor-
relation of clinical and EMG data, light- and electron-microscopic examination
of EP morphology, estimation of the number and distribution of AChRs at
the EP, in vitro eletrophysiologic analysis of parameters of neuromuscular
transmission, and molecular genetic studies when the preceding investigations
point to a candidate gene or protein.
Three presynaptic CMS have been recognized to date. All are recessively
inherited but their molecular genetic basis remains undeciphered. One syn-
drome, in which a paucity of synaptic vesicles causes reduced release of trans-
mitter quanta, closely mimics the clinical and EMG features of autoimmune
MG. In another syndrome, a defect in the resynthesis or vesicular packaging
of ACh causes episodic crises with apnea. In the third disorder, the electrophysi-
ologic features resemble those of the Lambert-Eaton syndrome and the putative
defect resides in the synaptic vesicle release complex.
A synaptic type of CMS is caused by absence or marked deficiency of
the asymmetric species of AChE in the synaptic basal lamina. The disease is
now known to be caused by recessive mutations in the collagenic tail subunit
of asymmetric AChE. The mutations prevent the association of the tail subunit
with catalytic subunits, or the insertion of the tail subunit into the synaptic
basal lamina.
All postsynaptic CMS recognized thus far stem from mutations in AChR
subunit genes that increase or decrease the synaptic response to ACh. An
increased response occurs in the slow-channel syndromes. Here dominant
mutations in different AChR subunits and in different domains of the subunits
prolong the activation episodes of AChR by altering channel gating, or by
increasing the affinity for ACh, or both.
A decreased synaptic response to ACh occurs in the fast-channel syn-
dromes where the mutated allele causing the kinetic abnormality is accompa-
nied by a null mutation in the second allele, so that the mutation causing the
kinetic abnormality dominates the clinical phenotype. The kinetic abnormali-
ties reduce the affinity for ACh, or primarily affect channel gating, or cause
mode-switching kinetics. In each instance, the AChR activation episodes are
abnormally brief and occur at a reduced probability.
Response to ACh is also reduced by low-expressor or null mutations in
AChR subunit genes that result in premature termination of the translational
chain or are missense mutations preventing subunit assembly or glycosylation.
These mutations are concentrated in the e subunit, probably because substitu-
tion of the fetal c for the adult e subunit can rescue humans from fatal null
mutations in e.
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S120.
Andrew G. Engel, MD, Department of Neurology, Mayo Clinic,

200 First Street SW, Rochester, MN 55905 (USA)
Tel. +1 507 284 5102, Fax +1 507 284 5831, E-Mail age@mayo.edu
............................
Juvenile and Late-Onset
Myasthenia gravis
zdemir
Feza Deymeer, Piraye Serdaroglu, Coskun O
Department of Neurology, University of Istanbul, Istanbul, Turkey
Myasthenia gravis (MG) is an autoimmune disease which can start at any

age, predominantly affecting young women and older men [1]. In early hospital-
based studies [2, 3], onset of the disease was found to be rare in the prepubertal
period; it made a peak during fertile years with a decline thereafter in women,
while the peak was in late life in men. Epidemiological studies in the last 2
decades have resulted in a change of concepts about the frequency of MG in
the elderly population [4], with the implication that MG may be disproportion-
ately a disease of later years [5], while basically confirming previously estab-
lished facts for other age groups.
Recently, the two extremes of age, the young and the old, have been
among topics of interest in MG, the former because it may have some clinical
peculiarities [6, 7] and the latter because of the ageing population [8] with a
high incidence of MG [9, 10]. In this chapter, we review the literature on
juvenile (JMG) and late-onset (LOMG) MG, concentrating on studies which
specifically deal with young and elderly patients, and add our experience with
733 patients attending the Neuromuscular Clinic of the University of Istanbul
(which will be referred to as UI).
Juvenile MG
JMG is defined as an acquired autoimmune disorder, not encompassing

neonatal MG and congenital myasthenic syndromes [6]. Onset is after the age
of 1 in JMG, while it is usually before the age of 1 in congenital myasthenic
syndromes and within the first few hours of life in neonatal MG [1, 11].
Table 1. JMG studies
Authors Number of Age FMR Classification Positive anti- TXb Remission

patientsa years AChR Ab, % % rate
Millichap and 35 ?1 to =12 years: 3.3 Ocular: 3% 60 29% (TX)

Dodge [12] (8 of 447) p16 q12 years: 10 Respiratory (0) 14% (no TX)
difficulty: 43%
Seybold et al. 102 ?1 to 2.6 Ocular: 9% 47 38 (TX)
[13] p16 Generalized: 91% (1) 24 (no TX)
Ryniewicz and 47 =15 2.4 I: 19% 60 43 % (TX)
Badurska [14] IIa: 17% (1)
IIb: 43
IIIc: 6
IV: 15%
Snead et al. [15] 32 q1 to 1.9 I: 40% 51 22 29% (TX)
p18 IIa: 21% (0) 12% (no TX)
IIb: 11%
IIcd: 28%
Rodriguez et al. 149 ?1 to =12 years: 2.7 I: 9% 57 53% (TX)
[16] p16 q12 years: 3.7 IIa: 16% 30% (no TX)
IIb: 73%
III: 2%
Wong et al. [7] 101 ?0 to 1.1 Ocular: 71% Low or absent 12 17% (TX)
(39 of 262) =16 Generalized: 29% (8) 34% (no TX)
Andrews et al. 115 ?1 to Pre: 1 Pre: 50 72 35% (early TX)
[6] =20 Peri and post: 4.5 Peri: 68 (1) 2% (late TX)
Pre: 20 Post: 91 9% (no TX)
Peri and post: 93
Unknown: 2
Anlar et al. [17] 30 p15 012 years: 1 Crises: 33% 34 37 20% (no TX)
q12 years: F1M
Lindner et al. 79 q12 to 4 I: 9% 81 82 60% (TX)
[18] p18 IIa: 45% (0) 29% (no TX)
IIb: 32%
III and IV: 14%
Evoli et al. [19] 133 11 to Pre 1.1 Pre Pre: 74 Pre: 47 Pre:
(16 of 817) 0 20 I: 26% (0) 0% (TX)
IIa: 21% 21% (no TX)
IIb: 11%
Pre: 19 III and IV: 42%
Peri and post: 114 Peri and post: 4.2 Peri and post Peri and post: Peri and Peri and post
I: 16% 86 post: 84 31% (TX)
IIa: 38% (4) 0% (no TX)
IIb: 31%
III and IV: 15%
zdemir
Deymeer/Serdaroglu/O 114
Table 1 (continued)
Authors Number of Age FMR Classification Positive anti- TXb Remission

patientsa years AChR Ab, % % rate
UI study 140 q1 to Pre: 0.9 Pre Pre: 64 Pre: 0 Pre

(19 of 733) 0 20 I: 50% (0) 25% (no TX)
IIa: 31%
IIIb: 13%
Pre: 19 III and IV: 6%
Peri and post: 121 Peri and post: 3 Peri and post Peri and post: Peri and Peri and post:
I: 13% 78 post: 55 8% (TX)
IIa: 27% (2) 28% (no TX)
IIb: 45%
III and IV: 15%
Pre, peri, and post refer to the stage of puberty. FMR>Female to male ratio, TX>thymectomy.
a
% of total number is given in parentheses.
b
Number of thymomas is given in parentheses.
c
Generalized weakness without ocular or bulbar signs.
d
Generalized disease with bulbar involvement.
In selecting JMG studies [6, 7, 1219], compiled in table 1, we preferred those

which gave some information on the whole group of juvenile patients even though
the main purpose might have been to evaluate thymectomy and we refrained from
including studies done with patients undergoing thymectomy only [20].
Most of the JMG studies appropriately excluded neonatal MG and con-
genital myasthenic syndromes. The main source of difficulty [21, 22] in the
comparison of different studies was the arbitrary choice of the upper age limit
for the juvenile period, ranging from 15 to 19.
The juvenile period is heterogenous with pubertal stages which have differ-
ent characteristics. When the juvenile period is considered as a unity, the
uniqueness of each stage is lost. Unfortunately, studies on JMG used different
cutoff ages to separate prepuberty from postpuberty and most of them confined
themselves to giving the female to male ratio (FMR) for the different stages.
Seybold et al. [13] were among the few who treated prepuberty separately. And-
rews et al. [6, 23] analyzed the different stages in more detail, applying Tanners
criteria [24] to their patient population and divided it into three stages: prepu-
berty (08.9 years in females and09.3 years in males), postpuberty (115.3 years
in females and 116.5 years in males) and peripuberty in between these ages.
Like Evoli et al. [19], we used these age brackets in our study for the three stages.
Demographic Features
We analyzed demographic data in three categories: (1) studies dealing
specifically with JMG patients (table 1), (2) large hospital-based studies [2, 3,
Juvenile and Late-Onset Myasthenia gravis 115

2529], (3) population-based (epidemiological) studies with incidence rates
[9, 10, 3034]. This separation was at times artificial and some studies, analyzed
within one category, actually belonged to more than one category.
JMG Studies (table 1). In Caucasian series, JMG made up less than 20%
of the total patient population. Prepubertal MG constituted 24% of the entire
group of patients [19] (UI). Females were affected more frequently than males,
with this predominance being much less during prepuberty as compared to
later juvenile years. In some studies, the two sexes were almost equally affected
during prepuberty. In black prepubertal children, the incidence of MG was
higher than in comparable white children and FMR was 2 both in pre- and
postpubertal stages [6, 35]. The Chinese exhibited unique features in relation
to puberty [7], the highest incidence for both sexes being in the 1st decade,
with a peak at 23 years of age.
Hospital-Based Studies. There are several large hospital-based studies
(listed above) which give an idea of the age- and sex-related characteristics of
MG even though the main focus is not any specific age group. In these studies,
prepubertal patients made up the smallest portion of the whole group, only
paralleled by the oldest age brackets. A review of the literature [36] revealed
that in 4.3% of all cases the onset was before the age of 10 and in 24% before
the age of 20. Again, female predominance was evident, less so in prepuberty
than in postpuberty. There were even some studies in which males predomi-
nated during prepuberty [2, 29].
Population-Based (Epidemiological ) Studies. More precise demographic
information was obtained in population-based studies (listed above), which
are not flawed by the selection bias inherent in hospital-based studies. Age-
and sex-specific incidence rates of some of these studies are compared in
table 2. Overall, it can be said that the incidence rate was one of the lowest
of all age groups in the 1st decade, where female preponderance was not very
striking. The rate increased for both sexes in the 2nd decade, but much more
so for females than for males. In the Japanese [37], the highest incidence for
both sexes was in the 1st decade of life, similar to the Chinese [7]. Females
and males were equally affected.
The apparent discrepancy in the FMR between Caucasian and Chinese-
Japanese populations in the juvenile period is probably just a function of the
over-representation of prepubertal patients in the Chinese and the Japanese
studies and tends to disappear when prepuberty is considered separately in
Caucasian studies.
Onset Symptoms
It is well-known that ocular presentation is very high in prepuberty in
the Chinese and the Japanese, being 71 and 90%, respectively, in the two
zdemir
Table 2. Age- and sex-specific incidence rates per million population per year
Age, years Storm-Mathisen Giagheddu et al. Ferrari and Lovaste Christensen et al.
[30] [31] [34] [9]
females males females males females males females males
010 0.53 0.10 0.5 4.1 1.5 0.7

1019 4.58 0.66 4.7 1.0 3.1 4.0 1.2
2029 8.34 2.09 4.4 0.9 14.8 7.0 2.2
3039 7.21 2.33 5.7 1.1 13.1 9.2 6.1 2.1
4049 4.61 2.99 5.2 4.0 10.2 6.9 5.1 2.1
5059 4.34 2.63 3.6 3.3 3.6 7.7 7.3 5.2
6069 4.69 7.02 5.9 2.9 20.1 10.1 9.7 15.1
7079 6.44 3.48 2.2 a 0.9 a 14.1 a 11.7 14.1
180 1.35 2.99 1.3 8.6
a
Over 70 years of age.
studies [7, 37]. JMG also commonly presents with ocular symptoms in Caucasi-
ans [12, 15, 17, 38]. Information provided in the study by Bundey [36] for
onset symptoms lends itself to the separation of pubertal stages [6]. Seybold
et al. [13] also give detailed information on onset symptoms for 35 prepubertal
patients. Two interesting features emerge for Caucasians from these two studies
together with the UI study: the striking predominance of ocular presentation
in prepuberty and the presence of a peculiar onset symptom, lower extremity
weakness, becoming most evident in peri- and postpuberty.
Prepuberty. Presentation was with purely ocular symptoms in prepuberty
in 64% [36] and 89% (UI) of the patients. In the study of Seybold et al. [13]
which, unlike the other two studies, includes many patients over the age of
10, ocular presentation was seen in 47%. Ptosis was more commonly the onset
symptom as compared to diplopia [36] (UI). The second mode of presentation
was leg weakness. Onset with bulbar weakness was rare, not being present in
any of the patients of Bundey [36] and UI.
Peri- and Postpuberty. When these two stages are considered together,
presentation was with ocular (about 40%), bulbar (about 20%) and lower
extremity symptoms (about 20%), followed by extremity (both or upper extrem-
ities) and combined symptoms [36] (UI). The importance of leg weakness as
an onset symptom in these stages was particularly noteworthy. Disease started
with leg weakness in 24% of the prepubertal patients of Seybold et al. [13];
88% of these were children with onset at 1014 years. In the other two studies,
of all juvenile patients with leg weakness onset 73% [36] and 96% (UI) were

in peri- and postpubertal stages. Rarely, difficulties in smiling or in closing
the eyes accompanied leg weakness.
Leg weakness was sometimes so severe as to result in frequent falls. In
some of our patients, this was the only symptom for a fairly long time and
predominated even when other symptoms were added so that differential
diagnosis from muscular dystrophies which can also present at this age became
an important issue. Oosterhuis [39] explains the frequent occurrence of leg
weakness, which he notes as an onset symptom in myasthenics below the age
of 30, on the basis of overactivity in young people putting an extra burden
upon the lower extremities.
Anti-Acetylcholine Receptor Antibodies and HLA Associations

The seropositivity rate and the titer of anti-acetylcholine receptor (anti-
AChR) antibodies were found to be low in prepubertal children [19, 23].
Interestingly, some seronegative children became seropositive at or after pu-
berty. In the postpubertal period, the seropositivity rate was not different from
that in myasthenic adults [23]. Not differing substantially from Caucasians in
the behavior of antibodies [7, 40], the Chinese showed a strong association
with HLA-DR9 and Bw46 [41] whereas Caucasians showed no specific HLA
association in the juvenile period.
Thymoma
Thymoma associated with MG was found to be very rare in the juvenile
period, particularly in prepuberty [19, 42]. Isolated cases, usually in postpu-
berty, were reported (table 1).
Associated Autoimmune Conditions

Thyroid disease was the most frequently associated autoimmune disease
in this age group [7, 12, 18] (UI). Rheumatoid arthritis, lupus erythematosus,
diabetes mellitus, asthma and vitiligo were other cited diseases in the JMG
studies. MG was not usually associated with other autoimmune conditions
during prepuberty [19]. In the UI series, all 8 patients with thyroid disease
and the 1 patient with rheumatoid arthritis were in peri- and postpuberty.
Severity and Outcome

Proper comparison of different studies is hampered by both assessment
and selection biases. Different classifications and improvement measures
[20, 21] or seemingly the same measures with different meanings attributed to
similar categories have been used [14, 15]. Remission was defined by some as
a state of total eradication of symptoms with no medications [6, 12, 1416, 19]
(UI) and by others as the same with medications [18]; others still [13] accepted
zdemir
minor symptoms as part of remission. Another major problem was the different
criteria in the selection for thymectomy [12, 22], such as exclusion of very
young children or patients responding to steroids. In the UI study, 75% of
thymectomized patients during peri- and postpuberty were moderately or
severely involved while the corresponding figure was only 36% in nonthymecto-
mized patients so that any comparison became meaningless. In addition, our
more severely involved patients were usually kept on a low-dose, alternate-
day steroid maintenance treatment making it impossible to judge remissions.
The confounding effect of immunosuppressants starting prior to thymectomy
further complicated evaluations [43]. Seybold [22] found only 5 studies [6, 12,
13, 16, 20] which allowed proper assessment of the value of thymectomy in
juvenile patients. We did include remission rates of thymectomized and non-
thymectomized patients for all studies in table 1, but the reader should evaluate
them with these reservations in mind.
Prepuberty. Prepubertal MG was not always found to have a benign
progression although it was associated with two benign features: a high percent-
age of purely ocular (Osserman class I) MG and long-lasting spontaneous
remissions. While about three quarters of Chinese [7] and Japanese [37] children
with MG had ocular symptoms only, this percentage was less but still high
(about one quarter to one half ) in some Caucasian series [19] (UI). Spontane-
ous remissions were more frequent in prepuberty [6, 16] and lasted as long
as 1213 years [13] (UI). Patients with ocular MG were more likely to have
spontaneous remissions [11]. The high remission rate made the evaluation of
all treatment modalities difficult.
When the disease progressed mildly, anticholinesterases were sufficient
to keep it under control [15]. However, respiratory difficulties occurred in as
many as 40% of the patients [12, 19], a fact which has to be kept in mind
even when the disease appears to be mild. Also, an acute fulminating form
has been described [11]. Immunosuppressant drugs and thymectomy have to
be considered when the disease assumes a severe form. Some studies [14, 20,
44, 45] with a relatively large number of prepubertal patients found thymec-
tomy beneficial, while others [6, 16] reported less favorable results as compared
to peri- and postpuberty. A review of the results of several studies showed
a lower remission rate after thymectomy as compared to spontaneous remis-
sion for children under 5 [6]. It is unlikely that more definite conclusions
will emerge since the number of prepubertal children is small, but it is useful
to know that thymectomy in very young children has no major deleterious
effect on the immune system [22]. Overall, with immunosuppressants and/
or thymectomy, where necessary, the outcome is good in most patients [6,
12, 46]. With purely ocular MG, low-dose, alternate-day steroids can resolve
the symptoms [35].

