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Molecular Methodologies in MDs 3
1
Application of Molecular
Methodologies in Muscular Dystrophies
Katharine M. D. Bushby and Louise V. B. Anderson
The term “muscular dystrophy” (MD) describes a group of primary genetic
disorders of muscle that often have a distinctive and recognizable clinical phe-
notype, accompanied by characteristic, but frequently not pathognemonic,
pathological features. Research into the molecular basis of the MDs by a com-
bination of positional cloning and candidate gene analysis has provided the
basis for a reclassification of these disorders, with genetic and protein data
augmenting traditional clinically based nomenclature (Table 1). These find-
ings have brought insights into the molecular pathogenesis of MD, with an
increasing number of potential pathways involved in arriving at a dystrophic
phenotype. Some common themes can be recognized, however, including the
involvement of five members of the dystrophin-associated complex (dystrophin
and four sarcoglycans) in different types of MD (Fig. 1: 1), and the involve-
ment of two nuclear envelope proteins in producing an Emery-Dreifuss MD
phenotype (2). Other disease-associated genes appear to cause MD in a com-
pletely unrelated way, such as the involvement of calpain 3 in a form of limb-
girdle muscular dystrophy (3).
Section 1 of this book reviews traditional strategies used to identify MDs.
Meantime, techniques developed as a result of the research strategies described
above have become an integral part of the management of many patients with
MD and their families, and these techniques are addressed in Sections 2 (DNA-
based tests) and 3 (protein-based analyses). The continued effort to translate
this enhanced understanding into a molecular cure or treatment for MD is
reviewed in Subheading 4.
This book concentrates on those methods most likely to be relevant to clini-
cal practice: thus, although linkage analysis has often been a crucial step in
From: Methods in Molecular Medicine, vol. 43: Muscular Dystrophy: Methods and Protocols
Edited by: K. M. D. Bushby and L. V. B. Anderson © Humana Press Inc., Totowa, NJ
3
4 Bushby and Anderson
Table 1
Muscular Dystrophy Genes and Proteins
Gene
Muscular dystrophy (MD) Abbreviation Alternative names location
Duchenne MD DMD Dystrophinopathy Xp21
Becker MD BMD Dystrophinopathy Xp21
Emery-Dreifuss MD EDMD Xq28
Dominant Emery-Dreifuss MD AD-EDMD 1q11
Limb-girdle MD type 1A LGMD1A 5q
Limb-girdle MD type 1B LGMD1B 1q11
Limb-girdle MD type 1C LGMD1C 3p25
Limb-hirdle MD type 1D LGMD1D 6q22
Limb-girdle MD type 1E LGMD1E 7q
Limb-girdle MD type 2A LGMD2A Calpainopathy 15q15
Limb-girdle MD type 2B LGMD2B Dysferlinopathy 2p13
Miyoshi myopathy MM Dysferlinopathy 2p13
Miyoshi-type MD MMD 10
α-Sarcoglycanopathy SGCA LGMD2D, SCARMD2 17q21
β-Sarcoglycanopathy SGCB LGMD2E 4q12
γ-Sarcoglycanopathy SGCC LGMD2C, SCARMD1 13q12
δ-Sarcoglycanopathy SGCD LGMD2F 5q33
Limb-girdle MD type 2G LGMD2G 17q11
Limb-girdle MD type 2H LGMD2H 9q31
Limb-girdle MD type 2I LGMD2I 19q13-3
Merosin-negative congenital MD 6q22
Congenital MD with rigid spine 1p35
Fukuyama congenital MD FCMD 9q31
Congenital Myopathy (or ?MD) 12q13
Facioscapulohumeral MD FSHD 4q35
Myotonic dystrophy DM 19q13
Myotonic dystrophy type 2 DM2 3q
Oculopharyngeal MD OPMD 14q11
Epidermolysis bullosa simplex MD-EBS 8q24
+ MD
Additional loci for many of these phenotypes (e.g., autosomal recessive and dominant LGMD,
merosin-positive CMD, FSHD) probably exist, as inferred from the exclusion of all the known
loci in additional large families.
