Professional Documents
Culture Documents
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Monographs in
Clinical Neuroscience
Vol. 18
Neuromuscular Diseases:
From Basic Mechanisms to
Clinical Management
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.
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VII Preface
Contents VI
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Preface
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
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.
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;
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
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vector that lacks all viral genes. Proc Natl Acad Sci USA 1997;94:16451650.
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1996;3:965972.
Cooper BJ, Winand NJ, Stedman H, Valentine BA, Hoffman EP, Kunkel LM, Scott MO, Fischbeck KH,
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locus is defective in X-linked muscular dystrophy of dogs. Nature 1988;334:154156.
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............................
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
First Experiments in MT
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
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
Use of Normal and Genetically Modified Myoblasts for the Treatment of Myopathies 15
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
Use of Normal and Genetically Modified Myoblasts for the Treatment of Myopathies 17
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
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.
Use of Normal and Genetically Modified Myoblasts for the Treatment of Myopathies 19
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
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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Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.
Monogr Clin Neurosci. Basel, Karger, 2000, vol 18, pp 2643
............................
Disorders of the Sarcoglycan Complex
(Sarcoglycanopathies)
Carsten G. Bonnemann
Department of Neuropediatrics, University Childrens Hospital, Gottingen, Germany
Dystrophin-Associated Proteins
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 ?
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
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
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
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
Bonnemann 36
Treatment
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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............................
Facioscapulohumeral
Muscular Dystrophy:
Diagnostic and Molecular Aspects
Peter W. Lunt
Clinical Genetics Unit, Bristol Royal Hospital for Sick Children, Bristol, UK
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,
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].
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
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.
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
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.
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.
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].
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
Future (Speculation)
References
Lunt 58
10 Tawil R, Forrester J, Griggs RC, Mendell J, Kissel J, NcDermott M, King W, Weiffenbach B,
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in facioscapulohumeral muscular dystrophy. Ann Neurol 1996;39:744748.
11 Funakoshi M, Goto K, Arahata K: Epilepsy and mental retardation in a subset of early onset
4q35-facioscapulohumeral muscular dystrophy. Neurology 1998;50:17911794.
12 Jardine PE, Upadhyaya M, Maynard J, Harper P, Lunt PW: A scapular onset muscular dystrophy
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muscul Disord l994;4:477482.
13 Zatz M, Marie SK, Cerqueira A, Vainzof M, Pavanello RCM, Passos-Bueno MR: The facioscapulo-
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14 Kohler J, Rupilius B, Otto M, Bathke K, Koch MC: Germline mosaicism in 4q35 facioscapulohu-
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485490.
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1994;2:225234.
16 Hewitt JE, Lyle R, Clark LN, Valleley EM, Wright TJ, Wijmenga C, van Deutekom JCT, Francis
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associated with facioscapulohumeral muscular dystrophy. Hum Mol Genet 1994;3:12871295.
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elements. Hum Mol Genet 1998;7:16811694.
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Clinical review of a new surgical method. J Shoulder Elbow Surg 1996;5:201205.
22 Kissel JT, McDermott MP, Natarajan R, Mendell JR, Pandya S, King WM, Griggs RC, Tawil R,
FSH- DY group: Pilot trial of albuterol in facioscapulohumeral muscular dystrophy. Neurology
1998;50:14021406.
23 Padberg GW, Lunt PW, Koch M, Fardeau M: Facioscapulohumeral muscular dystrophy; in Emery
AEH (ed): Diagnostic Criteria for Neuromuscular Disorders, ed 2. London, Royal Society of
Medicine Press, 1997, chap 3, pp 915.
24 Lunt PW, Compston DAS, Harper PS: Estimation of age-dependent penetrance in faciosca-
pulohumeral muscular dystrophy by minimising ascertainment bias. J Med Genet l989;26:755
760.
25 Awerbuch GI, Nigro MA, Wishnow R: Beevors sign and facioscapulohumeral dystrophy. Arch
Neurol 1990;47:12081209.