Peri- and Postpuberty. These patients are similar to myasthenics with early
onset [19] with minor differences. In some studies, males were found to be less
severely affected than females [6, 17] (UI). Thymectomy, done much more
frequently at this stage, was considered to be beneficial by most authors [6, 20,
44, 4749]. Despite the absence of randomized studies, probably unethical at
this point, thymectomy has a firm place in the treatment of peri- and postpuber-
tal JMG [39]. Remissions were more frequent with early thymectomy, done
within 12 years of disease onset [6, 16], although thymectomy done later also
produced satisfactory results [13, 14]. Again, the outcome is good with different
modalities of treatment including immunosuppressants [6, 18] and is possibly
superior to that in adults [12].
Conclusions
Prepubertal MG differs from MG in other age groups in sex prevalence,
racial characteristics, seropositivity, clinical severity and progression. Males are
often as frequently affected as females at this stage. The predominant presentation
is ocular and purely ocular forms are often seen. Both the percentage and the
titer of positive anti-AChR antibodies are low. Thymoma associated with MG
is extremely rare. A high remission rate is noteworthy, making evaluation of all
treatment modalities very difficult. This benign feature has to be weighed against
the fact that a fairly high percentage may have respiratory difficulties so that
thymectomy and immunosuppressants have to be seriously considered. Although
thymectomy appears to be effective in isolated cases, its value continues to be
disputed among authors and the issue will be difficult to resolve because of the
small number of patients. With appropriate treatment, the outcome is good.
The Chinese and the Japanese show striking differences from the Caucasi-
ans in prepuberty. It is a period with the highest incidence of MG for both
sexes and it is predominantly purely ocular.
With the advent of puberty, the characteristics of MG are very similar
to those of later fertile years. An important onset symptom of the peri- and
postpubertal years is leg weakness and distinction from muscular dystrophies
is of great importance. Only isolated cases of thymoma have been reported.
The beneficial effect of thymectomy in peri- and postpuberty, particularly if
done early, is stressed by many. The outcome is good with appropriate treat-
ment including immunosuppressants.
Late-Onset MG
Previous literature accepted the age of 40 as the beginning of the older

age group [50]. Somnier et al. [33], based on epidemiological data, suggested
zdemir
that onset age for LOMG should be set at 50 rather than earlier. Aarli [4]
recently defined LOMG as MG without evidence of thymoma, occurring after
the age of 50. In our attempt to adhere to this definition in selecting the
relevant papers dealing with elderly myasthenics [8, 5156], compiled in table 3,
we encountered several difficulties: neither was the age limit uniformly defined,
varying from 50 to 65 in the studies, nor was thymoma considered separately
within the elderly population.
Demographic Features
The same design and the same references (for hospital-based and epidemi-
ological studies) as in the JMG section were used.
LOMG Studies (table 3). When larger studies were considered, patients
over 50 years of age made up about 30% of the total patient population and
those over 60 years about 20%. Males outnumbered females or FMR was 1.
Hospital-Based Studies. Oosterhuis [50], compiling information from sev-
eral large hospital-based studies, reported that 3647% of the patients had
disease onset at or above the age of 40 except in a Japanese study with only
22%. In the hospital-based studies listed in the JMG section, LOMG was
more frequent in men as compared to women. Peak age of onset was in the
5th to 7th decades in men and in the 2nd to 3rd decades in women. In some
European studies, there was a relatively constant distribution of onset age for
men after the 1st decade [29] or the peak was at a younger age similar to
women [25, 57].
Population-Based (Epidemiological) Studies. Most population-based stud-
ies of Caucasians showed that the highest incidence rates of all age groups
occurred in the elderly (table 2). They confirmed what was already known for
men, but the results for women were very surprising. Either the highest inci-
dence rate for women was found to fall within older age brackets or a bimodal
distribution was present with a second peak late in life. FMR was 1 in the
elderly population. Chinese [7] and Japanese [37] populations were completely
different from the Caucasians in that they did not show a late peak.
Onset Symptoms
The most frequent presenting symptoms were ocular, occurring as an
isolated symptom in over 50% of the patients in the majority of the studies.
Onset with bulbar symptoms followed this type of presentation. In the UI
study, bulbar onset was more common in women as compared to men. Pre-
sentation with weakness in the extremeties only was uncommon in this age
group. An interesting onset symptom, easily confused with other etiologies in
this age group, was weakness in the neck (head drop) [55], occurring in 6%
in the UI study.

Table 3. LOMG studies
Authors Number of Age FMR Classification Positive anti- TXb Good outcome
patientsa years AChR Ab % (remission plus
% improvement)
Herishanu et al. [51] 15 q60 0.9 0

(45 of 33)
Evoli et al. [52] 37 160 0.6 I: 3% 94 27 78%
(13 of 278) IIa: 46% (5)
IIb: 24%
III and IV: 27%
Donaldson et al. [8] 55 q50 0.4 I: 11% 82 38 87%
(33 of 165) IIa: 20% (10)
IIb: 33%
III and IV: 36%
Antonini et al. [53] 25 160 0.7 Ocular: 8% 86 4 92%
(20 of 122) Generalized: 92% (1)
Schon et al. [54] 13 q60 0.4 85 8
(59 of 22) (1)
Bille-Turc et al. [55] 34 q65 0.6 Ocular: 15% 94 6 68%
(17 of 200) Generalized: 85% (2)
Slesak et al. [56] 113 ?60 1 Ocular: 32% 13 8793
(30 of ?) (10)
UI study 183 q50 0.7 I: 20% 86 16 81%
(25 of 733) IIa: 25% (18)
IIb: 44%
III and IV: 11%
FMR>Female to male ratio, TX>thymectomy.

a
% of total number is given in parentheses.
b
Number of thymomas is given in parentheses.
Anti-AChR Antibodies and HLA Associations

That LOMG may be a different disease from early-onset MG has been
suggested by several immunological and HLA antigen studies. HLA-A3 was
found to be more frequent in older patients [58] and HLA-B8 in young female
patients [59, 60]. A comparison of the characteristics of nonthymoma patients
below and above the age of 40 [61] showed that the older patients had a unique
profile: association with HLA-A3, B7, DRw2, lower anti-AChR antibody
zdemir
titers, higher antistriated muscle antibodies, higher autoantibodies other than
those known to be associated with MG and higher immune diseases other
than MG.
Although anti-AChR antibody titers were found to be low, the percentage
of positive anti-AChR antibodies was not any lower in the elderly than in
other subgroups [6164], with values at or above 85% in the majority of the
studies on LOMG (table 3).
Thymoma
Thymomas have the highest prevalence in older men and women [39].
The percentage of elderly patients with thymoma was about 510 % in the
studies on LOMG (table 3). The incidence in older women was twice as much
as that in older men in some studies [39] (UI).
Associated Autoimmune Conditions

Thyroid disease, particularly hypothyroidism, was the most common auto-
immune disease associated with LOMG [8, 55]. Hashimoto thyroiditis was
present in 6% of the UI patients. Rheumatoid arthritis, systemic lupus eryth-
ematosus, diabetes mellitus, pernicious anemia, ankylosing spondylitis and
idiopathic thrombocytopenic purpura were other cited autoimmune disorders.
In the UI study, there were 3 patients with asthma, 2 patients with pernicious
anemia and 1 each with systemic lupus erythematosus, idiopathic thrombo-
cytopenic purpura, pemphigus and psoriasis. Association with all autoimmune
diseases was more frequent in those with the onset around the age of 60 or
later (UI).
Severity and Outcome

About 1030 of the patients were classified as ocular MG (Osserman
class I) in the majority of the LOMG studies. Myasthenics with mild symptoms
(Osserman classes 1 and 2a) made up about 3050% of the patients. On the
other hand, about 30% had severe MG. Thus, LOMG can be mild, but one
has to be aware of the fact that it is a potentially very severe disease.
There was no consensus about the effect of thymectomy in this age group
although some authors reported a benefit [65, 66]. Thymectomy was performed
only in a minority of the cases unless the patient had a thymoma so that in
most studies remission and improvement can easily be attributed to the effect
of immunosuppressants.
With immunosuppressants, mainly steroids, 6892% of the patients im-
proved, including a minority who went into remission. We have noted as did
Bille-Turc et al. [55] that low-dose steroids can be very effective in selected
mildly affected patients. Yet, high doses are necessary in potentially severe cases.

Complications of steroids in the elderly, particularly cataracts and infection [8],
have been stressed by some [52], while others have not found the side effects
to be different from those in young people [53]. Because of these reservations,
azathioprine is increasingly used as an adjunct to steroids in the elderly [8, 56]
(UI). Azathioprine has fewer side effects than those of steroids [39, 67]. Our
experience with azathioprine in the elderly is also favorable.
Conclusions
Onset of MG in the elderly is more common than previously thought.
While it is well known that the peak age of onset is in the older age groups
in men, recent epidemiological studies have revealed a similar tendency in
women or at least a bimodal distribution with a second peak late in life. FMR
is close to the 1 in the older age group.
The disease often starts with ocular symptoms. Onset with weakness in
the extremeties is uncommon. An interesting onset symptom in LOMG is
weakness in the neck. Although anti-AChR titers are low in the elderly, the
seropositivity rate is similar to that in early-onset MG. A unique antigen and
HLA profile suggests that LOMG may be a different form of the disease. The
disease is mild in half the cases; however, it can be severe in a third. Treatment
is in many ways similar to that in the other age groups. The possibility of
response to low-dose steroids, yet the necessity to give high doses in potentially
severe cases and the possible beneficial effects of thymectomy in selected cases
should be considered. Azathioprine is increasingly used in the elderly. The
prognosis is good with appropriate treatment.
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Feza Deymeer, MD, Department of Neurology, University of Istanbul,

C
apa 34390, Istanbul (Turkey)

............................
Hereditary Peripheral Neuropathies
Peter De Jonghe a, b, Vincent Timmerman a, Eva Nelis a
a
Flanders Interuniversity Institute for Biotechnology (VIB), Born-Bunge
Foundation (BBS), Department of Biochemistry, University of Antwerp (UIA) and
b
Department of Neurology, University Hospital Antwerp (UZA), Antwerp, Belgium
At the end of the 1960s Dyck et al. [1] classified the inherited neuropathies
of the peripheral nervous system based on mode of inheritance, clinical fea-
tures, neuropathological and electrophysiological findings. Dyck and co-
workers distinguished three large groups, i.e. hereditary motor and sensory
neuropathies (HMSN), hereditary motor neuropathies (HMN) and hereditary
sensory neuropathies (HSN) or hereditary sensory and autonomic neuro-
pathies (HSAN). Each category is further subdivided into several types. Mo-
lecular genetic studies have confirmed this extensive heterogeneity. Currently,
22 inherited peripheral neuropathy loci have been mapped (table 1). However,
the genetic loci for many types still remain to be found and one can safely
predict that there must exist between fifty and one hundred genetically distinct
types. This huge number of loci stands in sharp contrast to the small number
of only four genes that, so far, have been found to be involved in the inherited
peripheral neuropathies. These genes are: peripheral myelin protein 22 gene
(PMP22) at chromosome 17p11.2, myelin protein zero gene (MPZ, P0) at
chromosome 1q22-q23, connexin 32 gene (Cx32,GJB1) at chromosome Xq13
and the early growth response element 2 gene (EGR2) at chromosome 10q21.1-
q22.1. At present, 332 different mutations within these four genes have been
detected, leading to a wide variety of clinical phenotypes [2]. A database,
containing all published and a number of unpublished mutations, can be
consulted at http://molgen-www.uia.ac.be/CMTmutations/. The results of
functional studies of the mutated proteins in human biopsy specimens, cellular
and animal models have been the subject of recent review papers and will not
be discussed here [3, 4]. We will focus on the inherited neuropathies in which
molecular genetics have provided new insights.
Clinical Phenotypes
Most patients affected by an inherited peripheral neuropathy have a clin-

ical phenotype closely resembling the patients originally described by Charcot
and Marie [5] and Tooth [6] in 1886. The eponym Charcot-Marie-Tooth disease
or CMT is still commonly used to designate this phenotype [7]. The disease
usually starts in the 2nd decade of life with a progressive paresis of the peroneal
muscles. Several years later some weakness of the intrinsic hand muscles also
develops. Sensory symptoms are usually discrete or absent and tendon reflexes
are often abolished. Skeletal abnormalities such as pes cavus or scoliosis are
frequently observed. The severity of the disease is variable. Most patients are
able to live an independent life even in old age while other patients become
wheelchair-bound due to the involvement of the proximal muscles of the legs.
Additional features may be present such as deafness, pupillary abnormalities,
vocal cord paralysis, and poorly healing ulcers [8]. A second phenotype is the
Dejerine-Sottas syndrome (DSS) [9]. Some authors have proposed to reserve
this eponym for patients with a severe, early onset demyelinating neuropathy
[10]. They further specified that nerve conduction velocities (NCV) should be
severely reduced to less than 12 m/s and the protein content of the cerebrospinal
fluid should be increased. This syndrome is usually considered to be inherited
as an autosomal recessive (AR) trait although most of the patients do not
have affected relatives [7]. In the recent literature, DSS is often used to designate
a severe, early onset inherited peripheral neuropathy without any reference to
pathology, NCVs or mode of inheritance. The difference between DSS and
severe CMT is therefore mainly a matter of taste. A third, rare phenotype is
congenital hypomyelination (CH). CH patients have a congenital onset of the
disease with breathing and feeding problems. The evolution is often rapidly
progressive with a fatal outcome in infancy. CH is, by definition, a demyelinat-
ing neuropathy. Molecular genetic studies have shown that each of these pheno-
types can be caused by mutations in different genes and therefore should be
considered as syndromes and not as separate disease entities [2].
Classification of HMSN
The HMSN are a heterogeneous group of disorders in which both motor

and sensory nerves are affected. The HMSNs are subdivided into seven sub-
types based on clinical phenotype, mode of inheritance, electrophysiological
and neuropathological characteristics [7]. We will only discuss types I, II and
III since molecular genetics have made progress in these types. HMSN type I,
also called CMT1, and HMSN type III or DSS are demyelinating neuropathies
Hereditary Peripheral Neuropathies 129

Table 1. Loci for CMT and related peripheral neuropathies
Locus Chromosome Mutation/gene
Autosomal dominant Charcot-Marie-Tooth type 1 (CMT1 HMSN type I )

CMT1A 17p11.2 1.5-Mb tandem duplication/
dosage of PMP22
CMT1A 17p11.2 PMP22 mutations
CMT1B 1q22-23 MPZ mutations
CMT1C ? ?
CMT1 10q21.1-22.1 EGR2 mutation
Autosomal recessive Charcot-Marie-Tooth type 1 (CMT1 HMSN type I )
CMT4A 8q13-21 ?
CMT4B 11q23 ?
CMT4 5q31-33 ?
HMSN-L 8q24 ?
Dominant X-linked Charcot-Marie-Tooth type 1 (CMT1 HMSN type I )
CMT1X Xq13.1 Cx32 mutations
Autosomal dominant Charcot-Marie-Tooth type 2 (CMT2 HMSN type II )
CMT2A 1p35-36 ?
CMT2B 3q13-22 ?
CMT2C ? ?
CMT2D 7p14-15 ?
Autosomal recessive Charcot-Marie-Tooth type 2 (CMT2 HMSN type II )
CMT2 1q21.2-21.3 ?
Axonal motor-sensory neuropathy with deafness and mental retardation
CMT2X Xq24-26 ?
Dejerine-Sottas Syndrome (DSS HMSN type III )
DSS 17p11.2 PMP22 mutations
DSS 1q22-23 MPZ mutations
DSS 10q21.1-22.1 EGR2 mutations
AD-DSS 8q23-24 ?
Congenital hypomyelination (CH )
CH 1q22-23 MPZ mutation
CH 10q21.1-22.1 EGR2 mutations
CH 17p11.2 PMP22 mutations
Distal hereditary motor neuropathy (Distal HMN )
Distal HMN II 12q24 ?
Distal HMN V 7p ?
Congenital non-progressive distal HMN 12q23-24 ?
Recessive distal HMN 9p12-21 ?
De Jonghe/Timmerman/Nelis 130
Table 1 (continued)
Locus Chromosome Mutation/gene
Hereditary sensory neuropathy (HSN )