Molecular Methodologies in MDs 5
Table 1 (continued)
Muscular Dystrophy Genes and Proteins
Recessive/ Other
dominant Protein names Location of protein
r Dystrophin Plasma membrane
r Dystrophin
r Emerin Nuclear membrane
d Lamin A/C Nuclear membrane
d Myotilin Sarcomeric
d Lamin A/C Nuclear membrane
d Caveolin 3 Plasma membrane
d ? Homolog of band 4.1 ? Plasma membrane
d ?
r Calpain 3 ? Cytoplasm/nucleus
r Dysferlin Plasma membrane
r Dysferlin
r ?
r α-Sarcoglycan adhalin, A2 Plasma membrane
50DAG,
r β-Sarcoglycan A3b Plasma membrane
r γ-Sarcoglycan 35DAG, A4 Plasma membrane
r δ-Sarcoglycan Plasma membrane
r Telethonin Sarcomeric
r ?
r ?
r Laminin α2-chain Merosin Basal lamina/
extracellular matrix
r ?
r Fukutin Secreted extracellularly ?
r Integrin α7-chain Plasma membrane
d ?
d Myotonin protein kinase DMPK Various, according to
antibody
d ?
d Poly(A) binding protein-2 PABP2 Nucleus
r Plectin 1 PLEC1 Basement membranes
6 Bushby and Anderson
Fig. 1. Schematic diagram of the proteins that are located at or near the muscle
plasma membrane, and are implicated in muscle disease (seeTable 1).
moving toward a molecular definition of these disorders, in the clinical context
it is generally of limited value in only a small number of defined situations.
Even when a disease gene has been identified, the types of mutation(s) encoun-
tered in a specific disease influence the practical availability of genetic testing
(Table 2). For example, in myotonic dystrophy a single type of mutation (trip-
let-repeat expansion) is present in most affected individuals. Most patients with
facioscapulohumeral MD can be shown to carry a DNA deletion, variable in
size, but detectable using a single DNA probe (seeChap. 18). In both of these
disorders, therefore, a relatively straightforward analysis can be employed to
identify the majority of affected individuals, although in neither case is the
molecular pathology of the disease understood. In the dystrophinopathies,
the predominance of a particular type of mutation (intragenic deletion) simpli-
fies the analysis in the majority of families (seeChap. 6). The continued need
to apply a range of different tests, to provide as complete an answer as possible
to the questions of carrier testing and prenatal diagnosis in these conditions
means that, although such testing is widely available, it may remain complex
(Chaps. 19 to 23). In other disorders, e.g., the limb-girdle muscular dystrophies
(4), a range of point mutations have been described. This emphasizes the need
to integrate DNA and protein-based diagnosis in these disorders whenever pos-
sible; techniques for finding unknown point mutations in multiexon genes will
undoubtedly continue to improve, but the current situation is that protein analy-
sis, in the first instance, usually on muscle biopsy samples, is much more prac-
tical for most individuals in the clinical setting (5; Section 2). Mutation
Molecular Methodologies in MDs 7
Table 2
Types of Mutation Found in the Muscular Dystrophies:
Whole gene/adjacent genes deleted Rare cause of DMD
Single/multiple exons deleted Common in DMD: rare in other MDs
Single/multiple exons duplicated Seen in DMD
Single/few base pairs removed Common in LGMDs, EDMD, CMD,
rare in DMD
Single/few base pairs inserted Common in LGMDs, EDMD, CMD,
rare in DMD
Base substitution Common in LGMDs, EDMD, CMD,
(variable in type and position) rare in DMD
Expanded triplet motif DM (all cases)
Contracted repetitive elements FSHD (but gene responsible not known)
detection directed by the results of such protein analysis is a much more viable
option, in order to define the mutation in an individual case, to confirm the
diagnosis and offer genetic counseling or prenatal diagnosis. If treatments
based on the modification of specific gene defects ever come into clinical prac-
tice, then the requirement to define the mutation in every case will become
absolute.
However, there should be a degree of caution applied to the use of the ever-
increasing number of molecular tests available. The advisedness of proceeding
with testing is a concern faced regularly by those involved in counseling
patients and families affected with genetic diseases. Specific discussions in
this area relate to the issue of carrier testing in childhood or to the application
of presymptomatic testing for late-onset disorders, especially given the contin-
ued imprecision of such techniques in predicting the timing of onset or severity
of the disorder in question. Just because a test may be technically possible, it is
not necessarily appropriate to everyone’s situation, needs, or wishes, and cer-
tainly should not be applied without informed counseling and consent. Genetic
issues may be uniquely sensitive, and concerns about confidentiality and auto-
nomy of decision-making need to be taken seriously. The rush to employ these
new methodologies, with their emphasis so often on prevention, should not be
used to appear to devalue the people affected with these conditions or to dis-
tract the clinician, or indeed the broader society, from the need to address other
aspects of their rights.