26 Yamamoto K, Ikeda S, Hanyu N, Takeda S, Yanagisawa N: A pedigree analysis with minimised
ascertainment bias shows anticipation in Met30-transthyretin related familial amyloid polyneurop-
athy J Med Genet 1998;35:2330.
27 Deidda G, Cacurri S, Piazzo N, Felicetti L: Direct detection of 4q35 rearrangements implicated in
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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(FSHD) aetiology and diagnosis. Hum Mol Genet 1998;7:12071214.
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
Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.
Monogr Clin Neurosci. Basel, Karger, 2000, vol 18, pp 6178
............................
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
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
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.
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.
Myotonic Dystrophy 65
[CTG]n Repeat Mosaicism in DM: Its Impact on Clinical Features and
Treatment
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
Myotonic Dystrophy 67
or cell death. These ideas and other theories about the genetic pathomechanism
in DM are under investigation.
Moxley/Meola 68
Table 3. Management of DM in infancy and childhood [adapted from 1]
Problem Management
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
Should physical activity be restricted Not feasible or justified; obtain yearly ECG to search
for signs of cardiac conduction disturbance
Myotonic Dystrophy 69
Table 4. Treatment of skeletal muscle problems in DM
Problem Management
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
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
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
Myotonic Dystrophy 71
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)
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.
Myotonic Dystrophy 73
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].
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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Myotonic Dystrophy 75
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............................
Muscle Ion Channel Diseases
Reinhardt Rudel, Karin Jurkat-Rott, Frank Lehmann-Horn
Department of Physiology, University of Ulm, Germany
Sodium Channelopathies
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
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.
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.
Rudel/Jurkat-Rott/Lehmann-Horn 82
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].
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).
Rudel/Jurkat-Rott/Lehmann-Horn 84
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
Rudel/Jurkat-Rott/Lehmann-Horn 86
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.
Rudel/Jurkat-Rott/Lehmann-Horn 88
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].
Chloride Channelopathies
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
Rudel/Jurkat-Rott/Lehmann-Horn 90
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
Rudel/Jurkat-Rott/Lehmann-Horn 92
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].
Acknowledgments
References
1 Lehmann-Horn F, Engel AG, Ricker K, Rudel R: The periodic paralyses and paramyotonia congen-
ita; in Engel AG, Franzini-Armstrong C (eds): Myology, ed 2. New York, McGraw-Hill, 1994,
vol 2, pp 13031334.
2 Lehmann-Horn F, Rudel R: Molecular pathophysiology of voltage-gated ion channels. Rev Physiol
Biochem Pharmacol 1996;128:195268.
3 Rudel R, Lehmann-Horn F, Ricker K: The nondystrophic myotonias; in Engel AG, Franzini-
Armstrong C (eds): Myology, ed 2. New York, McGraw-Hill, 1994, vol 2, pp 12911302.
4 Cannon SC: From mutation to myotonia in sodium channel disorders. Neuromuscul Disord 1997;
7:241249.
5 Mitrovic N, George AL Jr, Lerche H, Wagner S, Fahlke C, Lehmann-Horn F: Different effects on
gating of three myotonia-causing mutations in the inactivation gate of the human muscle sodium
channel. J Physiol (Lond) 1995;487:107114.
6 Lehmann-Horn F, Jurkat-Rott K: Voltage-gated ion channels and hereditary disease. Physiol Rev
1999;79:13171371.
7 Melzer W, Herrmann-Frank A, Luttgau HC: The role of Ca2+ ions in excitation-contraction coupling
of skeletal muscle fibres. Biochim Biophys Acta 1995;1241:59116.
8 Ptacek LJ: The familial periodic paralyses and nondystrophic myotonias. Am J Med 1998;105:
5870.
9 Tricarico D, Servidei S, Tonali P, Jurkat-Rott K, Camerino DC: Impairment of skeletal muscle
adenosine triphosphate-sensitive K+ channels in patients with hypokalemic periodic paralysis. J Clin
Invest 1999;103:675682.
Rudel/Jurkat-Rott/Lehmann-Horn 94
10 Loke J, MacLennan DH: Malignant hyperthermia and central core disease: Disorders of Ca2+
release channels. Am J Med 1998;104:470486.