HSN-I 9q22 ?
Congenital cataracts facial dysmorphism neuropathy (CCFDN )
CCFDN 18qter ?
Hereditary neuropathy with liability to pressure palsies (HNPP )
HNPP 17p11.2 1.5-Mb deletion/dosage of PMP22
HNPP 17p11.2 PMP22 mutations
Hereditary neuralgic amyotrophy (HNA ) or familial brachial plexus neuropathy
HNA 17q25 ?
that show signs of de- and remyelination on nerve biopsy studies. CMT1
presents with a classical CMT phenotype and can be inherited as an autosomal
dominant (AD), AR or X-linked trait. HMSN type III patients have the more
severe DSS phenotype. Since many of the DSS patients have normal parents,
it was suggested that DSS is mainly inherited as an AR trait. HMSN type II
or CMT2 is an axonal neuropathy showing loss of nerve fibers and signs of
regeneration without overt signs of de- and remyelination. CMT2 patients
have a classical CMT phenotype and cannot be distinguished from CMT1
patients on clinical examination only. Electrophysiological studies demonstrate
that CMT2 patients have normal or slightly reduced NCVs while CMT1
patients have severely reduced NCVs, usually less than 38 m/s for the motor
median nerve [11]. In most families all patients can be diagnosed as either
CMT1 or CMT2. However, some families seem to have a puzzling mixture
of CMT1 and CMT2 patients and for these families the term intermediate
CMT was proposed without gaining wide acceptance [12].
CMT1
AD CMT1
AD CMT1 represents the most common form of the inherited peripheral
neuropathies. Molecular genetic studies have identified mutations in the genes
coding for peripheral myelin protein 22 (PMP22) at chromosome 17p11.2
(CMT1A) [13,14], myelin protein zero (MPZ, P0) at chromosome 1q22-q23
(CMT1B) [15] and the early growth response element 2 gene (EGR2) at chromo-

some 10q21.1-q22.1 (the name of this locus is still pending) [16] as causes for
the majority of AD CMT1 cases. Some AD CMT1 families have been excluded
for linkage to the known AD CMT1 loci (preliminarily designated CMT1C)
[17].
CMT1A. The majority of AD CMT1 families showed linkage to chromo-
some 17p11.2 (CMT1A) [18]. In 1991, two research groups independently
demonstrated that some CMT1A patients have a 1.5-Mb tandem duplication
in one of their chromosome 17 homologues [13, 14]. This duplication contains
the PMP22 gene [19, 20]. Occasionally alternatively sized duplications are
found but they always contain the PMP22 gene [21]. The detection of a PMP22
point mutation in Trembler mice provided solid evidence that PMP22 is indeed
the CMT1A gene [22]. Epidemiological studies showed a high prevalence of
the 1.5-Mb CMT1A tandem duplication, reaching 71% in very rigorously
selected unrelated CMT1 families [23]. The CMT1A duplication is therefore,
by far, the most frequent mutation in CMT1. An unexpected finding was the
high rate of de novo CMT1A duplications [24]. Diagnosing a CMT1A de
novo mutation has important consequences for genetic counseling since it will
be transmitted as an AD trait to the next generations. Some CMT1A families
do not have the CMT1A duplication but carry mutations in the PMP22 gene
[25, 26]. These PMP22 mutations are present in the heterozygous state and
are thus AD-inherited. Many of these PMP22 mutations, resulting in a severe
DSS phenotype, are de novo mutations [27]. Recently, a recessive PMP22
mutation was reported in DSS siblings born to normal consanguineous parents
who were heterozygous for this mutation [28]. So far 39 distinct PMP22
mutations have been observed. These include three polymorphisms, 29 muta-
tions leading to CMT1A or DSS and seven mutations associated with heredi-
tary neuropathy with liability to pressure palsies (HNPP), a disorder that will
be discussed later in this chapter.
CMT1A duplication patients have a classical CMT phenotype. The sever-
ity varies between families, within families and even between monozygous
twins [29]. Most patients are able to lead an independent life even at an old
age. In general, CMT1A with the duplication can be considered to be mild
to moderately severe. A few patients homozygous for the CMT1A duplication
have been described [13, 30]. They usually have a more severe phenotype. All
patients, regardless of the severity of the clinical phenotype, have slowed NCVs,
which is thus a completely penetrant trait. Therefore, NCV testing is a 100%
reliable test to confirm or to rule out the disease in CMT1A families. As we
will discuss later this finding cannot be extrapolated to CMT1B and CMT1X.
NCVs are severely slowed to 2030 m/s. However, a few of our CMT1A
duplication patients had a NCV in the motor median nerve of 42 m/s. These
exceptional cases would be misdiagnosed using a strict cutoff value of 38 m/s
to differentiate between CMT1 and CMT2. Follow-up studies show that NCVs
do not dramatically change over a long period of time [31]. Also, there is a
rather poor correlation between severity of the disease and NCV slowing [32].
CMT1A patients with PMP22 mutations usually have a more severe phenotype
and several patients with a DSS phenotype have been reported. Neuropathol-
ogical studies in CMT1A, due to the CMT1A duplication or PMP22 muta-
tions, show loss of large myelinated fibers and the presence of classical onion
bulbs consisting of concentric layers of Schwann cell processes. Recently,
biopsies of two family members with a peculiar PMP22 mutation showed
loosening of the compaction of myelin sheaths resembling the abnormalities
seen in some patients with MPZ mutations [33].
CMT1B. CMT1B is caused by mutations in the myelin protein zero gene
(MPZ, P0) localized at chromosome 1q22-23. So far, 69 distinct disease-
causing mutations and 3 polymorphisms have been reported in MPZ. All
the disease-causing mutations behave like AD mutations and lead to clinical
symptoms when present in the heterozygous state. A few homozygous patients,
born to consanguineous CMT1B parents, have been reported. The parents
were asymptomatic or had a mild phenotype while the homozygous children
were severely affected [34, 35]. Recently, a case of germ-line mosaicism was
reported for a MPZ mutation. The mutation was present in 2 affected children
and absent in the DNA extracted from lymphocytes of both parents [36].
MPZ mutations are associated with a variety of clinical phenotypes, i.e. clas-
sical CMT1, DSS, CH and surprisingly a CMT2-like phenotype [2]. Initially,
MPZ mutations were detected in families with a classical CMT1 phenotype
and this is still the most common phenotype associated with MPZ mutations.
On average, CMT1B patients are somewhat more severely affected than
CMT1A patients with the 1.5-Mb CMT1A duplication. Some MPZ mutations
result in a DSS phenotype. These DSS patients usually represent de novo
MPZ mutations since reproductive fitness is decreased in these severely affected
individuals. Also, CH patients due to MPZ mutations have been described
[34]. All these CMT1B, DSS and CH patients with MPZ mutations have
severely slowed NCVs to less than 38 m/s. Two distinct neuropathological
phenotypes have been described in CMT1B patients [37]. Some biopsies show
classical onion bulbs consisting of concentric layers of myelin sheaths while
decompaction of the myelin sheaths is a striking feature in others. Surprisingly,
MPZ mutations can also be associated with a CMT2 like phenotype, i.e.
slightly reduced to normal NCVs and neuropathological features of an axonal
neuropathy. The first two CMT1B families with a CMT2 phenotype were
actually reported for other reasons and their peculiar phenotype remained
almost unnoticed [34, 35]. In both instances, these patients were the consan-
guineous parents of severely affected children who were homozygous for an

MPZ mutation. In 1998, Marrosu et al. [38] reported a large Italian family
with a CMT2 phenotype due to a MPZ mutation. Subsequently, other MPZ
mutations with a similar phenotype have been described, including several
families affected by the peculiar Thr124Met mutation [39, 40]. Interestingly,
all families with the Thr124Met mutation have a similar CMT2 phenotype
characterized by late onset age, severe gait disturbances, lancinating pains,
pupillary abnormalities and deafness [39]. Neuropatholocial examination of
sural nerve biopsy specimens showed clusters of regenerating fibers in the
absence of myelin abnormalities. NCVs ranged from normal values in asymp-
tomatic young mutation carriers to severely reduced values in older patients.
EGR2. Recently, Warner et al. [16], using a candidate gene approach,
detected three mutations in the early growth response 2 (EGR2) gene localized
at chromosome 10q21.1-q22.1, in patients with a CMT1 and CH phenotype.
Some patients were heterozygous for a mutation while others were homozy-
gous. Subsequently a fourth EGR2 mutation was reported in a DSS patient
[41]. At present six distinct EGR2 mutations have been reported [2].
X-Linked CMT1
The X-linked form of CMT1 (CMT1X) is caused by mutations in the
Cx32 gene localized at chromosome Xq13 [42]. So far, 221 distinct disease-
causing mutations and 7 polymorphisms in the coding region have been re-
ported [2]. In two families, pathogenic mutations in the promoter region of
Cx32 have been found. Most CMT1X patients have a positive family history,
but a genuine de novo mutation has recently been described [43]. All Cx32
mutations seem to result in a CMT phenotype although marked variability
in severity exists between and within families. In general, male patients are
more severely affected than females but female patients can be severely affected
and become wheelchair-dependent. At least in male patients, CMT1X is more
severe than the AD forms of CMT1. A subclinical involvement of central
nervous system (CNS) pathways has been detected by brainstem auditory
evoked potentials CMT1X families [44]. It has been suggested that the detec-
tion of these CNS abnormalities by brainstem auditory evoked potentials can
help to select families for mutation screening of the Cx32 gene. Surprisingly
Cx32 mutations are the second most common mutation in CMT1 only sur-
passed by the CMT1A duplication. The explanation for this discrepancy is
the fact that the presence of severely affected females can easily obscure the
X-linked inheritance pattern. Only the absence of a male-to-male transmission
then points to a possible X-linked mode of inheritance. NCV studies in large
families with Cx32 mutations have finally solved some controversies in the
classification of CMT types. Male CMT1X patients have reduced motor and
sensory NCVs but values higher than 38 m/s for the motor median nerve are
common. NCVs in female patients or female mutation carriers can be normal
or slightly reduced. Based on a cutoff value of 38 m/s for the motor median
nerve, some patients (especially males) will be diagnosed as CMT1 and others
(mainly females) as CMT2 [45]. In retrospect, most of the intermediate CMT
families turned out to be X-linked families with Cx32 mutations. Neuropatho-
logical studies in CMT1X have shown a mixture of axonal pathology and the
presence of small onion bulbs [46].
AR CMT1
AR CMT1 is rare in outbred populations but large pedigrees have been
studied in populations with a high rate of consanguineous marriages such as
North Africa. An AR CMT1 type has been described in Gypsies (HMSN-L),
an ethnically isolated group. AR CMT1 patients are, in general, more severely
affected than AD CMT1 patients. Additional features such as deafness and
scoliosis seem to be an integral part of the phenotype in some AR CMT1
types. All patients with AR CMT1 have severely slowed NCVs. The neuro-
pathology of AR CMT1 seems to be more complex than in AD CMT1. Besides
classical onion bulbs consisting of concentric Schwann cell lamellae, other,
previously unrecognized alterations of the myelin sheaths have been described
such as myelin outfoldings and basal lamina onion bulbs. Four loci for AR
CMT1 have been mapped but the genes involved are not known. To complicate
matters, the designation CMT4 is used for the AR CMT loci. The AR CMT1
loci are: CMT 4A at chromosome 8q13-q21, CMT4B at chromosome 11q23, a
locus at chromosome 5q31-33 the name of which is pending and HMSN-L
at chromosome 8q24.
CMT4A. The first AR CMT1 locus (CMT4A) was mapped to chromo-
some 8q13-q21 in inbred Tunisian families [47]. The clinical phenotype is
severe early onset CMT. Many patients in these families became wheelchair-
dependent. Nerve biopsy studies showed the presence of classical onion bulbs.
CMT4B. The CMT4B locus has been mapped to chromosome 11q23 in
a large inbred Italian family [48]. Patients have a severe early onset CMT.
Nerve biopsy examination shows irregular outfoldings of the myelin sheaths.
AR CMT1 with myelin outfoldings is a genetically heterogeneous disorder
since some families with this phenotype have been excluded for linkage to the
CMT4B locus [49].
AR CMT1 with Basal Lamina Onion Bulbs. LeGuern et al. [50] mapped a
third AR CMT1 locus to chromosome 5q23-33 in Algerian inbred families. They
refrained from giving a name to this new locus. The clinical phenotype in these
families is very peculiar. Patients develop a severe scoliosis as the presenting sign
of the disease. Only later they develop signs of a peripheral neuropathy. Onion
bulbs consisting of basal laminae are found in nerve biopsies [51].

HMSN-L. This AR CMT1 locus has been described in Bulgarian Gypsy
families and maps to chromosome 8q24 [52]. To make HMSN classifications
even more confusing, the name of this new locus refers to the town Lom were
these families live. Patients have classical CMT but hearing loss is a constant
additional feature.
HMSN Type III or DSS
HMSN type III or DSS is a genetically heterogeneous group of disorders.

There is a tendency in the recent literature to use the terminology of DSS as
a mere synonym of early onset severe CMT. We have already discussed that
mutations in PMP22, MPZ and EGR2 can lead to DSS. These patients are
usually isolated and represent de novo cases of dominant mutations in these
genes. Occasionally, DSS patients are homozygous for the CMT1A duplication
or mutations in MPZ [2]. Recently, the first example of an MPZ germ-line
mosaicism was reported in a DSS sib [36]. A locus for AD DSS was assigned
to 8q23-q24 in a single pedigree [53]. No mutations in the known CMT genes
were found in a small AD DSS family that could also be excluded for linkage to
the known loci [54]. This implies further genetic heterogeneity in this syndrome.
CMT2
CMT2 is usually inherited in an AD or AR mode [7]. An X-linked CMT2

type has been described but additional features, such as hearing loss and
mental retardation, were also present [55]. Molecular genetic studies in CMT2
have been much less productive than in CMT1. One of the reasons, for which
we have no obvious explanation, is that large CMT2 families suitable for
linkage studies seem to be much rarer than extended CMT1 pedigrees. A
major disadvantage for linkage studies in CMT2 is the lack of a reliable
biological marker. In CMT1 families, NCV studies can score each individual,
including asymptomatic young persons, as either affected or unaffected since
this trait is 100% penetrant in most CMT1 variants. In CMT2, NCVs can
still be normal in clinically affected individuals. Molecular genetic studies have
demonstrated that CMT2 is very heterogeneous.
AD CMT2
Three AD CMT2 loci have been mapped: CMT2A at chromosome 1p35-
p36 [56], CMT2B at chromosome 3q21q22 [57] and CMT2D at chromosome
7p14 [58]. CMT2C is reserved for the locus of AD CMT2 with vocal cord
paralysis [59]. These CMT2C families have been excluded for linkage to the
AD CMT2 loci. No genes for CMT2 have been found so far. For the clinician
it is very important to keep in mind that the rigorous use of a cutoff value of
38 m/s to differentiate between CMT1 and CMT2 will classify some CMT1A,
CMT1B and CMT1X patients as CMT2 as we have already mentioned. There-
fore, we screen our CMT2 patients for MPZ and Cx32 (if no male-to-male
transmission is present) mutations [39, 60].
CMT2A. The first AD CMT2 locus (CMT2A) was mapped to chromosome
1p35-36 [56] in 1993. CMT2A is associated with a classical CMT phenotype.
CMT2B. In 1995, Kwon et al. [57] reported linkage to chromosome 3q13-
q22 in a family with an axonal neuropathy and both sensory and motor deficits.
They designated this new locus CMT2B. However, sensory abnormalities were
much more pronounced than one normally observes in CMT2 patients. Several
patients in the family of Kwon et al. had poorly healing ulcers that necessitated
amputations of distal parts of the limbs. Linkage to the CMT2B locus has
been confirmed in a second family with a similar phenotype [61].
CMT2D. The CMT2D locus was mapped to chromosome 7p14 [58]. A
striking feature in these CMT2D families is the unusual evolution and distribu-
tion of the motor symptoms. Unlike other CMT types, which invariably start
with weakness in the feet, CMT2D begins in the hands where weakness still
predominates later in the evolution of the disease. This unusual phenotype
was observed in the families that were originally used to map the CMT2D
locus. However, it might be that also families with classical CMT2 map to
the same locus.
AR CMT2
Recently, the first locus for AR CMT2 was mapped to chromosome
1q21.2-q21.3 in a large consanguineous Moroccan family [62]. Patients had
a severe neuropathy sometimes with proximal involvement.
X-Linked CMT2
A locus for X-linked CMT2 has also been mapped to chromosome Xq24-
26 [55]. The phenotype, however, is not a pure axonal peripheral neuropathy
since additional features such as deafness and mental retardation are also
present, suggesting a concomitant involvement of the CNS.
Distal Hereditary Motor Neuropathies
The distal HMN mainly present with a classical CMT phenotype. The
main discriminative clinical factor between CMT1/CMT2 and distal HMN is

the absence of sensory symptoms and signs in the latter. Sensory signs, however,
are often discrete in CMT1 and CMT2 and, therefore, testing of the sensory
NCVs is obligatory to rule out subclinical sensory involvement. Neuropatho-
logical findings in distal HMN are scarce and demonstrate an axonal neuro-
pathy of the motor nerves while the sensory nerves remain intact. Motor
NCVs in distal HMN are normal or slightly reduced when severe muscle
atrophy is present. Sensory NCVs are normal and the sensory nerve action
potentials have normal amplitudes. Concentric needle EMG shows chronic
neurogenic alterations. This latter observation is important since some distal
myopathies can mimic distal HMN. Distal HMN is clinically and genetically
very heterogeneous and a classification into seven subtypes has been made
based on mode of inheritance, age at onset, severity and the presence of
additional symptoms [63]. This already extensive classification, however, is not
complete and some clinical entities were not included or have been described
later on [6466]. Distal HMN is usually inherited in an AD or AR mode.
X-linked families have not been described so far. Molecular genetic studies
have recently confirmed that these clinical distinct phenotypes represent sepa-
rate genetic entities.
Distal HMN Type II

The locus for distal HMN type II also known as spinal CMT was
mapped to chromosome 12q24 in a single Belgian family [67, 68]. The disease
starts between the age of 1520 years and presents with a classical, though
rather severe CMT phenotype since some older patients become wheelchair-
bound.
Distal HMN Type V

A locus for distal HMN type V was localized on chromosome 7p14 in
a single large Bulgarian family [69]. The linkage region shows a large overlap
with the CMT2D region. Interestingly, distal HMN type V also starts in
the hands where it continues to predominate. Families with a similar pheno-
type have been excluded from this region indicating genetic heterogeneity
[70].
Congenital Nonprogressive Distal HMN

This congenital form of distal HMN was not included in the original distal
HMN classification. Its locus was recently mapped to chromosome12q23-q24.
Although distal HMN type II also maps to chromosome 12q24 there is no
overlap between these two linkage regions [71]. This dominantly inherited
disorder presents at birth and is associated with arthrogryposis. The course
is benign with almost no progression [65, 66].
Recessive Distal HMN
The first locus for AR distal HMN was mapped to chromosome 9p21-
p12 in Jordanian families from the Jerash region [72]. Clinical features include
pyramidal signs within the early stages of the disease pointing to an additional
involvement of CNS pathways.
Hereditary Sensory Neuropathies
The HSN also called HSAN are rare diseases [73]. Clinically they present
with prominent sensory abnormalities sometimes leading to poorly healing
ulcers that result in osteomyelitis and eventually necessitate the amputation
of distal parts of the limbs. Good foot care can largely prevent these complica-
tions. Varying degrees of autonomic disturbances may be present. The HSNs
are mostly axonal neuropathies. Electrophysiologically, the sensory nerve ac-
tion potentials have reduced amplitudes or are not obtainable. A classification
in several subtypes has been proposed based on clinical and genetic data. Both
AD and AR variants have been reported.
AD HSN Type I
Molecular genetic linkage studies have mapped the gene for AD HSN
type I to chromosome 9q22.1-q22.3 in Australian families [74]. These families
originate from England and the ancestors were deported as convicts. Patients
in these chromosome 9-linked HSN type I families have striking sensory
abnormalities and occasionally develop poorly healing ulcers in the distal
parts of the limbs. However, most of the patients also have some weakness of
the distal muscles of the legs. Therefore, HSN type I is not an exclusively
sensory neuropathy. There must exist a third locus for AD HSN or AD CMT2
with prominent sensory signs since a large family with an ulceromutilating
neuropathy was excluded for linkage to the CMT2B and HSN I locus [75].
Hereditary Recurrent Neuropathies
Hereditary Neuropathy with Liability to Pressure Palsies