Accepting these caveats, and the need for these tests to be approached with
counseling and sensitivity, the authors have drawn together in this book meth-
ods that may be of use in each specific disease. Faced with a diagnosis of a
genetic disease, a number of questions are commonly encountered, relating to
8 Bushby and Anderson
the exact nature of the disorder, its cause and likely progression, the risk of it
happening again either to the individual or to the wider family, and the pros-
pects for prevention and cure. The methods described here are those most com-
monly applied to try to answer these questions in families with MD.
References
1. Ozawa E., Noguchi, S., Mizuno, Y., Hagiwara, Y., and Yoshida, M. (1998) From
dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dys-
trophy.Muscle Nerve21, 421–438.
2. Bonne, G., di Barletta, M. R., Varnous, S., Becane, H., Hammouda, E., Merlini,
L., et al. (1999) Mutations in the gene encoding lamin A/C cause autosomal domi-
nant Emery-Dreifuss muscular dystrophy. Nature Genet. 21,285–288.
3. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Bourg,
N., et al. (1995) Mutations in the proteolytic enzyme calpain 3 cause limb-girdle
muscular dystrophy type 2A. Cell81, 27–40.
4. Bushby, K. (1999) Making sense of the limb-girdle muscular dystrophies. Brain
122, 1403–1420.
5. Anderson, L. V. B. and Davison, K. (1999) Multiplex western blotting for the
analysis of muscular dystrophy proteins. Am. J. Pathol.154, 1017–1022.
Clinical Exam in Diagnosis 9
2
Clinical Examination as a Tool
for Diagnosis
Historical Perspective
David Gardner-Medwin
The purpose of diagnosis, since the days of classical Greece, when the con-
cept was introduced, has been to provide a basis for prognosis and for the pre-
scription of a regimen of management. Prognosis, i.e., explaining to the patient
and family what the future holds, remains the central purpose of medicine,
encapsulated in the private consultation. All other matters, including such im-
portant things as investigation as an aid to diagnosis and treatment as a compo-
nent of management, are peripheral to this.
It is only in the past 50 yr that serious attention has been given to genetic
prognosis, and for only half that period have most clinicians devoted the scien-
tific care and compassion to this aspect of prognosis that justify the use of the
term “genetic counseling.” Indeed, as far as the muscular dystrophies (MDs)
are concerned, it is the need for accurate genetic counseling that has been the
spur to defining the diagnostic categories with precision.
Only occasional case reports of what is now called MD can be identified in
the literature before the 1850s. Then Meryon in England, Aran and Duchenne
in France, and Wachsmuth and Griesinger in Germany began to recognize the
progressive degenerative diseases of muscle, distinguished neuropathic from
myopathic muscle disease and developed systems of clinical, pathological,
and electrical examination of muscles, which enabled them to begin to classify
and name disorders. The term “progressive muscular dystrophy” was intro-
duced by Erb in 1891 (1) for progressive myopathic degenerations. Although
these early authors clearly recognized the hereditary nature of many of their
cases, the concept of genetically determined disease did not become central to
From: Methods in Molecular Medicine, vol. 43: Muscular Dystrophy: Methods and Protocols
Edited by: K. M. D. Bushby and L. V. B. Anderson © Humana Press Inc., Totowa, NJ
9
10 Gardner-Medwin
the understanding of these disorders until the rediscovery of Mendel’s work
and the defining of the Mendelian modes of inheritance, in the first decade of
this century; the idea that sporadic cases were somehow different in nature
lingered on in the minds of some doctors until the 1960s. By about 1910, most
of the commoner types of MD had been approximately identified, and then, for
more than 30 yr, no further significant progress was made. Bell (1943) (2)
attempted a genetic classification, revealing an unsatisfactory correlation
between modes of inheritance and the then-recognized clinical types. Walton
and Nattrass (1954) (3) began the modern process of attempting to combine
clinical and genetic criteria in their classification. The introduction of the esti-
mation of blood aldolase and creatine kinase (CK) activity, in the 1960s, and,
shortly afterwards, the introduction of histochemical examination of muscle
biopsies (which brought to light several previously unidentifiable congenital
myopathies), provided two valuable new tools for differential diagnosis. Fur-
thermore, the use of CK estimation in carriers of the Duchenne gene intro-
duced a useful (though fallible) technique for the scientific study of the carrier
state and its genetic implications.