11 Jurkat-Rott K, McCarthy TV, Lehmann-Horn F: Genetics and pathogenesis of malignant hyper-
thermia. Muscle Nerve 2000;23:417.
12 Hudson AJ, Ebers GC, Bulman DE: The skeletal muscle sodium and chloride channel diseases.
Brain 1995;118:547563.
13 Pusch M, Jentsch TJ: Molecular physiology of voltage-gated chloride channels. Physiol Rev 1994;
74:813827.
14 Wagner S, Deymeer F, Kurz LL, Benz S, Schleithoff L, Lehmann-Horn F, Serdaroglu P, O zdemir
C, Rudel R: The dominant chloride channel mutant G200R causing fluctuating myotonia: Clinical
findings, electrophysiology, and channel pathology. Muscle Nerve 1998;21:11221128.
15 West JW, Patton DE, Scheuer T, Wang Y, Goldin AL, Catterall WA: A cluster of hydrophobic
amino acid residues required for fast Na+-channel inactivation. Proc Natl Acad Sci USA 1992;89:
1091010914.
16 Mitrovic N, Lerche H, Heine R, Fleischhauer R, Pika-Hartlaub U, Hartlaub U, George AL Jr,
Lehmann-Horn F: Role in fast inactivation of conserved amino acids in the IV/S4-S5 loop of the
human muscle Na+ channel. Neurosci Lett 1996;214:912.
............................
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
Classification of CMS
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
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
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
Postsynaptic Syndromes
Engel/Ohno/Stans 102
a
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
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.
Engel/Ohno/Stans 104
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
Engel/Ohno/Stans 106
Table 3. Twenty-nine low-expressor or null mutations in the AChR e
subunit
Conclusion
Engel/Ohno/Stans 108
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.
References
1 Engel AG: Myasthenic Syndromes; in Engel AG, Franzini-Armstrong C (eds): Myology: Basic and
Clinical, ed 2. New York, McGraw-Hill, 1994, pp 17981835.
2 Engel AG, Ohno K, Sine SM: Congenital myasthenic syndromes; in Engel AG (ed): Myasthenia
gravis and Myasthenic Disorders. New York, Oxford University Press, 1999, pp 251297.
3 Rothbart HB: Myasthenia gravis. Familial occurrence. JAMA 1937;108:715717.
4 Engel AG, Lambert EH, Gomez MR: A new myasthenic syndrome with end-plate acetylcholinester-
ase deficiency, small nerve terminals, and reduced acetylcholine release. Ann Neurol 1977;1:315
330.
5 Engel AG, Lambert EH, Mulder DM, Torres CF, Sahashi K, Bertorini TE, Whitaker JN: A newly
recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine-
induced ion channel. Ann Neurol 1982;11:553569.
6 Mora M, Lambert EH, Engel AG: Synaptic vesicle abnormality in familial infantile myasthenia.
Neurology 1987;37:206214.
7 Walls TJ, Engel AG, Nagel AS, Harper CM, Trastek VF: Congenital myasthenic syndrome associat-
ed with paucity of synaptic vesicles and reduced quantal release. Ann NY Acad Sci 1993;681:461
468.
8 Engel AG, Hutchinson DO, Nakano S, Murphy L, Griggs RC, Gu Y, Hall ZW, Lindstrom J:
Myasthenic syndromes attributed to mutations affecting the epsilon subunit of the acetylcholine
receptor. Ann NY Acad Sci 1993;681:496508.
9 Vincent A, Newsom-Davis J, Wray D, Shillito P, Harrison J, Betty M, Beeson D, Mills K, Palace
J, Molenaar P, Murray N: Clinical and experimental observations in patients with congenital
myasthenic syndromes. Ann NY Acad Sci 1993;681:451460.
10 Milone M, Hutchinson DO, Engel AG: Patch-clamp analysis of the properties of acetylcholine
receptor channels at the normal human endplate. Muscle Nerve 1994;17:13641369.