HNPP is an AD-inherited peripheral neuropathy characterized by recurrent
palsies of individual nerve trunks [76, 77]. The palsies are typically painless and
both motor and sensory nerves can be involved. Peroneal, ulnar and radial nerve
palsies are common in HNPP. Palsies usually follow minor trauma to the affected
nerve such as prolonged pressure at common entrapment sites. Recovery is often
complete within minutes or months although some patients develop residual

deficits. Molecular genetic studies have shown that HNPP is a genetically homo-
geneous disorder linked to the CMT1A locus at chromosome 17p11.2. Most
HNPP patients have a 1.5-Mb deletion in the region that is duplicated in CMT1A
patients. Thus, most HNPP patients have only one copy of the PMP22 gene [78].
Only a few HNPP patients have a point mutation in PMP22. These mutations
are either nonsense mutations leading to truncated protein or frameshift muta-
tions that result in a severely altered and probably nonfunctional PMP22 protein
[2]. Initially, it was suggested that HNPP is genetically heterogeneous since some
families were excluded for linkage to chromosome 17p11.2 [79]. However, reanal-
ysis of the data showed that two different peripheral neuropathies were segregat-
ing in a single family. Family members with HNPP were subsequently shown to
harbor a PMP22 point mutation [70]. Extensive clinical and electrophysiological
studies of large groups of patients, diagnosed as HNPP based on DNA testing,
have recently been reported [80]. Four clinical phenotypes emerge. Most patients
have a typical HNPP phenotype with recurrent palsies. However, almost 30% of
mutation carriers remain asymptomatic. We have also seen patients who almost
immediately developed paraesthesias after slight pressure but never had pro-
longed motor or sensory deficits. Finally, some HNPP patients have a progressive
sensorimotor neuropathy that closely resembles CMT. Electrophysiological
studies have clearly demonstrated that there exists a widespread involvement in
HNPP. This is an important diagnostic tool to differentiate HNPP from acquired
mononeuropathies. More than 90% of HNPP patients have prolonged terminal
latencies of the motor median nerve and reduced sural nerve conductions even
in the absence of clinical signs of damage to these nerves. Nerve biopsies in HNPP
show focal myelin thickening called tomacula. HNPP patients with PMP22
point mutations tend to have more severe clinical, electrophysiological and
neuropathological abnormalities although there exists a large overlap with
HNPP due to the common 1.5-Mb deletion [81].
Hereditary Neuralgic Amyotrophy

Hereditary neuralgic amyotrophy (HNA) is a rare disorder characterized
by recurrent episodes of brachial plexus palsies [77]. In contrast to HNPP,
these HNA episodes are always preceded by intense pain in the affected arm.
Intriguingly, HNA episodes are often triggered by recent viral infections,
immunizations or parturition suggesting some autoimmune disease mecha-
nism. Motor symptoms predominate but minor sensory symptoms may be
present. Occasionally the lumbar plexus, the phrenic nerve or individual cranial
nerves can be involved. Symptoms usually recover within weeks to months.
Recovery is usually good but can be incomplete. The number of episodes
ranges from a single episode to more then ten attacks. The attacks can be
uni- or bilateral. HNA is inherited as an AD trait. Dysmorphic features such
as epicanthus, hypotelorism, cleft palate and short stature have been described
in some HNA families but these features usually do not cosegregate with the
HNA trait [77]. The HNA gene was recently mapped to chromosome 17q25
[82, 83]. The HNA gene, however, is not yet found. Most HNA families show
linkage to the chromosome 17q25 HNA locus but some families have been
excluded. HNA is thus a genetically heterogeneous disorder.
Conclusions
Molecular genetic investigations had a direct impact on the study of

inherited peripheral neuropathies by mapping the disease loci and identifying
causative gene mutations. This paved the way for DNA diagnosis that can
now be offered to patients and their relatives. DNA diagnosis is essential for
prenatal and preimplantation diagnoses [84, 85]. Indirectly, these molecular
genetic studies have greatly enhanced our knowledge about the variability in
clinical, electrophysiological and neuropathological features. Initially, the need
for large families or homogeneous groups of families, suitable for linkage
studies, has forced researchers to carefully document the phenotypes in their
patients and families. Later on, genotype-phenotype correlations in patients
with known mutations or families with proven linkage to a certain locus made
it possible to delineate new disease entities.
Acknowledgments
Our research is funded by grants of the Fund for Scientific Research Flanders (FWO),
the Geneeskundige Stichting Koningin Elisabeth (GSKE), a Special Research Fund of the
University of Antwerp, the Association Francaise contre les Myopathies (AFM, France),
and the Muscular Dystrophy Association (MDA, USA). V.T. and E.N. are research assistants
of the FWO.
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Peter De Jonghe, Laboratory of Molecular Genetics, Department of Biochemistry,

University of Antwerp (UIA), Universiteitsplein 1, B2610 Antwerp (Belgium)
E-Mail dejonghe@uia.ua.ac.be
............................
Acute Inflammatory Demyelinating
Polyradiculoneuropathy
Isabelita R. Bella
Department of Neurology, UMass Memorial Health Care, Worcester, Mass., USA
Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is an

immunologically mediated disorder producing multifocal demyelination of
peripheral nerves, nerve roots and cranial nerves. In recent years, neurologists
have gained a better understanding of the pathophysiology of this disorder
that has lead to advances in therapeutic modalities.
The eponym, Guillain-Barre syndrome (GBS), has been used synony-
mously with AIDP, in recognition of the authors who provided descriptions
of this condition. In recent years, the term GBS has come to encompass a
number of disorders of different pathophysiological patterns comprised of
demyelinating and axonal variants. AIDP is the most common subtype in
developed countries, while axonal variants acute motor axonal neuropathy
(AMAN) and acute motor sensory axonal neuropathy (AMSAN) are found
more commonly in northern China [1].
AIDP is the most common cause of rapidly progressive weakness with
an annual incidence of 0.61.9 cases per 100,000 population [2]. It occurs at
all ages [2]. The potential to cause respiratory failure and autonomic nervous
system instability make this disorder a potential neurologic emergency.
Clinical Features
The major clinical features of GBS are progressive weakness and areflexia.
Weakness evolves rapidly (usually over days) and is heralded by paresthesias
of the feet and hands. The legs are often affected first (approximately 50% of
cases) with subsequent spread to the arms. Weakness may, however, start in
the cranial nerves or arms and descend to the legs (approximately 14% of
cases), or start simultaneously in the arms and legs (approximately one third
of cases) [2]. Unlike typical axonal neuropathies, proximal muscles of the arms
and legs are affected early in the course of the disease. Paresthesias described
as pins and needles and tingling sensations often occur in the distal muscu-
lature and ascend as the disease progresses.
Moderate to severe deep aching pain in the back and leg simulating
sciatica and/or dysesthesias of the limbs was seen in approximately 50% of
patients at presentation in one series [3]. Pain may be exacerbated by straight
leg raising, suggesting that the pain may be due to traction of an inflamed
nerve root [3].
On examination, there is symmetric proximal and distal muscle weakness
and loss or attenuation of reflexes. Objective signs of sensory loss are usually
mild, often involving only diminished vibratory sensation despite complaints
of significant pain and dysesthesias. Widespread and profound sensory loss
however has been described as a variant of GBS [4]; presumably the brunt of
the pathology falls on sensory nerves. Respiratory muscle involvement may
occur, with up to one third of patients requiring mechanical ventilation [2]
within 18 days after onset of symptoms [5]. Bilateral facial and oropharyngeal
weakness are seen in more than half the patients with AIDP [6] while ophthal-
moparesis occurs in less than 20% [2]. Ophthalmoplegia is rare but may be
seen in association with ataxia and areflexia in the Miller-Fisher variant [2].
Pupillary abnormalities have been described primarily in those with severe
ophthalmoparesis and ptosis, presumably secondary to involvement of post-
ganglionic parasympathetic and sympathetic nerves. Rarely, papilledema with
pseudotumor cerebri occurs in the setting of markedly elevated cerebrospinal
fluid (CSF) protein (?200 mg/dl) [2].
Autonomic nervous system abnormalities are potentially lethal and may
be seen in as many as 65% of patients [2]. They are more frequent in patients
with severe motor dysfunction and respiratory failure. Manifestations include
cardiac arrhythmias (sinus tachycardia, bradycardia, ventricular tachycardia,
atrial flutter, atrial fibrillation, and asystole), orthostatic hypotension, and
hypertension [2, 7]. Transient bladder paralysis, sweating abnormalities, and
paralytic ileus may also occur [2].
Weakness typically does not progress for longer than 4 weeks; 50% of pa-
tients will reach the nadir of their clinical course in 2 weeks, and 90% in 4 weeks
[4]. Weakness progressing longer than 4 weeks may be seen in a variant of GBS
[4] but progression of a demyelinating polyneuropathy for more than 2 months
suggests chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)
[810]. CIDP shares similar clinical, electrophysiological, and pathological fea-
tures to AIDP but differs in its temporal evolution, course, and response to
treatment [11]. Two to five percent of patients have recurrent GBS [12].
Bella 148
The severity of GBS is variable. Symptoms may vary from mild face and
limb weakness to quadriplegia and requirement for mechanical ventilation
within a few days of onset.
Variations to the typical clinical picture of GBS include pure motor GBS,
pure sensory GBS, Miller-Fisher variant, pandysautonomia, and axonal forms
(see below).
Axonal Variants
Until recently, GBS has been used interchangeably with AIDP, referring
to its predominant pathological features of demyelination and inflammation.
Axonal degeneration, when encountered, was felt to be secondary to intense
inflammation a bystander effect [13, 14]. The recognition of primary axonal
forms of GBS, AMSAN and AMAN, has broadened the clinical spectrum of
GBS. In 1986, Feasby et al. [14] reported 5 patients with severe motor and sensory
neuropathy that fulfilled the clinical criteria for GBS. These patients differed
clinically in that the time to peak severity was much shorter (1 week) and symp-
toms were more severe with more than half requiring mechanical ventilation; all
had inexcitable motor nerves, and all but 1 had poor recovery. Autopsy of 1
patient revealed an acute axonal neuropathy without evidence of demyelination,
suggesting that the primary insult was to the axon. Griffin et al. [1] described
similar cases of GBS in northern China and introduced the term AMSAN to
describe these patients. They also discovered another distinct pattern of GBS
prominent in northern China that of an acute AMAN occurring primarily in
the summer months among rural children and young adults [15]. Autopsy studies
showed wallerian-like degeneration of motor fibers. Recovery was good, similar
to that of traditional AIDP, suggesting that degeneration primarily affects the
motor nerve terminals and intramuscular axons [16].
Antecedent Events
In approximately two thirds of patients, there is an acute flu-like illness

13 weeks prior to the onset of the neuropathy, most commonly with upper
respiratory symptoms, or a gastroenteritis. A number of infectious agents have
been associated with GBS [2, 17] including cytomegalovirus, Epstein-Barr
virus, hepatitis, influenza, mycoplasma, herpes virus, and Campylobacter jejuni.
C. jejuni, in particular, has been been found to be an important antecedent
infectious agent. Culture or serological evidence of C. jejuni occurs in 2640%
of sporadic GBS in developed countries [18, 19]. In contrast, 6674% of GBS
Acute Inflammatory Demyelinating Polyradiculoneuropathy 149

patients in China, where the motor variant is more common, are seropositive
for C. jejuni [20, 21]. Although C. jejuni is found in both axonal and demyelinating
forms of GBS in addition to the Fisher variant, some studies show it occurs more
frequently in the motor axonal form (AMAN) [19, 22]. There are conflicting
reports as to whether C. jejuni infection correlates with a more severe neuropathy
with a poor prognosis [18, 19]. Because the convalescent excretion of C. jejuni
lasts less than 3 weeks, serology may be more sensitive than stool culture in
identifying infection in a patient who later develops GBS [22].
Visser et al. [23] suggest that GBS related to CMV infection produces a
different clinical pattern compared to GBS patients with C. jejuni infection
or GBS patients without either infection. They found CMV-associated GBS
patients were younger, developed severe sensory loss, more often developed
cranial nerve involvement (most commonly facial paresis), and more often
required mechanical ventilation [23].
Other antecedent events include immunization, renal transplantation and
surgery. GBS may also occur in the setting of an underlying systemic disease
such as systemic lupus erythematosus, Hodgkins disease, and in early HIV
infection. Rarely, Lyme polyradiculoneuropathy may mimic GBS.
Epidemiological studies determined that there was an increased risk of
developing GBS up to 6 weeks after immunization with the 19761977 swine
flu vaccine [24]. Recent influenza vaccination programs (19921993 and 1993
1994) were associated with only a small risk of GBS (12 additional cases
per 1 million vaccinated persons) [25]. Many physicians are understandably
concerned about administering influenza vaccinations to GBS patients. It
appears reasonable to follow the recommendation of Hughes et al. [26] of
delaying immunization for 1 year after the onset of GBS, to prevent the small
chance of relapse of symptoms with vaccination [26]. Patients should avoid
reimmunization if the initial vaccination precipitated GBS, especially with
tetanus toxoid [26].
Laboratory Features
Albuminocytologic dissociation (elevated CSF protein without pleo-

cytosis), electrodiagnostic studies revealing evidence of demyelination, and
nerve biopsy revealing inflammation and demyelination are the characteristic
laboratory findings in GBS.
Routine Laboratory Studies

Hematologic studies are typically normal but may show a mild increase
in erythrocyte sedimentation and white blood cell count [2]. Creatine kinase
Bella 150
levels are often mildly elevated in those with severe pain, perhaps reflecting
myonecrosis [2]. Hyponatremia may be seen on occasion due to inappropriate
secretion of antidiuretic hormone possibly due to a resetting of the osmorecep-
tor response [17].
Cerebrospinal Fluid
Albuminocytologic dissociation in the CSF is characteristic of GBS. A
breakdown in the blood-CSF barrier may account for these abnormalities [2].
In the first 48 h after symptom onset, CSF protein may be normal, but after
the 1st week of symptoms, CSF protein is elevated [4], peaks in 34 weeks, at
times reaching as high as 1,800 mg/dl [2]. Rarely, no rise in CSF protein is
seen even after several weeks. The presence of oligoclonal bands has also
been described in GBS [2]. The CSF is typically acellular with 10 or fewer
mononuclear leukocytes/mm3; however, 1150 mononuclear leukocytes/mm3
may rarely be encountered [4]. CSF pleocytosis raises the possibility that GBS
may be occurring in the setting of HIV or Lyme disease.
Autoantibodies to Glycoconjugates
A number of antiglycoconjugate antibodies have been described in GBS.
The strongest association is between IgG anti-GQ1b and the Miller-Fisher
syndrome [21]. Approximately 90% of Miller-Fisher syndrome patients have
high titers of GQ1b antibodies in the acute phase which disappear with clinical
recovery [27].
The association between other ganglioside antibodies and GBS, however,
is variable. Ganglioside antibodies which have been described in GBS include
LM1, GM1, GD1b, GD1a and GT1b [28]. Anti-GM1 antibodies have been
reported to occur in 2560% of GBS patients [18, 21]. The significance of
these antibodies is unclear. Several studies suggest anti-GM1 antibodies occur
more frequently in the pure motor form of GBS [21, 29], in those with axonal
variants [29, 30], and in those with a poor prognosis [29], while others have
found no correlation between the presence of anti-GM1 antibodies and out-
come or pattern of disease (axonal vs. demyelinating) [18, 19].
Kuwabara et al. [30] shed light into the prognostic value of anti-GM1
antibodies. They found the presence of anti-GM1 antibodies to correlate with
the acute motor axonal pattern of GBS. However, there were two patterns of
clinical recovery: those who improved quickly over 2 weeks and those who
had a slower recovery than the anti-GM1-negative patients, requiring several
months for recovery and for whom prognosis was poor. The rapid recovery
of the first group of patients suggests that a mechanism other than axonal
degeneration must be in place. Perhaps reversible immune-mediated changes
at the nodes of Ranvier in motor fibers [31] produce conduction block and

subsequent weakness. Anti-GM1 antibodies have been reported to affect so-
dium and potassium currents [32] and to block conduction, lending support
to this mechanism [31]. Degeneration and regeneration of intramuscular motor
nerve terminals may be another mechanism that could account for rapid
recovery in AMAN patients [31]. Poor outcomes in the second group of
patients suggest an axonal pathogenesis.
More recently, IgG anti-GD1a antibodies have been strongly associated
with the AMAN subtype of GBS found in China [21].
Electrodiagnostic Studies
In AIDP. Electrodiagnostic studies in AIDP patients reveal evidence of
a demyelinating neuropathy. Conduction velocity is reduced (080% of lower
limit of normal) and sensory and motor distal latencies are prolonged [4].
Characteristic findings of segmental demyelination such as partial conduction
block and temporal dispersion due to differential slowing help differentiate
acquired from hereditary demyelinating disorders [33]. Similar to CSF protein
abnormalities, routine electrical studies may be normal early in the disease;
in these cases, F wave responses are often prolonged [33] presumably due to
proximal nerve or nerve root involvement. The compound motor action poten-
tial amplitude may be reduced due to axon loss or distal conduction block.
Needle electromyography may only show reduced recruitment early in course
of GBS, but after several weeks, fibrillation potentials and positive sharp waves
may be seen if there has been axon loss.
In Axonal Variants. In the severe axonal form of GBS, AMSAN, motor
and sensory nerves may be electrically inexcitable [14]. In AMAN patients,
electrodiagnostic studies disclose low or absent motor responses with normal
conduction velocities and normal sensory responses [1]. Needle examination
in both axonal variants may reveal denervation potentials [34], within 34
weeks of onset of symptoms [35].
Pathogenesis
It now appears that the varied histopathological patterns seen in GBS