By the early 1980s, before the molecular genetics revolution, matters stood
thus: The MDs formed a discrete group of muscle disorders, readily distinguish-
able on clinical, histological, enzyme assay, and electromyographic grounds
from the neuropathic disorders, the histochemically identifiable congenital
myopathies, the endocrine myopathies, polymyositis, and various rarer myo-
pathies. Within the group of true MDs, the clinical diagnosis was based on
three criteria: the precise distribution of affected muscles, weak and wasted or,
in some cases, hypertrophic; the related criteria of the age at the onset of symp-
toms and the rate of progression of the symptoms; and the mode of inheritance,
when this was evident from the family history. Sporadic cases were naturally
relatively difficult to identify. Muscle histology and serum CK activity were
valuable adjuncts, electromyography was less so.
All these criteria, by then refined by much study and scientific discourse,
resulted in a classification that included the X-linked Duchenne (DMD), Becker
and Emery-Dreifuss muscular dystrophy (EDMD) types, the autosomal domi-
nant facioscapulohumeral (FSHD), scapulohumeral, distal and oculopharyn-
geal types, as well as the multisystem disorder, myotonic dystrophy. It was
among the autosomal recessive (AR) types, so often presenting as sporadic
cases, that the most difficulty was encountered; the provisional categories of
these were the limb-girdle muscular dystrophy (LGMD) types, presenting in
childhood or adult life, and the congenital muscular dystrophy (CMD) types,
together with some rarer forms, distal and Fukuyama CMDs.
The names of many of these types clearly indicate the primary diagnostic
importance of detailed recording of the distribution of affected muscles; the
Clinical Exam in Diagnosis 11
distinction between DMD and BMD, and between the CMD and LGMD types,
depended on the timing and progression of symptoms. The problem with the
AR types was that there seemed to be no consistent or clearly definable pat-
terns of selective muscle involvement by which these might be positively iden-
tified or subdivided, nor was laboratory investigation sufficiently helpful to
solve the problem. Indeed, it was recognized that at least one autosomal domi-
nant form of LGMD existed, although its identification in the sporadic case
was never secure. Distinction of LGMD from BMD or from the Duchenne
carrier state, in which proximal muscular weakness sometimes occurs, was a
particular problem, and serious errors in the consequent genetic advice were
not uncommon.
In recent years, many new categories of AR MD have been identified by
their genetic linkage to particular loci, and, more recently still, by their specific
molecular pathology. In many cases, this has made it possible to recognize for
the first time characteristic patterns of selective muscle involvement, not pre-
viously discernible among the muddled group of LGMDs. Whether every
LGMD type will ultimately prove to be clinically recognizable, it is too early
to say, but, at present, this looks unlikely. Because most of the MDs have char-
acteristic patterns of selective muscle involvement, it should not be assumed to
be axiomatic that all of them must.
This is not the place to describe in detail the precise patterns of muscle
involvement that characterize the various classical types of MD. These may be
found in clinical texts such as those by Walton, Karpati, and Hilton-Jones
(1994) (4). A resumé would be valueless and potentially dangerous, because
the essence of clinical diagnosis of these disorders lies in the details. For inspi-
ration in the techniques involved the works of Duchenne (1870) (5) and Gowers
(1881)(6) can be recommended.
The advent of molecular genetics has transformed the diagnosis and classi-
fication of the MDs as later chapters in this book show. The chief consequences
can be listed as follows:
1. Molecular diagnosis has confirmed the validity of many previously identified
entities, for example, DMD, BMD, EDMD, FSHD and myotonic dystrophies.
2. It has provided a firm basis for identifying carriers of the X-linked types, and for
achieving preclinical and prenatal diagnosis.
3. It has resulted in the recognition of many new types of MD previously consigned
to wrong categories or to the unsatisfactory limb-girdle group.
4. It has revealed the molecular basis or cause of many of these disorders for the
first time, providing a means not only of identifying but of defining the different
types of MD. Incidentally, this also provides a logical direction for the search for
a cure.