Engel/Ohno/Stans 110
35 Donger C, Krejci E, Serradell P, Eymard B, Bon S, Nicole S, Chateau D, Gary F, Fardeau M,
Massoulie J, Guicheney P: Mutation in the human acetylcholinesterase-associated gene, COLQ, is
responsible for congenital myasthenic syndrome with end-plate acetylcholinesterase deficiency. Am
J Hum Genet 1998;63:967975.
36 Ohno K, Brengman JM, Milone M, Shen X-M, Tsujino A, Anlar B, Engel AG: Congenital endplate
acetylcholinesterase deficiency: Novel missense and null mutations in the collagen-like tail subunit
of the asymmetric enzyme (abstract). Am J Hum Genet 1998;63:A377.
37 Ohno K, Brengman JM, Nakano S, Walsh P, Heidenreich FR, Vincent A, Nagwane S, Engel AG:
Congenital endplate acetylcholinesterase deficiency caused by nonsense, splice-site, and missense
mutations in the collagenic tail subunit of asymmetric AChE (abstract). Neurology 1999;52(suppl 2):
A184.
38 Ohno K, Engel AG, Brengman JM, Harper CM, Shen X-M, Heidenreich FR, Vincent A, Milone
M, Tan E, Demirci M, Walsh P, Nakano S, Akiguchi I: The spectrum of mutations causing endplate
acetylcholinesterase deficiency. Ann Neurol 2000;47:162170.
39 Ohno K, Brengman JM, Felice KJ, Cornblath DR, Engel AG: Congenital endplate acetylcholinester-
ase deficiency caused by a nonsense mutation and an A-to-G splice donor site mutation at position
+3 of the collagen-like tail subunit gene (COLQ): How does G at position +3 result in aberrant
splicing? Am J Hum Genet 1999;65:635644.
40 Ohno K, Hutchinson DO, Milone M, Brengman JM, Bouzat C, Sine SM, Engel AG: Congenital
myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a muta-
tion in the M2 domain of the e subunit. Proc Natl Acad Sci USA 1995;92:758762.
41 Sine SM, Ohno K, Bouzat C, Auerbach A, Milone M, Pruitt JN, Engel AG: Mutation of the
acetylcholine receptor a subunit causes a slow-channel myasthenic syndrome by enhancing agonist
binding affinity. Neuron 1995;15:229239.
42 Engel AG, Ohno K, Milone M, Wang H-L, Nakano S, Bouzat C, Pruitt JN, Hutchinson DO,
Brengman JM, Bren N, Sieb JP, Sine SM: New mutations in acetylcholine receptor subunit genes
reveal heterogeneity in the slow-channel congenital myasthenic syndrome. Hum Mol Genet 1996;
5:12171227.
43 Ohno K, Hutchinson DO, Milone M, Nakano S, Sieb JP, Brengman JM, Engel AG: Molecular
genetic basis of a slow channel syndrome (abstract). Muscle Nerve 1995;18:463.
44 Wang H-L, Auerbach A, Bren N, Ohno K, Engel AG, Sine SM: Mutation in the M1 domain of
the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation. J Gen Physiol
1997;109:757766.
45 Milone M, Wang H-L, Ohno K, Fukudome T, Pruitt JN, Bren N, Sine SM, Engel AG: Slow-channel
syndrome caused by enhanced activation, desensitization, and agonist binding affinity due to mutation
in the M2 domain of the acetylcholine receptor alpha subunit. J Neurosci 1997;17:56515665.
46 Gomez CM, Maselli R, Gammack J, Lasalde J, Tamamizu S, Cornblath DR, Lehar M, McNamee
M, Kuncl R: A beta-subunit mutation in the acetylcholine receptor gate causes severe slow-channel
syndrome. Ann Neurol 1996;39:712723.
47 Croxen R, Newland C, Beeson D, Oosterhuis H, Chauplanaz G, Vincent A, Newsom-Davis J:
Mutations in different functional domains of the human muscle acetylcholine receptor a subunit
in patients with the slow-channel congenital myasthenic syndrome. Hum Mol Genet 1997;6:767773.