may stem from different pathogenetic mechanisms. All are immune-mediated
but the immune response may be targeting different portions of the peripheral
nerve to account for the various presentations of GBS. In AIDP the immune
attack appears to be directed to epitopes on the Schwann cell while in axonal
variants, the axon is preferentially targeted. The exact antigenic determinants
to which the immune response is directed, however, are not known [2]. Gang-
liosides or glycoconjugates, located at the abaxonal surface of the Schwann
Bella 152
cell in AIDP or at the motor nodes of Ranvier and internodal axolemma in
AMAN, have been proposed as the targets of the immune attack [36, 37].
In AIDP
Both cellular and humoral components of the immune system appear to
play a role in the demyelinating lesion of GBS. Observations in the animal
model of GBS called experimental autoimmune neuritis (EAN) lend support
to a cellular-mediated immune response in the pathogenesis of GBS [38].
Waksman and Adams [39] injected rabbit nerve/ganglia and Freunds adjuvant
into rabbits which produced quadriplegia within 2 weeks. Transfer of specific
T cell lines reactive to P2/PO produced the classical clinical, electrophysiolo-
gical, and pathological features of EAN in rats [38]. Further evidence of T cell
activation comes from the finding of elevated levels of soluble IL-2 receptors in
GBS [40]. Equally important findings support a humoral mediated immune
response in the pathogenesis of GBS. Koski et al. [41] found anti-peripheral-
nerve myelin antibodies in the sera of GBS patients with clinical improvement
correlating with a reduction of the anti-peripheral-nerve myelin titer. Sera
from EAN animals and GBS patients injected intraneurally or subperineurally
in animals have also produced demyelination [42, 43]. Lastly, the effectiveness
of plasmapheresis in GBS supports a major role for humoral factors [44].
Recent evidence suggest that the target of the immune attack in AIDP is
the Schwann cell. Hafer-Macko et al. [36] found complement activation
markers (C3d and C5b-9) along the outer surface of the Schwann cell, sup-
porting a complement-mediated immune mechanism directed towards the
Schwann cell [36]. They speculate that complement-fixing antibodies bind to
epitopes on the outer surface of the Schwann cell, thereby activating comple-
ment and leading to myelin vesiculation.
In Axonal Forms
Evidence that axonal constituents are the target of the immune attack in
axonal forms of GBS comes from work by Hafer-Macko et al. [37]. They
examined peripheral nerves from 7 AMAN patients who came to autopsy.
They found IgG and C3d (a complement activation product) bound to the
nodal and internodal axolemma in motor fibers which had not yet begun
wallerian-like degeneration [37]. Macrophages were also found in internodal
periaxonal space displacing the axon from the Schwann cell and myelin sheath.
This is in contrast to AIDP, in which complement activation markers were
found on the Schwann cell itself. The above findings suggest that axonal forms
of GBS may be complement- and IgG-mediated, and that the immune attack
is directed to epitopes of the axolemma. The specific epitope again is not
known, but the presence of high titers of anti-GD1a antibodies in AMAN

but not AIDP patients suggests that a GD1a-like epitope may be the target
of the immune attack [21].
As mentioned earlier, the rapid improvement seen in AMAN patients
may be due to reversible changes at the node. In their study using rat nerve
fibers, Takigawa et al. [32] found that anti-GM1 antibodies, in the presence
of complement, decreases Na+ current and induces membrane leakage. This
may lead to blocking of Na+ channels and prevent further impulse conduction.
Molecular Mimicry
The presence of antiganglioside antibodies, particularly anti-GM1 anti-
bodies, in both demyelinating and axonal forms of GBS and the finding of
ganglioside-like epitopes on some strains of C. jejuni have led to the concept
of molecular mimicry [21, 45], in which an immune attack occurs on the
epitope shared by the nerve fiber and infectious organism [46], as a possible
mechanism for C. jejuni-associated GBS. Yuki et al. [45] demonstrated the
presence of a terminal tetrasaccharide located in lipopolysaccharides of
C. jejuni serotype (PEN 19) to be identical to that of GM1 [45]. The
authors speculate that infection by C. jejuni induces production of anti-
GM1 antibodies which may bind to motor nerve terminals causing nerve
inexcitability and subsequent weakness [45]. Anti-GQ1b-like epitopes have
also been demonstrated in C. jejuni isolates associated with the Fisher
syndrome [47].
Infection with a C. jejuni strain containing the same ganglioside-like
epitopes shared with nerve fibers and the presence of sera containing antigan-
glioside antibodies against these specific ganglioside-like epitopes are not in
itself sufficient to cause GBS [46]. Perhaps differences in host susceptibility
(genetic predisposition, intercurrent illness) or differences in organisms may
account for the varied immune responses [46].
Pathology
The predominant pathological features of AIDP are a mononuclear in-

flammatory infiltrate of the endoneurium often in a perivenular distribution,
and segmental demyelination [13]. The inflammatory changes are present
throughout the peripheral nervous system, affecting sensory and motor nerve
roots, spinal nerves, plexuses, proximal and distal peripheral nerve trunks,
and even intramuscular nerve twigs [13]. Areas of segmental demyelination
characterized by retraction of the myelin sheath in the node of Ranvier in
early lesions and denuded axons in more developed lesions are found adjacent
to inflammatory areas [13]. Nerve biopsy, however, is rarely indicated as the
Bella 154
clinical, electrodiagnostic, and CSF abnormalities of AIDP reliably support
the diagnosis.
Differential Diagnosis
Table 1 contains a number of conditions which cause rapidly progressive

weakness and must be differentiated from AIDP.
Management and Treatment
GBS patients should be observed in the hospital, preferably in an intensive

care unit, for potential respiratory and autonomic nervous system compromise
(see table 2). Patients with neck flexor weakness (correlates with diaphragmatic
muscle weakness), in particular, require close monitoring. Forced vital capacity
and maximum inspiratory pressure should be monitored around the clock as
weakness may rapidly progress to involve respiratory muscles. Obtaining an
arterial blood gas is helpful in determining adequate oxygenation and possible
hypercapnia. Clinically, however, patients may become restless, tachycardic,
tachypneic, and sedated before changes in blood gases are detected. A normal
forced vital capacity is 65 ml/kg; at a level of 30 ml/kg, a poor cough with
accumulation of secretions may be seen which can be managed with chest
physical therapy and supplemental oxygen. At a level of 25 ml/kg, the sigh
mechanism is compromised producing atelectasis and hypoxia; incentive spiro-
metry and deep breathing can minimize atelectasis [48]. Criteria for intubation
have been suggested by Ropper and Kehne [49] (see table 1). Tracheostomy
should be delayed for 710 days as up to one-third of patients may improve
rapidly and can be extubated after the first few days [49].
Signs of autonomic nervous system dysfunction should be monitored by
measuring blood pressure, fluid status, and cardiac rhythm. Hypotension can
be managed by infusion of fluids; hypertension treated only if persistent and
severe, using short-acting a-adrenergic blocking agents, and bradyarrhythmias
with atropine [7].
Care should also be taken to avoid complications of a bedridden patient,
such as deep venous thrombosis and pulmonary embolism, compression neuro-
pathies, and pressure sores. Frequent turning of the patient, physical therapy,
insulating pads over potential compression sites, and subcutaneous heparin
can help prevent these complications.
Patients with GBS may be treated with either plasmapheresis (PE) or
intravenous immunoglobulin (IVIG). Several large, multicenter controlled

Table 1. Conditions that may mimic GBS [adapted from 64, with permission]
Disorder Major distinguishing features
Myasthenia gravis Reflexes are spared

Ocular weakness predominates
Positive response to edrophonium
EMG: decremental motor response
Botulism Predominant bulbar involvement
Autonomic abnormalities (pupils)
EMG: normal velocities, low amplitudes, incremental response
(with HFRNS)
Tick paralysis Rapid progression (l2 days)
Tick present
Shellfish poisoning Rapid onset (face, finger, toe numbness)
Follows consumption of mussels/clams
Toxic neuropathies EMG: usually axon loss
Organophosphorus toxicity Acute cholinergic reaction
Porphyric neuropathy Mental disturbance
Abdominal pain
Diphtheritic neuropathy Prior pharyngitis
Slower evolution
Palatal/accommodation paralysis
Myocarditis
Poliomyelitis Weakness, pain and tenderness
Preserved sensation
CSF: protein and cell count elevated
Periodic paralysis Reflexes normal
Cranial nerves and respiration spared
Abnormal serum [K+]
Critical illness neuropathy Sepsis and multiorgan failure ?2 weeks
EMG: axon loss
Acute myopathy of Tetraparesis and areflexia
intensive care Follows prolonged treatment with NMBA and corticosteroids
Trauma; status asthmaticus; and organ transplantation-
associated
Clinical and EMG features of myopathy
Vasculitic neuropathy Systemic signs
Asymmetry in sensory and motor loss
Reflexes reduced proportionate to weakness
Rabies Fever
Asymmetric sensory and motor signs
Muscle spasms, agitation, abnormal mental status
EMG>Electromyogram; HFRNS>high-frequency repetitive nerve stimulation; NMBA>

neuromuscular blocking agent.
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Table 2. Management of GBS [adapted from 65, with permission]
Clinical status Management
Very mild GBS Admit to ward

Ambulatory without assistance Monitor VC every 8 h
and no ventilatory compromise Observation
Consider PE or IVIG1, 2
Ambulatory only with assistance Admit to ICU if possible or to floor with frequent monitoring of VC
and no ventilatory compromise Check ABG
IVIG1 or PE (4 exchanges)
Not ambulatory and Admit to ICU
mild ventilatory compromise Monitory VC frequently
Check ABG
Intubate if [49]
VC =1215 ml/kg
Falling VC over 46 h
Oropharyngeal paresis with aspiration
Respiratory fatigue with VC of 15 ml/kg
Not ambulatory and Admit to ICU
requires assisted ventilation Monitor for autonomic nervous system dysfunction [7]
Treat hypotension with infusion of fluids
Use short-acting a-adrenergic blocking agents to treat persistent
hypertension
Treat bradyarrhythmias with atropine
Frequent turns to avoid pressure sores
Avoid compression neuropathies
Physical therapy
ABG>Arterial blood gases.

1
IVIG is preferable if there is poor vascular access or cardiovascular instability. The dose administered
is 400 mg/kg/day for 5 consecutive days.
2
One study [53] demonstrated improvement with 2 plasma exchanges compared to none in mild GBS
patients. There has been no controlled study showing benefit of IVIG in mild GBS patients. Because
efficacy of IVIG and plasma exchange has been demonstrated in patients with more severe GBS, IVIG
would appear to be just as helpful for mild GBS.
trials [50, 51] demonstrated the beneficial effect of PE when used within the
first 2 weeks of disease onset, even in those with poor prognostic signs [52].
On average, patients treated with PE were able to walk 1 month earlier than
untreated patients; respiratory-dependent patients treated with PE walked 3
months earlier than those untreated [50]. The GBS study group recommends

exchanging 200250 ml/kg in 35 sessions over 714 days [50]. PE, until
recently, was utilized in patients who were not ambulatory, had evidence of
respiratory compromise, or had bulbar difficulties. Results from the French
Cooperative Group on Plasma Exchange in Guillain-Barre Syndrome study
revealed that mildly affected patients who remain ambulatory also benefit
from 2 plasma exchanges while moderate to severely affected patients (those
not ambulatory or requiring mechanical ventilation) benefit from 4 plasma
exchanges; severely affected patients did not benefit from 2 additional ex-
changes [53]. Spontaneous relapse may occur in a small number of patients
(310%) within days to weeks after improvement; additional treatment with
plasma exchange benefits these patients [54]. PE requires good venous access
and is available in major medical centers. Patients who have cardiovascular
instability or autonomic dysfunction may tolerate this poorly due to its poten-
tial for inducing hypotension. These limitations prompted a search for alterna-
tive treatments.
The favorable results of several uncontrolled studies suggested IVIG may
be a therapeutic alternative to PE. A large randomized trial was subsequently
performed by Dutch investigators [55] and found that IVIG given in the first 2
weeks of the disease was as efficacious as PE. Following this study, two small
uncontrolled studies [56, 57] reported a high relapse rate of more than 50% com-
pared to 10% seen in PE-treated patients [54]. Subsequently, a large randomized
controlled trial conducted by The Plasma Exchange/Sandoglobulin Guillain-
Barre Syndrome Trial Group confirmed the equivalence of IVIG and PE; there
was not a substantial benefit in using a combination of PE followed by IVIG
although there was a nonsignificant trend favoring the use of combination treat-
ment in some outcome measures [58]. Minor side effects of IVIG including flu-
like symptoms, headache, nausea and fatigue occur in less than 10% of patients
[59]. Care must be taken to avoid administering IVIG to those with IgA defi-
ciency or renal disease as it may cause anaphylaxis in the former [59] and renal
failure in the latter [60]. Because of its relative ease of administration, IVIG has
increasingly become the initial treatment in AIDP.
The use of corticosteroids in AIDP is generally ineffective. The results of
a pilot study suggest that treatment with IVIG and methylprednisolone may
be more advantageous than treatment with IVIG alone [61]. Larger, controlled
trials are needed to confirm this.
Outcome
The majority of patients have a good outcome. Recovery typically occurs

over weeks to months and patients are usually able to return to normal life
Bella 158
within 612 months [2]. The length of time to full recovery is related to the
severity of the illness. It may take 1.52 years of intense rehabilitation to attain
maximal improvement [2]. Several factors are associated with a poor outcome:
age greater than 60 years, rapidly progressive weakness in less than 7 days,
need for mechanical ventilation, and the most powerful predictor a mean
distal motor amplitude of 20% of normal or less [52]. The presence of all four
factors is associated with a less than 20% probability of walking unassisted
at 6 months without treatment. Treatment with plasmapheresis, however, in-
creases this probability to 40% [52].
Approximately 75% of patients make a good recovery; of these, less than
15% have no residual deficits and the remainder are able to return to work
with mild residual deficits manifesting as paresthesias or minimal distal weak-
ness [2]. Severe residual deficits impairing ambulation occur in approximately
10% of patients despite therapy [62]. Although mortality has improved with
the institution of mechanical ventilation and respiratory care, 212% of pa-
tients with AIDP die from the disorder. Causes include sepsis, adult respiratory
distress syndrome, autonomic dysfunction, pulmonary embolism, and most
commonly, ventilator-associated pneumonia [63].
Conclusion
GBS comprises a group of disorders of different pathophysiological pat-

terns AIDP, AMSAN, and AMAN. Patients present similarly with rapidly
progressive weakness, areflexia, and albuminocytologic dissociation in the
CSF. In AIDP, evidence of a primary demyelinating neuropathy is seen on
electrodiagnostic studies. Although rarely indicated, nerve biopsies reveal en-
doneurial mononuclear inflammatory infiltrates along with evidence of demy-
elination. The majority of patients make a good recovery, especially with IVIG
or PE treatment. In the axonal variants, AMSAN and AMAN, a primary
axonal process is evident on electrodiagnostic and pathological studies. AMSAN
patients differ from AIDP by their more severe course and longer time to
recovery. AMAN patients, on the other hand, lack sensory abnormalities
typically present in AIDP, while their prognosis is good. Recent work has
suggested that molecular mimicry may be a possible pathogenetic mechanism
for some patients with GBS. Targeting epitopes located in different portions
of the peripheral nerve (Schwann cell in AIDP and axolemma in axonal
variants) may account for the various presentations of GBS.

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Isabelita R. Bella, MD, Department of Neurology, UMass Memorial Health Care,

55 Lake Avenue North, Worcester, MA 01655 (USA)
Tel. +1 508 856 4147, Fax +1 508 856 6778
Bella 162
............................
Proximal Spinal Muscular Atrophy of
Childhood
Irena Hausmanowa-Petrusewicz a, Jacek Zaremba b
a
Neuromuscular Unit, Medical Research Centre, Polish Academy of Sciences and
b
Department of Genetics, Institute of Psychiatry and Neurology, Warsaw,
Poland
History
Proximal childhood spinal muscular atrophy (SMA) is one of the most

common and severe autosomal recessive diseases of children. In all spinal
atrophies, dysfunction and loss of anterior horn cells leads to muscle atrophy
and weakness sparing the sensory system and the central nervous system.
The first description of SMA is attributed to the paper by Sevestre [1] in
Paris. The affected child was symptomatic from birth, and severely paralyzed.
In 1891, an Austrian neurologist Werdnig [2] described two brothers who
had weak limbs from the age of 10 months and abnormal breathing; the
legs were more affected than the arms. Werdnig also described the postmortem
examination of 23 muscles, peripheral nerves and central nervous system.
In 1893, Hoffmann [3], a neurologist in Heidelberg, described two families
with similar symptoms which started 2 months after birth and led to death
at the ages of 1, 2, 3 or 5 years. Hoffmann noticed general floppiness and
more severely affected legs than arms. As opposed to the case of Sevestre
both Werdnigs and Hoffmanns infantile cases had a more chronic course
but were equally severe. The introduction of clinical electromyography (EMG)
after World War II extended the concepts of SMA: the dystrophy with
fasciculations was proved to be juvenile spinal atrophy (Kugelberg and We-
lander syndrome) [4]. Subsequently there was a vivid debate between lumpers
and splitters whether SMA represented two (or even three) different diseases.
This uncertainty was later resolved by molecular geneticists.
Table 1. Three forms of proximal childhood SMA
Onset Survival
Form 1
1a in utero =2 years
(overt at birth)
1b within 6 months 24 years
810% of these children survive much
longer but do not improve
Form 2 =18 months several years
90% up to 10 years
Form 3
3a =3 years normal life expectancy
3b 317 years normal life expectancy
Clinical picture: form 1a infants: floppy, frog-like position, legs more para-
lyzed than arms, areflexia, abdominal breathing, weak cry, finger tremor, alert;
form 1b: similar symptoms but progress slower than in group 1a; unable to
lift the head and sit without support; form 2 (intermediate): proximal limb
weakness and atrophy, joint contractures, scoliosis, chest deformities; achieve
unaided sitting but cannot stand; form 3: proximal weakness, mostly in the
legs; form 3a: slow progress leading to wheelchair after 57 years, and form
3b: ambulatory for a very long time.
Classifications of Childhood SMA and Clinical Features
The main criteria used to classify SMA are age at onset of symptoms,
course of the disease and age at death. We recognize three forms of proximal
childhood SMA [5, 6] (table 1).
Dubowitz [7] distinguished ten classes of varieties within each form; he
also indicated the possibility of an SMA form 0: the most severe with a history
of reduced fetal movement in utero, with asphyxia and severe universal muscle
weakness at birth (including facial weakness) [8]. The inclusion and exclusion
criteria of childhood SMA were approved by the International SMA Consor-
tium at the European Neuromuscular Center [9]. The inclusion criteria corre-
spond to clinical features described above and confirmed by neurogenic EMG
and biopsy. Exclusion criteria summarized signs and symptoms considered
incompatible with SMA such as involvement of diaphragm or extraocular
muscles, mental retardation, marked slowing of nerve conduction velocity
(CV), sensory deficit, congenital joint contractures, metabolic abnormalities
and high CK activity.
Hausmanowa-Petrusewicz/Zaremba 164
Epidemiology
The current incidence varies between 10 and 15 per 100,000 live births,
which is higher than in the past because the detectability has improved. On
the basis of these estimates the frequency of heterozygosity for autosomal
recessive SMA would be 1:50. The prevalence of a chronic, milder form of
SMA3 is about 1:83,000 of the general population [10]. In some countries
consanguineous marriages are more frequent and the incidence of SMA is
very high, e.g. in some Muslim countries. An exceptionally high incidence of
infantile SMA was found in the Karaite community in Israel [11] the preva-
lence of SMA there is 1:400, and carrier frequency 1:10.
Laboratory Findings
No biochemical markers of SMA are known. Generally CK activity in

SMA children is in the normal range.
In small infants with SMA1, electromyographic studies (EMG) show
fibrillations and fasciculations which are not very prominent and a typical
spontaneous activity which is motor unit firing at a rate of 515 Hz [12].
During slight contraction, two populations of motor unit action potentials
were found one short and low and the second long and high [13]. There are
no complex potentials. In SMA2 and 3, the spontaneous activity includes
fibrillations, long positive waves and fasciculations. Quantitative EMG is char-
acterized by long, high motor unit action potentials which are often complex
(up to 75%) [13]. Nerve conduction studies in SMA2 and 3 have shown normal
motor and sensory CV; after a few years it may slightly slow down. However,
motor CV is slow in SMA1: the delay in maturation of CV in SMA1 children
is obvious [14].
Morphological investigations of muscle are needed to recognize the neu-
rogenic character of the weakness. In spite of the progress in molecular genetics
muscle biopsy is necessary in the non-deleted cases or in some exceptional
cases. Light microscopy in SMA1 shows clusters of small muscle fibers of 5-
to 8-lm diameter as well as normal and/or hypertrophic fibers. The proportion
of small fibers is higher in severe cases [15]. They are different from the small
atrophic denervated fibers in other neurogenic disorders: in SMA they are
round, with a well-preserved architecture, centrally placed single nuclei and
have a high activity of desmin, fetal and neonatal myosin, and are surrounded
by N-CAM [16] (fig. 1). The fetal-like character of these changes is confirmed
by electron microscopy which shows myotube-like fibers with large centrally
placed nuclei and scanty sarcoplasm, and by the presence of immature fibers
Proximal Spinal Muscular Atrophy of Childhood 165

1
Fig. 1. SMA1b: musculus quadriceps femoris. Round small muscle fibers and a group
of fibers of normal diameter. HE.420.
Fig. 2. SMA1a: musculus quadriceps femoris. Myotube-like cell.9,400.
consisting of 23 cells at different stages of maturation surrounded by a com-

mon basal lamina [16] (fig. 2). Undifferentiated histochemical fiber types persist
beyond the myotube stage. In SMA1, apoptosis of muscle is observed, which
does not occur in the muscles of normal neonates. Nucleus and sarcoplasm
show condensation, fragmentation and formation of apoptotic bodies [17].
Fig. 3. SMA3: musculus quadriceps femoris. Muscle fibers of normal diameter among
the hypertrophic and atrophic fibers. HE.390.
Intramuscular nerves also reveal some signs of immaturity with multiaxonal

and premyelinated fibers.
The histopathological pattern in SMA3 is different and resembles the
chronic denervation of mature muscle. The process in SMA3 begins in the
perinatal period or later. Small mononucleated muscle fibers are among the
normal and hypertrophic ones. The atrophic fibers are irregular, often angular;
sometimes, only groups of nuclei are seen. Splitting and other secondary
changes are superimposed. Target or targetoid fibers can be seen (fig. 3).
In peripheral nerves, demyelination and wallerian degeneration or thin
myelin sheaths are found. A higher percentage of thin fibers and a uniform
length of their internodes regardless of fiber diameter [18] are described.
Changes in ventral roots are similar to those in peripheral nerves [19].
Motoneurons of the spinal cord and motor nuclei of VIXII cranial nerves
show both atrophic and degenerative changes mostly expressed in lumbar and
cervical parts of the spinal cord. Degenerating cells are shrunken, angulated and
darkly stained. In electron microscopy, they show the presence of apoptosis.
Some investigators suggest that immaturity of muscle, motoneurons, or
both in SMA [2022] is evidenced by the myotube-like appearance of muscle,
undifferentiated metabolically, small muscle fibers, the multiaxonal character
of intramuscular innervation, delay of nerve maturation, excessive death of
motoneurons and apoptosis seen in muscles of SMA babies.

The hypothesis of a disturbed interaction between nerve and muscle in
SMA was proposed by Vrbova and Lowrie [23]. They suggested that improper
nerve-muscle interactions in SMA inhibit the conversion of motoneurons from
growing to transmitting cells. They cannot cope with functional demands and
are activated too early, before maturation, which leads to death. This happens
extensively in the most severe form of SMA. If a motoneuron has more time
for maturation before it becomes active, survival is better.
As an alternative hypothesis to the arrest of maturation, Morrison [20]
discussed the slow progressive death of neurons beginning in utero and continu-
ing until death. In spite of many pieces of evidence that in nerve-muscle
interaction the motoneuron defect is primary, some data suggest a possible
primary role of the muscle in the SMA pathology [17, 22, 24]; thus, the problem
is still unsolved.
Genetics
The locus of all three forms of SMA is 5q11.213.3 on the basis of linkage
studies [25]. In 1995, Melki et al. [26] detected deletions in SMA individuals
within 5q13 and identified a candidate gene, the survival motor neuron (SMN)
gene. Simultaneously, a Canadian group identified another candidate gene,
the neuronal apoptosis inhibitory protein (NAIP) gene [27]. The region encom-
passing both SMN and NAIP contains an inverted duplication; the genes and
polymorphic markers within this region have two copies telomeric and
centromeric (fig. 4). The region is particularly unstable, prone to deletions and
other rearrangements. Another gene mapped to the 5q13 region is p44, which
is a subunit of a basal transcription factor TFIIH [28].
The SMN gene contains eight exons that span 20 kb; the telomeric SMNt
and centromeric SMNc (now named SMN1 and SMN2, respectively) differ
only by five base pairs. SMN1 and SMN2 produce a transcript encoding 294
amino acids. Lefebvre et al. [26] found that 98.6% of patients had a deletion
of SMN1 with the loss of exons 7 and 8 or exon 7 alone; the remaining patients
had a single base and other small mutations. It is now a well-established fact
that mutations in the SMN1 gene are the major determinants of the SMA
phenotype. The NAIP gene containing 16 exons is extending over 60 kb and
also has two copies. In the series of Roy et al. [27] 45% of SMA1 and 18%
of SMA2 and SMA3 patients had a deletion of the NAIP gene.
NAIPc and NAIPt differ, NAIPc does not contain exon 5 (present in
NAIPt) and it is a pseudogene (unable to transcribe).
The telomeric copy of the p44 gene is deleted in SMA at a frequency
similar to that observed for the NAIP gene. Both the NAIP gene and the p44
Fig. 4. SMA duplicated chromosomal region according to Lefebvre et al. [33]. Cen>
Centromere; Tel>telomere.
gene do not seem to be critical for the development of SMA although they
are detected more frequently in the most severe form of SMA1, and NAIP is
a good candidate for a modifier because of its antiapoptotic function. Scharf
et al. [29] recently found a novel gene H4F5 situated very close to SMN1 and
deleted in over 90% of SMA1. Therefore, the authors proposed that H4F5
may be a good candidate for a phenotypic modifier in SMA. There is now
evidence that SMA2 and SMA3 may not be due to deletions but to gene
conversion events in which SMN1 is replaced by SMN2. Normal individuals
usually have two copies of SMN2 and two copies of SMN1 (one copy of each
gene per chromosome). Conversion of SMN1 to SMN2 provides an increase
of copies of SMN2 [3032]. McAndrew et al. [30] found three (instead of two)
copies of SMN2 in 1.9% of normals, 19% of SMA1 carriers and 34.5% of
SMA2 and SMA3 carriers. Thus, the SMN2 product appears to partly com-
pensate the absence of SMN1 [33]. However, an increased number of SMN2
copies have also been found in the severe form of SMA, and this can indicate
that apart from SMN2 there are other factors which may modulate the SMA
phenotype [33a].
The SMN protein is located in nuclear structures called gems which are
found in vicinity of coiled bodies (hence the term gems: gemini of coiled
bodies) [34]. Interactions were shown of SMN protein with RNA-binding
protein and with antiapoptotic protein Bcl-2 suggesting an antiapoptotic role
of SMN [35]. It was also shown that the SMN protein was associated and
colocalized in the cytoplasm and in gems with a novel SMN-interactive
protein 1. The SMN-interactive protein 1 complex is involved in the cyto-
plasmic assembly of small nuclear ribonucleoproteins to spliceosomal RNAs
and with the nuclear import of the small nuclear ribonucleoprotein complex
[36]. These facts provide evidence of a likely part played by the SMN protein
in the metabolism of RNA [reviewed in 24, 33, 37]. Immunohistochemical
analysis revealed a marked deficiency of SMN protein in motor neurons in
SMA1 and a reduction in SMA3 [38].

The role of the SMN protein in the pathogenesis of SMA is not known.
An important clue to this problem may be the finding of the SMN protein
self-oligomerization defect in SMA and evidence that the severity of SMA
correlates with the intracellular concentration of oligomerization-competent
SMN proteins [39]. In spite of a difference of five nucleotides between SMN1
and SMN2 genes, they encode an identical protein. The question arose, there-
fore, why SMN2 was unable to fully compensate the loss of SMN1. The answer
was provided only recently by Lorson et al. [40] who found that it was due
to one nucleotide exchange CKT at codon 280 a change which dictates
exon 7 alternative splicing and production of SMN2-defective mRNA which
lacks exon 7. This was a very important step forward in the elucidation of
SMA pathogenesis and a finding which according to the authors may be
helpful in designing a gene therapy targeted at the repair of the SMN2 gene
transcript. The above data show remarkable progress in the studies on the
molecular basis of SMA.
There remains, however, a number of phenomena which seem to be also
related to epigenetic factors, i.e. those altering the activity of the SMN gene
without changing its structure. These are in particular incomplete penetrance
of the mutated genes and gender influence, which are characteristic mainly of
the mildest form of SMA.
Investigations based on traditional segregation analysis showed a predom-
inance of males in the mildest SMA3 group and revealed incomplete penetrance
of the mutated gene, particularly in females after an age at onset of 8 years.
These findings could suggest the existence of a female sparing factor possibly
connected with some milestones of female hormonal development [41, 42].
More recent investigations not only confirmed the predominance of males in
the mildest forms of SMA [43] but also provided molecular evidence of incom-
plete penetrance of the mutated gene in some families: unaffected individuals
were found in SMA families among carriers of homozygous deletions of exons
7 and 8 of the SMN gene, and the majority of them were females [44, 45]. It
is worthwhile noting that the influence of sex is also characteristic of some
other motor neuron diseases, such as the distal form of SMA [46] and amy-
otrophic lateral sclerosis [47, 48].
It is tempting to speculate that some sex steroids, possibly owing to a
mechanism involving their nuclear receptors, may be capable of upregulat-
ing transcription of SMN2 and/or antiapoptotic genes, particularly in the
mildest SMA3 form of the disease. Obviously other mechanisms of the posi-
tive influence of sex steroids on the clinical picture of SMA are also con-
ceivable.
Differential Diagnosis
(A) With other diseases: The differential diagnosis of SMA1 includes all
conditions that cause the floppy baby syndrome: general (e.g. system disorders),
cerebellar, congenital neuromuscular diseases such as myasthenia gravis, nema-
line myopathy, myotubular myopathy, congenital mitochondrial myopathies,
congenital myotonic dystrophy, congenital neuropathies. In SMA3, the differ-
entiation with muscular dystrophy or polymyositis is most important. (B) The
differentiation between SMA and related (apparently similar) neurogenic dis-
orders is more difficult. This large group includes: (1) monomelic forms of SMA,
e.g. Ryukyuan disease still not mapped [49], a juvenile distal form of Hirayama
et al. [50], an asymmetric, nongenetic syndrome, (2) distal SMA a heteroge-
neous group [51], particularly important is diaphragmatic SMA [52] with a char-
acteristic involvement of the diaphragm; it does not map to 5q, and (3) bulbar
forms, including the Fazio-Londe syndrome [53, 54]. (C) SMA-plus: Cases not
mapped to chromosome 5q with some classical clinical features of SMA but also
with some symptoms considered as exclusion criteria in the diagnosis of SMA,
e.g. SMA with mental retardation, with olivopontocerebellar features [55, 56],
with ophthalmoplegia or oculo-pharyngeal syndrome [57].
Most important, however, are the descriptions of disorders presenting the
same mutations as in classical SMA but entirely distinct phenotypes considered
until recently to be incompatible with SMA. SMN deletions have been found
in some cases of congenital axonal neuropathies with extremely slow CV [58]
or in some cases of arthrogryposis [59, 60] which is against the formerly
accepted exclusion criteria [9]. Obviously, not all cases of congenital neuropathy
or arthrogryposis map to chromosome 5q. The neurogenic form of arthro-
gryposis has been considered by some authors a variant of SMA but con-
tractures are not found in typical SMA (except minor contractures of some
joints). Moreover, SMA is familial while even severe arthrogryposis is mostly
sporadic and unlike SMA improves with time.
Infrequent cases of familial arthrogryposis (autosomal recessive or
X-linked) with fractures of the long bones were previously diagnosed as SMA
[61]. The severe subset of arthrogryposis seems to be allelic with SMA. Some
cases of congenital heart disease coexisting sometimes with classical SMA
show SMN deletion [62].
Treatment
In clinical practice, attention is given to ventilatory support, physical

therapy, orthopedic devices and surgery. Physical and orthopedic treatment

maintains late ambulation and prevents contractures. The scoliosis sometimes
requires surgery (fusion). Some neurotrophic factors enhance motoneuron
survival in vitro and therapeutic trials are being conducted. Until now no
clinical application has been tried. Regarding the gene therapy of SMA, some
hope may be attached to already established molecular characteristics of SMA
and to the role played by the modifying genes. Understanding basic molecular
features of SMN2 which are alternative splicing of exon 7 [40] and special
ability of SMN2 to convert to SMA1 seems to make SMN2 the best candidate
and a target for gene therapy. Finding ways of suppressing apoptosis of the
motoneurons could be a possible therapeutic approach, e.g. through activation
of NAIP and/or other antiapoptotic genes, such as Bcl-2. Disclosing molecular
mechanisms of the lack of clinical expression in homozygous carriers of exon
7 and 8 deletion of SMN1 [44, 45] might provide a clue for the future approach
to gene therapy of SMA.
Genetic Counselling and Prenatal Diagnosis
SMA conforms to the autosomal recessive pattern of inheritance, which

makes estimation of the genetic risk a simple matter. The genetic risk value
1 in 4 should be given, although in SMA3 it was found to be smaller, particu-
larly for females due to the incomplete penetrance of the mutated gene and
to the sex influence [41, 42].
The risk for a heterozygous healthy sibling of an affected homozygous
patient of having an affected child is 2/31/501/4>1/300, and the risk of
having such a child for a midly affected or unaffected (nonmanifesting) indi-
vidual with a homozygous mutation of the SMN1 gene is 1:100 (assuming a
frequency of heterozygotes in the general population of 1:50). Sometimes the
genetic risk is not high even for the couples who previously gave birth to an
affected child, since 2% of the cases are due to new mutations of the SMN1
gene in one of the parents [63]. Detection of such cases is more complicated
than simply finding a homozygous deletion, because it is based on linkage
analysis in unaffected siblings of the proband. Moreover, one should bear in
mind that even if we find in a healthy child the same haplotype as in an
affected one, there is still a possibility of germinal mosaicism in the mother.
The prenatal diagnosis is based on the test which identifies homozygous
deletion of exon 7 of SMN1 gene [see commentary, 64]. False-negative results
occur in ~5% of the cases. Most of them must be compound heterozygotes
(with deletion of SMN1 in one chromosome and with another mutation in
the other) and the remaining, very infrequent cases are homozygotes for small
mutations in the SMN1 gene [65].
The prenatal test should be offered to all couples having a genetic risk
of 1/4, but should also be available to those with an increased but much
lower risk up to 1/300. One important condition, however, is a previous
identification of a homozygous deletion in the affected individual from the
same family. This condition does not have to be treated as absolute, if there
are good grounds for assuming that the clinical diagnosis of SMA in the
proband whose DNA is not available was correct. Absence of homozygous
deletion in such cases reduces the risk of SMA from 25 to 2% [65]; the 2%
risk is due to the infrequent occurrence of small mutations and, possibly, a
wrong clinical diagnosis of SMA in some cases.
Detection of carriers and compound heterozygotes is feasible [30, 66] but
difficult. Therefore, the SMA Consortium at a meeting in 1997 acknowledged
the fact that prenatal diagnosis in the absence of homozygous deletion of
SMN1 in the proband is hazardous and advised to refrain from prenatal tests
in such cases [64].
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Irena Hausmanowa-Petrusewicz, Neuromuscular Unit, Polish Academy of Sciences,