48 Ohno K, Milone M, Brengman JM, Lo Monaco M, Evoli A, Tonali P, Engel AG: Slow-channel
congenital myasthenic syndrome caused by a novel mutation in the acetylcholine receptor e subunit
(abstract). Neurology 1998;50:A432.
49 Sieb JP, Milone M, Engel AG: Effects of the quinoline derivatives quinine, quinidine, and chloroquine
on neuromuscular transmission. Brain Res 1996;712:179189.
50 Fukudome T, Ohno K, Brengman JM, Engel AG: Quinidine normalizes the open duration of slow-
channel mutants of the acetylcholine receptor. Neuroreport 1998;9:19071911.
51 Harper CM, Engel AG: Quinidine sulfate therapy for the slow-channel congenital myasthenic
syndrome. Ann Neurol 1998;43:480484.
52 Ohno K, Wang H-L, Milone M, Bren N, Brengman JM, Nakano S, Quiram P, Pruitt JN, Sine
SM, Engel AG: Congenital myasthenic syndrome caused by decreased agonist binding affinity due
to a mutation in the acetylcholine receptor e subunit. Neuron 1996;17:157170.
Engel/Ohno/Stans 112
Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.
Monogr Clin Neurosci. Basel, Karger, 2000, vol 18, pp 113127
............................
Juvenile and Late-Onset
Myasthenia gravis
zdemir
Feza Deymeer, Piraye Serdaroglu, Coskun O
Department of Neurology, University of Istanbul, Istanbul, Turkey
Juvenile MG
zdemir
Deymeer/Serdaroglu/O 114
Table 1 (continued)
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.
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,
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
Deymeer/Serdaroglu/O 116
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
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
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).
zdemir
Deymeer/Serdaroglu/O 118
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].
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
zdemir
Deymeer/Serdaroglu/O 120
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.
Authors Number of Age FMR Classification Positive anti- TXb Good outcome
patientsa years AChR Ab % (remission plus
% improvement)
zdemir
Deymeer/Serdaroglu/O 122
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).
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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Deymeer/Serdaroglu/O 124
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............................
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
Classification of HMSN
De Jonghe/Timmerman/Nelis 130
Table 1 (continued)
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-
De Jonghe/Timmerman/Nelis 132
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
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
De Jonghe/Timmerman/Nelis 134
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].
CMT2
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
De Jonghe/Timmerman/Nelis 136
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.
The distal HMN mainly present with a classical CMT phenotype. The
main discriminative clinical factor between CMT1/CMT2 and distal HMN is
De Jonghe/Timmerman/Nelis 138
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.
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].
De Jonghe/Timmerman/Nelis 140
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
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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............................
Acute Inflammatory Demyelinating
Polyradiculoneuropathy
Isabelita R. Bella
Department of Neurology, UMass Memorial Health Care, Worcester, Mass., USA
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
Laboratory Features
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
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
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
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
Bella 154
clinical, electrodiagnostic, and CSF abnormalities of AIDP reliably support
the diagnosis.
Differential Diagnosis
Bella 156
Table 2. Management of GBS [adapted from 65, with permission]
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
Outcome
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
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Bella 162
Deymeer F (ed): Neuromuscular Diseases: From Basic Mechanisms to Clinical Management.
Monogr Clin Neurosci. Basel, Karger, 2000, vol 18, pp 163176
............................
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
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.
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
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.
Hausmanowa-Petrusewicz/Zaremba 166
Fig. 3. SMA3: musculus quadriceps femoris. Muscle fibers of normal diameter among
the hypertrophic and atrophic fibers. HE.390.
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
Hausmanowa-Petrusewicz/Zaremba 168
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].
Hausmanowa-Petrusewicz/Zaremba 170
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
Hausmanowa-Petrusewicz/Zaremba 172
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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............................
Familial Amyotrophic Lateral Sclerosis:
A Review
Mitsuya Morita, Robert H. Brown, Jr.
Day Neuromuscular Research Center, Charlestown, Mass., USA
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].
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
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].
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
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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190
............................
Subject Index
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