Banacha 1a, bl.D, Room 718, PL02097 Warsaw (Poland)
E-Mail: neurmyol@cmdik.pan.pl
............................
Familial Amyotrophic Lateral Sclerosis:
A Review
Mitsuya Morita, Robert H. Brown, Jr.
Day Neuromuscular Research Center, Charlestown, Mass., USA
Amyotrophic lateral sclerosis (ALS), a degenerative disorder of cortical

and spinal motor neurons, causes a progressive paralysis that is usually fatal
because of respiratory weakness within 5 years of onset. The cause of the
progressive degeneration of motor neurons in this disease remains unknown
and these is no definitive treatment. The prevalence of ALS is about 5:100,000
and its incidence is approximately 1:100,000. In a subset of cases (approxi-
mately 10% of ALS cases), it is apparent that the disease is inherited, often
as a dominant trait [1]. Since the clinical and pathological characteristics of
familial ALS (FALS) and sporadic ALS (SALS) are almost identical, we and
others have presumed that understanding of the molecular defects that cause
FALS will provide insights into the pathogenesis of both FALS and SALS.
Although several loci have been linked to FALS, the only well-established
cause of FALS are defects in the gene encoding Cu/Zn-superoxide dismutase
(CuZn-SOD; SOD1). In this chapter, we summarize recent developments in
FALS research, consider how mutant SOD1 causes motor neuron cell death
and evaluate possible therapies for ALS.
Genetic Linkage Analysis in FALS
In 1991, we and collaborators [2] established that the gene defect in some
FALS pedigrees maps to chromosome 21q22.1-22.2. Because some families
were not linked to this locus, this investigation indicated that there is genetic
heterogeneity in FALS [2]. In 1993, Rosen et al. [3] in the same collaborative
group reported that some cases of FALS are associated with mutations in the
gene at this locus encoding SOD1. The first report described 11 different
missense mutations in the SOD1 gene in 13 FALS pedigrees. We now recognize
that approximately one quarter of FALS pedigrees arises from mutations in
the SOD1 gene [4, 5]. As of this writing, more than 70 mutations have been
reported among FALS and (to a considerably lesser extent) SALS cases [6];
these are detected exclusively in individuals with motor neuron disease. That
these mutations can cause ALS is strikingly underscored by the observation
that several lines of mice with transgenes for mutant SOD1 develop a progres-
sive, lethal paralysis that is clinically and pathologically highly homologous
to human ALS [710].
An unusual, juvenile-onset form of ALS was described in seventeen Tuni-
sian families by Ben Hamida et al. [11] in 1990. This was characterized by
recessive inheritance, early onset (typically at about 12 years), and very slow
disease progression. To date, the neuropathology of this ALS variant has not
been defined. Ben Hamida et al. recognized three forms of juvenile ALS. Type
1, the most common form, is characterized by distinctive lower motor neuron
involvement early in the disease, with denervational atrophy and weakness in
distal limb muscles and ultimately the tongue as well. This form was genetically
associated with a locus at 15q15-q22 [12]. Clinically, more prominent upper
and less evident lower motor neuron features mark type 2 juvenile ALS. A
locus for type 2 juvenile ALS has not been mapped. In type 3 juvenile ALS,
the distinctive feature is prominent spasticity of both face and limb muscles;
hand and peroneal atrophy are mild. Type 3 ALS has been mapped to 2q33-
q35 [13, 14] in a region spanning 1.7 cM between markers D2S116 and
D2S2237 [15]. A clinically distinct ALS-like disorder with juvenile onset (late
teen years) and very slow progression has been described in a large American
pedigree of English descent. Affected individuals develop distal limb denerva-
tion and wasting associated with corticospinal signs, but do not develop respir-
atory or bulbar paralysis. This disease is transmitted as an autosomal dominant
trait that has recently been linked to 9q34 [16, 17].
X-linked dominant ALS with clinical and neuropathological features in-
distinguishable from other types of ALS has been found in a single large
American pedigree. The causative gene defect is not known [18].
Pathophysiology of FALS Linked to the Mutant SOD1
SOD1 is an abundant, cytoplasmic enzyme consisting of 153 amino acids

encoded by five small exons. Each SOD1 molecule binds one atom of copper
and one of zinc. Functional SOD1 is homodimeric. The enzyme is extremely
stable, perhaps because the two SOD1 molecules are tightly packed together
via powerful hydrophobic interactions [19]. The biological importance of
Morita/Brown 178
SOD1 may be gauged by its wide expression pattern (it is expressed in all
eukaryotic cells), its abundance (about 0.52% of the soluble proteins in the
human brain [20, 21]) and its high degree of conservation during evolution.
Through cyclic reduction and oxidation of Cu, SOD1 detoxifies or dismutates
intracellular superoxide anion to form hydrogen peroxide (H2O2). H2O2 is
subsequently converted to H2O by catalase or glutathione peroxidase. Although
the mutant forms of SOD1 have been investigated in several laboratories, the
molecular basis for the neurotoxicity of mutant SOD1 protein is not well
understood. However, several lines of evidence suggest that the mutant SOD1
molecule has acquired adverse, cytotoxic properties. First, some of the SOD1
mutations (e.g. G37R, D90A) do not substantially reduce dismutation activity
[22, 23]. Second, mice that are totally devoid of SOD1 (SOD1 knockout mice)
do not show developmental defects and, in contrast with mice with mutant
SOD1 transgenes, do not subsequently develop motor neuron cell death [24].
Finally, for many of the mutant mouse strains, findings of motor neuron cell
death develop in the face of above-normal total dismutation activity [710].
In contrast, control mice expressing equivalent levels of wild-type SOD1 do
not develop such paralysis.
Despite the fact that the age of onset in FALS pedigrees with high pene-
trance may be earlier than in SALS [25, 26], FALS patients with SOD1 defects
are clinically indistinguishable from SALS or FALS patients without a known
SOD1 mutation [15]. In pedigrees with diminished disease penetrance, there
may be intra- or interfamilial phenotypic differences [27]. For some mutations,
the phenotype tends to be rather stereotypical. The A4V (alanine changed to
valine at position 4) mutation, detected in about half of all North American
families with SOD1 mutations [15, 28], triggers a disease with a survival time
of about 1218 months [15]. Also, on clinical and pathological study, A4V
patients reveal almost exclusively lower motor neuron involvement [29]. In
A4V cases, there is more widespread nonmotor pathology than SALS [29, 30].
In contrast with the A4V cases, others (including E21G [31], G37R [15, 28],
G41D [15], H46R [28, 32], D90A [33], G93C [15], L144S [34], L144F [28],
I151T [35]) may occasionally survive for 1015 years.
Particularly striking is the D90A mutation, most commonly encountered
in northern Scandinavia. In that setting, the resulting motor neuron disease
is inherited as a recessive trait; individuals heterozygous for D90A are asymp-
tomatic. Moreover, the disorder that results when D90A is homozygous is
highly atypical for ALS, with a very slow onset over several years characterized
by pain in the lower body and cramps in the muscles of the lower extremities
[23, 33]. Those patients typically survive 20 years or more. In contrast, indi-
viduals in Europe or the United States who are heterozygous for D90A survive
no longer than other ALS patients in those regions. These observations raise
Familial Amyotrophic Lateral Sclerosis 179

the exciting possibility that there is a genetic modifying factor within the
Scandinavian population that in some manner mitigates the neurotoxic influ-
ences of the mutant SOD1 allele.
Given the phenotypic diversity that can be encountered in families with
different SOD1 mutations, one might anticipate that subtle but important
differences in the biophysical properties of the different mutant proteins could
explain phenotypic differences. However, studies to date have failed to support
this possibility. Thus, Ratovitski et al. [36] examined the relationship between
several biochemical properties (e.g. dismutation activity, polypeptide half-time,
resistance to proteolytic degradation, solubility, etc.) of mutant SOD1 proteins
and disease phenotype; no specific biophysical parameter correlated with clin-
ical phenotype [36].
Neurotoxicity of Mutant SOD1 Protein
Several hypotheses have been proposed to explain the toxicity of mutant

SOD1 protein. Each proposed mechanism is based on the observation that
the mutant SOD1 protein can become unstable and misfold. Two hypotheses
are predicated on aberrant copper catalysis within the mutant protein. One
argument is that the SOD1 mutations render the copper more accessible to
peroxynitrite, allowing the formation of reactive nitronium-like intermediates
that can nitrate tyrosine residues [37]. Some studies documented the elevation
of nitrotyrosine in G37R transgenic mice [38] and SOD1-related FALS patients
[39]. Another catalytic mechanism suggests that the mutations increase the
peroxidase activity of SOD1, leading to the formation of more hydroxyl rad-
icals from hydrogen peroxide [40, 41].
A second, broad hypothesis to explain the injurious properties of the
mutant SOD1 molecule is that it is sufficiently unstable that, under some
circumstances, it misfolds and forms aggregates that are in some manner toxic.
Aggregated protein containing SOD1 has been detected in different transgenic
ALS mice; it is possible that the mutant protein coaggregates with unidentified,
essential component(s). These aggregates are often ubiquitinated.
In a recent report, Bruijn et al. [42] genetically manipulated the levels of
wild-type human SOD1 in ALS mice expressing the mutant SOD1 protein
(G85R). These investigators found that changing the level of wild-type SOD1
(none to 6 times of the normal level of SOD1) did not affect the pathology, onset,
or progression of disease and concluded that the aggregation of mutant SOD1
and its neurotoxicity are independent of the activity of the wild-type molecule.
This finding has not been repeated in other strains of mice, and thus the generality
of the conclusions from these experiments is not established as yet.
Morita/Brown 180
Autopsy studies have revealed aggregation and abnormal assembly of
neurofilaments in the perikarya and axons of motor neurons in both SALS
[4345] and FALS with posterior column and spinocerebellar tract involvement
[46]. Similar findings have been reproduced in transgenic mice with wild-type
mouse NF-L subunits [47], wild-type human NF-H subunits [48], or mutant
NF-L subunits [49]. Moreover, mutations in NF-H gene have been reported
among sporadic ALS patients [5052]. These findings raise the possibility that
abnormalities in neurofilament organization may be involved in the pathogen-
esis of ALS.
Marked neurofilamentous pathology is evident in FALS patients with the
A4V [53], I113T [54] and H48Q [55] mutations in SOD1 as well as in transgenic
mice expressing the G93A [56] protein. The evidence that disruption of NF-L
or overexpression of human NF-H in mutant SOD1 transgenic mice delay
onset of disease and/or extend life span suggests the possibility that neurofila-
ments modulate SOD1-mediated toxicity [57, 58]. However, it is uncertain
whether accumulation of neurofilaments itself directly causes motor neuron
cell death; in contrast with the above studies of mouse NH-L and human
NF-H, forced expression of mouse NF-H does not result in any overt pheno-
type or enhanced motor neurodegeneration, despite severe perikaryal accumu-
lation of neurofilaments and proximal axonal swellings in motor neuron [59].
Furthermore, recent reports that impairment of axonal transport was observed
at an early, asymptomatic stage before any pathological changes in SOD1
transgenic mice [6063] also suggest that accumulation of neurofilaments might
be caused as a second phenomenon.
Another important pathogenetic mechanism in SOD1-mediated ALS
implicates the excitatory neurotransmitter glutamate. The extracellular concen-
tration of glutamate in the central nervous system must be kept low to prevent
neuronal damage from excessive activation of glutamate receptors [64]. One
mechanism for this involves reuptake of glutamate by one or more astroglial
transporters. EAAT2/GLT-1 in astrocytes is believed to be the predominant
glutamate transporter involved in this regulation of extracellular glutamate
levels in the nervous system [65]. Diminished EAAT2 function has been re-
ported in ALS patients [66] and SOD1 transgenic mice [10]. An increase of
glutamate in extracellular space was also reported in some patients [67]. The
basis for this putative loss of glutamate transport activity is not clear. However,
in one detailed study, variant mRNA transcripts for the EAAT2 glutamate
transporter were detected in affected central nervous system tissue of 60%
of autopsied SALS patients [68]. In vitro analyses indicate that the variant
transcripts reduce expression of the EAAT2 protein, and thereby augment
glutamate activity at synapses. As yet, these findings have not been duplicated
in other studies [69, 70].

A recent report by Trotti et al. [71] demonstrated that FALS-linked SOD1
mutants can induce loss of function of EAAT2, particularly in the face of
superimposed oxidative stress (e.g. addition of iron). These investigators argued
that the mechanism for this inhibition of EAAT2 involves peroxidase activity
of the mutant SOD1, because the chelation of the copper ion prevented EAAT2
inhibition, and because a similar pattern of EAAT2 inactivation was obtained
by injecting Fe2+. Fe2+ catalyzes the production of highly reactive hydroxyl
radicals via the Fenton reaction. In both human and mouse ALS, riluzole,
which blocks glutamate release from presynaptic terminals, slows the disease
course, albeit moderately. This also supports the possibility that glutamate-
mediated toxicity contributes to the pathogenesis of ALS.
Other studies of ALS pathogenesis point to a role for mitochondrial
dysfunction in this disease. Thus, abnormal mitochondria are evident early in
the course of motor neuron degeneration in the G37R ALS mice [8]. Other
studies show that the G93A SOD1 mutation alters electron transport enzymes
in mitochondria and induces elevation of cytosolic calcium concentration
[72]. Moreover, both in humans and mice with ALS, calcium is observed to
accumulate in mitochondria within distal motor neuron terminals present in
motor point biopsies in muscle [73, 74]. On these grounds, it is conceivable that
mitochondrial dysfunction contributes to the pathogenesis of SOD1 transgenic
mice.
Two closely related concepts are that the mutant SOD1 protein alters the
oxidative milieu within the motor neuron cytoplasm, setting the stage for
oxidative injury to critical cellular constituents [7577], and thereby triggering
such adverse events as the aggregation of proteins and the activation of pro-
grammed cell death. Indeed, there are now several reports that mutant SOD1
protein can activate cell death genes [7880]. It is clear, for example, that
caspase-1 is activated both in the spinal cords of ALS mice and in serum-
deprived, oxidatively stressed neuroblastoma cells that express mutant SOD1
protein [30].
Therapeutic Trials in ALS
Transgenic mice with mutant SOD1 provide an excellent opportunity to

dissect the etiology of this disease and to explore new avenues for ALS therapy.
Although the exact mechanism how mutant SOD1 induces motor neuron cell
death is unclear, several of the foregoing hypotheses lead directly to drug trials
in the ALS mice. Thus, the hypothesis that oxidative metabolism is aberrant
led to a trial of vitamin E in the ALS mice and then in ALS patients. As
reported by Gurney et al. [81], dietary supplementation with vitamin E and
Morita/Brown 182
selenium causes a subtle delay in disease onset in the G93A mice but does
not prolong survival once the disease begins. More recently, a French trial of
vitamin E in ALS patients also showed a modest but significant benefit [82].
The hypothesis that glutamate toxicity is a factor in this disease also led
Gurney et al. to explore the effect of two putative modulators of the gluta-
matergic system, riluzole and gabapentin. Both showed a modest benefit [81].
Based on the hypothesis that the mutant SOD1 molecule is toxic because
of increased exposure to copper, the copper-chelating compound d-penicillam-
ine was administered orally to G93A SOD1 transgenic mice; a modest delay
in onset was seen [83]. The data that mitochondrial function, and hence ATP
generation, are abnormal in ALS prompted Beal and colleagues [84] to try
oral administration of creatine. At either 1 or 2% of the mouse diet, this
compound improves the survival of G93A mice and prevents oxidative damage.
Creatine is thought to help to buffer intracellular energy stores and inhibit
mitochondrial transition pore opening. Since mutant SOD1 has been thought
to be proapoptotic in motor neurons, two antiapoptotic genetic manipulations
were tried in mice. These entailed (1) breeding the G93A animals to mice that
overexpressed a dominant negative inhibitor of caspase-1 and (2) overexpres-
sing the antiapoptotic protein, bcl-2, in the G93A mice. Bcl-2 is a well-described
inhibitor of several cell death genes, including the interleukin-1b-converting
enzyme (ICE). Friedlander et al. [85] crossed transgenic mice expressing a
dominant negative inhibitor of ICE with a G93A mouse. The resulting F1
animals showed no difference in disease onset relative to the G93A mice
without the ICE inhibitor. However, once the disease started, the doubly
transgenic animals survive significantly longer from the onset of the disease
(27 days) than the mutant SOD1 mice (11.7 days). Kostic et al. [86] crossed
G93A mice with transgenic bcl-2 mice to determine whether the overexpression
of human bcl-2 protects these ALS mice. Disease onset was significantly later
(203 days) in the G93A/bcl-2 mice as compared with the G93A mice (170
days). On the other hand, the duration of the disease did not differ between
these two groups.
In this context, it is of considerable interest that some compounds that
have not worked in human trials in ALS appear to have failed in the ALS
mice as well. These prominently include the growth factors BDNF (brain-
derived neurotrophic factor), GDNF (glial-derived neurotrophic factor),
CNTF (ciliary neurotrophic factor) and IGF-1 (insulin-like growth factor-1).
As a final point, in considering the targets for therapy in ALS, it must be
recalled that the death process may involve cell types other than the motor
neuron itself. As the above discussion of EAAT2 indicates, one suspects that
dysfunction within astrocytes may be important in this process. Several other
lines of argument incriminate astrocytic and microglial dysfunction in ALS

[8789]. Ultimately, strategies for slowing or reversing this disease may target
both these nonneural cells and motor neurons.
An extrapolation from this consideration is the more general point that
in the long term, one also anticipates that effective therapy for ALS will require
multiple agents, perhaps administered at different times in the illness. That is,
if the evolution of the disease involves multiple stages (initiation, propagation,
spreading or dissemination) then different agents may act differentially on
these phases. Of course, this possibility that there will be polydrug therapy
for this illness has precedence in cancer chemotherapy.
Two final points should be noted regarding ALS therapeutics. First, for
those cases that arise from inherited, abnormal proteins that are cytotoxic,
the ultimate therapy will be to devise methods such as ribozyme or somatic
cell gene mutation therapies to inactivate the toxic allele. It is conceivable that
this might obviate multiple therapies that target events downstream from
synthesis of the offending protein. Second, there is a growing interest in the
use of neural stem cells in neurodegenerative diseases. These cells have a
remarkable capacity to migrate within the brain, and an extraordinary ability
to differentiate into diverse types of neural tissue cells. It is conceivable that
such cells might migrate to sites of dying motor neurons and provide trophic
support by becoming new populations of normal astrocytes, avidly taking up
glutamate or secreting neurotrophic substances. It is also possible that these
cells might even differentiate into motor neurons and thereby have at least the
potential of reinnervating denervated muscle. At all events, it is now clear that
a variety of potential therapeutic strategies exist to treat both inherited and
sporadic ALS. The existence of a mouse model for this disease will undoubtedly
assist greatly in testing these therapeutic options.
Acknowledgement
This work was supported by the National Institutes of Health, the ALS Association, the
Muscular Dystrophy Association, the Pierre L. de Bourgknecht ALS Research Association,
and Project ALS. Dr. Browns laboratory receives support from the C.B. Day Foundation.
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Robert H. Brown, Jr., MD, Day Neuromuscular Research Center,

MGH, East, Room 6627, Building 149, 13th Street, Navy Yard, Charlestown, MA 02129 (USA)
Tel. +1 617 726 5750, Fax +1 617 726 8543, E-Mail brown@helix.mgh.harvard.edu

............................
Author Index
Bonnemann, C.G. 26 Meola, G. 61

Bella, I.R. 147 Morita, M. 177
Brown, R.H. Jr 177 Moxley, R.T. 61
Buyse, G.M. 1
Nelis, E. 128
De Jonghe, P. 128
Deymeer, F. 113 Ohno, K. 96
zdemir, C. 113
O
Engel, A.G. 96
Rudel, R. 79
Hausmanowa-Petrusewicz, I.
163 Serdaroglu, P. 113
Hoffman, E.P. 1 Skuk, D. 12
Stans, A.A. 96
Jurkat-Rott, K. 79
Timmerman, V. 128
Lehmann-Horn, F. 79 Tremblay, J.P. 12
Lunt, P.W. 44
Zaremba, J. 163
190
............................
Subject Index
Acetylcholine receptor classification 147, 149

antibodies in myasthenia gravis clinical features 149
juvenile disease 118 electrodiagnostic studies 152, 159
late-onset disease 122, 123 infection role in onset 149, 150
mutations, see Congenital myasthenic pathogenesis 153, 154
syndromes Acute motor sensory axonal neuropathy
Acetylcholinesterase deficiency, see classification 147, 149
Congenital myasthenic syndromes clinical features 149
Acute inflammatory demyelinating electrodiagnostic studies 152, 159
polyradiculoneuropathy infection role in onset 149, 150
clinical features 147149 pathogenesis 153, 154
differential diagnosis 155, 156 Adeno-associated virus, gene therapy
epidemiology 147 vector 48
infection role in onset 149, 150 Adenovirus, gene therapy vector 46
laboratory findings Amyotrophic lateral sclerosis
antiglycoconjugate autoantibodies epidemiology 177
151, 152 hereditary form, see Familial amyotrophic
blood analysis 150, 151 lateral sclerosis
cerebrospinal fluid analysis 151 Anticipation
electrodiagnostic studies 152, 159 facioscapulohumeral muscular dystrophy
management 48
complications 155 myotonic dystrophy 64, 65
intravenous immunoglobulin 158 Azathioprine, myasthenia gravis
overview 155, 157 treatment 124
plasmapheresis 155, 157, 158
molecular mimicry 154 Basal lamina onion bulbs,
nomenclature 147, 149 Charcot-Marie-Tooth disease 135
outcomes 158, 159
pathogenesis 152154 Campylobacter jejuni, infection role in
pathology 154, 155 Guillain-Barre syndrome 149, 150, 154
Acute motor axonal neuropathy Carbamazepine, myotonic dystrophy
antiglycoconjugate autoantibodies 152 trials 74
191
Central core disease overview 96
clinical features 89, 90 postsynaptic syndromes
genetics 90 fast-channel syndrome
heredity 8 gating abnormality 105
Charcot-Marie-Tooth disease low-affinity fast-channel
classification 128131 syndrome 105
clinical phenotypes 129 mode-switching kinetics 105, 106
CMT1 overview 105
autosomal dominant mutations causing acetylcholine
CMT1A 132, 133 receptor deficiency with or without
CMT1B 133, 134 minor kinetic abnormalities
EGR2 mutations 134 106109
overview 131, 132 slow-channel syndrome
autosomal recessive gene mutations 102, 104
basal lamina onion bulbs 135 pathology 102
CMT4A 135 quinidine treatment 104
CMT4B 135 presynaptic syndromes
HMSN-L 136 episodic apnea 99, 100
overview 135 heredity 108
X-linked 134, 135 paucity of synaptic vesicles and reduced
CMT2 quantal release 99
autosomal dominant resembling Lambert-Eaton myasthenic
CMT2A 137 syndrome 100
CMT2B 137 synaptic acetylcholinesterase deficiency
CMT2D 137 pathology 100, 101
loci 136, 137 structure and mutations 101, 102, 108
autosomal recessive 137 Creatine, amyotrophic lateral sclerosis
overview 136 treatment 183
X-linked 137 Creatinine, muscle strength enhancement
gene mutations, overview 128, 130132 4
CLC-1 Cytomegalovirus, infection role in
channelopathy 90, 91, 93, 94 Guillain-Barre syndrome 150
structure and function 94
ColQ, structure and mutations 101, 102 Dehydroepiandrosterone sulfate, myotonic
Congenital hypomyelination dystrophy trials 73
clinical phenotype 129 Dejerine-Sottas syndrome
gene mutations 130 classification 136
Congenital myasthenic syndromes clinical phenotype 129
candidate genes 99 gene mutations 130, 136
classification 96, 97 Dihydropyridine receptor
diagnosis channelopathies 85, 86, 89
clinical history 97 functions 85
differential diagnosis 98 Disopyramide, myotonic dystrophy trials
electromyography 98 74
molecular analysis 99 Distal hereditary motor neuropathies
physical examination 97, 98 clinical presentation 137, 138
investigation 98, 99, 108 congenital nonprogressive disease 138
Subject Index 192

recessive disease 139 regulator role of repeats 57, 58
type II 138 repeat length effects 46, 52
type IV 138 penetrance and risk to offspring 55
Drug screening, animal models 3, 4 prenatal testing 56
Duchenne muscular dystrophy prognosis 53
animal models 13 prospects for research 58
myoblast transplantation, see Myoblast Familial amyotrophic lateral sclerosis
transplantation clinical features 177
Dystroglycan juvenile forms 178
dystrophin binding 27, 28 linkage analysis 177, 178
gene 27 SOD1 mutations
sarcoglycan complex 30 glutamate transporter effects
Dystrophin 181, 182
deficiency animal models 2, 3 mitochondrial dysfunction 182
expression in sarcoglycanopathy 33 neurotoxicity of mutant proteins
neuronal nitric oxide synthase localization 180182
role 2 phenotypes 179, 180
Dystrophin-associated proteins structure and function 178, 179
dystrophin complexes 26, 27 types 177, 178
sarcoglycan complex 29, 30 therapeutic trials
types 27, 28 combination therapy 184
copper chelators 183
EAAT2, mutant SOD1 effects 181, 182 creatine 183
EGR2, mutations 134 growth factors 183
prospects 184
Facioscapulohumeral muscular dystrophy vitamin E 182, 183
anticipation 48 X-linked dominant form 178
childhood testing 56 FK506, immunosuppression for myoblast
course 44, 47 transplantation 15, 18, 19
diagnosis
index case 4648 Gene therapy
linkage analysis 52, 53, 56 myoblast modification 15, 16
molecular testing sarcoglycanopathy 37
distribution of fragment size and vectors
type 49, 50 adeno-associated virus 48
false-positives 51 adenovirus 46
sensitivity and specificity 51, 52 Glutamine, muscle strength enhancement
family studies 54, 55 4
probes 48, 49, 51 Growth hormone, myotonic dystrophy
pulsed-field gel electrophoresis trials 72
4951 Guillain-Barre syndrome
epidemiology 44 axonal variants, see Acute motor axonal
family history 48 neuropathy, Acute motor sensory
gene mutation 44, 45 axonal neuropathy
management 53, 54 demyelinating variant, see Acute
molecular pathogenesis inflammatory demyelinating
position effects 45, 56, 57 polyradiculoneuropathy
Subject Index 193

Hereditary neuralgic amyotrophy Myasthenia gravis
clinical features 140, 141 age of onset 113
linkage studies 141 juvenile disease
Hereditary neuropathy with liability to anti-acetylcholine receptor antibodies
pressure palsies 118
clinical features 139, 140 autoimmune disease association 118
gene defects 140 definition 113
Hereditary peripheral neuropathies, see also demographic features 115, 116
Charcot-Marie-Tooth disease, Congenital onset symptoms
hypomyelination, Dejerine-Sottas ocular symptoms 116, 117
syndrome, Distal hereditary motor prepuberty 117, 120
neuropathies puberty and postpuberty
classification 128131 117, 118, 120
clinical phenotypes 129 racial differences 116, 120
gene mutations 128, 130, 131 severity and outcome
hereditary recurrent neuropathies prepuberty 119
139141 puberty and postpuberty 120
hereditary sensory neuropathies 139 study design 118, 119
Hyperkalemic periodic paralysis studies for analysis 114, 115
clinical features 80, 81 thymoma 118
electrophysiological basis 81, 82 late-onset disease
gene mutation 79 anti-acetylcholine receptor antibodies
pathogenesis of symptoms 84, 85 122, 123
Hypokalemic periodic paralysis autoimmune disease association 123
electrophysiology 86 azathioprine treatment 124
gene defect 86 definition 120, 121
pathogenesis 86, 87 demographic features 121
onset symptoms 121, 124
Influenza vaccine, role in Guillain-Barre severity and outcome 123, 124
syndrome 150 thymoma 123
Insulin, myotonic dystrophy trials 73 Myoblast transplantation
Insulin-like growth factor-1 animal studies
muscle strength enhancement 4 dog 18
myotonic dystrophy trials 73 monkey 19
Intravenous immunoglobulin, mouse 13, 19
Guillain-Barre syndrome management efficacy
158 congenital dystrophy 20
Duchenne muscular dystrophy
Limb-girdle muscular dystrophy, see benefits 19, 20
Sarcoglycanopathy early clinical trials 13, 14
metabolic myopathy 20
Malignant hyperthermia susceptibility genetically modified myoblasts 15, 16
gene mutations 88, 89 immune response 14, 15
management 87, 88 immunosuppression therapy 15
overview 87 migration of cells 17, 18
Mexiletine, myotonic dystrophy trials rationale 12, 13
74 survival factors 17
Subject Index 194

MyoD, deficiency animal models 2, 3 Pentoxifylline, muscle strength
Myotonia congenita enhancement 4
molecular pathology 91, 93, 94 Phenytoin, myotonic dystrophy trials 74
transfected cell studies of channel Plasmapheresis, Guillain-Barre syndrome
mutants 91, 93 management 155, 157, 158
types 90, 91 Potassium-aggravated myotonia
Myotonic dystrophy clinical features 80, 81
animal models 74, 75 electrophysiological basis 81, 82
anticipation 64, 65 gene mutation 79
cardiac manifestations 67 pathogenesis of symptoms 84, 85
clinical features 61, 62, 66, 68 Procainamide, myotonic dystrophy trials 74
diagnosis 6163
DMPK deficiency 61, 68 Quinidine, slow-channel congenital
genetic counseling 63 myasthenic syndrome treatment 104
management and trials
antimyotonia therapy 73, 74 Ryanodine receptor
dehydroepiandrosterone 73 channelopathies 85, 86, 89
growth hormone 72 functions 85
infancy and childhood 68, 69
insulin 73 Sarcoglycan
insulin-like growth factor-1 73 dystrophin complex 29, 30, 37
maternal-obstetric complications functions 30
68, 71 genes 29
skeletal muscle problems 68, 70 structures 29
surgical management of patients 68, 71 Sarcoglycanopathy
testosterone 68, 72 animal models 35, 36
natural history 68, 72 clinical features 31, 32
sex differences 65 diagnosis 32, 33
trinucleotide expansion limb-girdle muscular dystrophy
developmental changes 67, 68 classification 26, 27
enlargement 65 mutations
locus 61 a-sarcoglycan 33, 34
molecular testing 6163 b-sarcoglycan 34
mosaicism and clinical presentation c-sarcoglycan 34, 35
6668 d-sarcoglycan 35
protein expression patterns 33
Neuronal nitric oxide synthase, treatment 37
localization role of dystrophin 2 types and distribution 30, 31, 33
SCN4A, mutation 79
Oxatomide, muscle strength enhancement 4 SMN protein, function and mutations
169, 170
Paramyotonia congenita Spinal muscular atrophy, proximal disease
clinical features 80, 81 of childhood
electrophysiological basis 81, 82 differential diagnosis 171
gene mutation 79 epidemiology 165
pathogenesis of symptoms 84, 85 forms and clinical features 164
temperature sensitivity 85 gene mutations 168170
Subject Index 195

Spinal muscular atrophy (continued) neurotoxicity of mutant proteins
genetic counseling 172 180182
history of study 163 phenotypes 179, 180
laboratory findings structure and function 178, 179
electromyography 165 types 177, 178
histology 166, 167
muscle morphology 165167 Testosterone, myotonic dystrophy trials
linkage analysis 168 68, 72
pathophysiology 167, 168 Thymoma, myasthenia gravis association
prenatal diagnosis 172, 173 juvenile disease 118
sex differences 170 late-onset disease 123
SMN protein function 169, 170 Tocainide, myotonic dystrophy trials 74
treatment 171, 172
Superoxide dismutase, mutations in familial Utrophin, deficiency animal models 2, 3
amyotrophic lateral sclerosis
glutamate transporter effects 181, 182 Vitamin E, amyotrophic lateral sclerosis
mitochondrial dysfunction 182 treatment 182, 183
Subject Index 196

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Neuromuscular Diseases: From Basic Mechanisms to Clinical Management

Series Editor M. Fisher, Worcester, Mass.

Volume Editor F. Deymeer, Istanbul

16 figures and 21 tables, 2000

Library of Congress Cataloging-in-Publication Data

Neuromuscular diseases: From basic mechanisms to clinical management / volume editor,

Copyright 2000 by S. Karger AG, P.O. Box, CH4009 Basel (Switzerland)

1 Novel Approaches to Therapeutics of the Muscular Dystrophies

190 Author Index

This volume of Monographs in Clinical Neuroscience is devoted to neuro-

Recessively inherited muscular dystrophies are diseases caused by a lack

Animal Models as an Experimental Platform for

The identification of the dystrophin gene and protein as causative of

Drug Screening in Animal Models

Drug screening of animal models of muscular dystrophy has been pursued

Novel Approaches to Therapeutics of the Muscular Dystrophies 3

Gene Delivery to Dystrophic Muscle

All pharmacological approaches to treatment of muscular dystrophy hinge

Novel Approaches to Therapeutics of the Muscular Dystrophies 5

Novel Approaches to Therapeutics of the Muscular Dystrophies 7

Novel Approaches to Therapeutics of the Muscular Dystrophies 9

Dr. Eric P. Hoffman, Research Center for Genetic Medicine,

Novel Approaches to Therapeutics of the Muscular Dystrophies 11

The Rationale of Myoblast Transplantation

Genetic myopathies are characterized by different degrees of muscle weak-

As early as in 1978, Partridge et al. [5] suggested that the intramuscular

Early Clinical Trials

Inspired by these first experiments, several clinical MT trials were soon

The role of the immune system in MT was underestimated in some of

Following the demonstration of the immune response after MT, attention

Transplantation of Genetically Modified Autologous Myoblasts

Although used clinically for immunosuppression, the administration of

Most of the observations on myoblast survival after intramuscular injec-

Diffusion of Transplanted Myoblasts

MT is also confronted with the limited capacity of myoblasts to migrate

MT must prove to be effective under conditions approaching the clinical

Could MT Improve Patients?

The effectiveness of MT must not only be demonstrated by the presence of

Although most of the attention on MT was given to the treatment of

Dr. Jacques P. Tremblay, Unite Recherche en Genetique Humaine, Rc 9300,

Limb-Girdle Muscular Dystrophies

After years of inconsistent use, the diagnostic category of limb-girdle

The SG are part of the larger group of dystrophin-associated proteins

Designation Genetic Gene Protein Number

along with dystrophin in digitonin-solublized skeletal muscle membrane prep-

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 27

C-terminus at the cysteine-rich domain, spans the membrane and extracellu-

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 29

Sarcoglycanopathies: Clinical Features

Deficiency of SG proteins was demonstrated in dystrophin-positive

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 31

Diagnosis, Protein Expression Patterns and Mutational Spectrum

The diagnosis of a SGpathy rests on history and clinical examination,

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 33

The first known animal model of an SGpathy is the cardiomyopathic

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 35

There are a few observations in the literature suggesting that in the

Note Added in Proof

A muscle-specific protein, filamin 2, has recently been identified as an

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 37

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 39

Disorders of the Sarcoglycan Complex (Sarcoglycanopathies) 41