Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Humana Press Methods and Protocols Muscular Dystrophy Edited by Katherine M. D. Bushby Louise V. B. Anderson Methods and Protocols Muscular Dystrophy Edited by Katherine M. D. Bushby Louise V. B. Anderson Molecular Methodologies in MDs 3 1 3 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 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 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 Recessive/ Other dominant Protein names Location of protein r Dystrophin Plasma membrane rDystrophin 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 rDysferlin Plasma membrane rDysferlin 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? dMyotonin protein kinase DMPK Various, according to antibody d? d Poly(A) binding protein-2 PABP2 Nucleus r Plectin 1 PLEC1 Basement membranes Table 1 (continued) Muscular Dystrophy Genes and Proteins 6 Bushby and Anderson 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 (see Chap. 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 (see Chap. 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 Fig. 1. Schematic diagram of the proteins that are located at or near the muscle plasma membrane, and are implicated in muscle disease (see Table 1). Molecular Methodologies in MDs 7 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 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) 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 Nerve 21, 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. Cell 81, 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 9 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 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 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. [...]... other hand, in order to compare values obtained in different laboratories, through different methods, results of SCK are classified as 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 31 32 Zatz, Vainzof, and Passos-Bueno normal (when in the normal range for age and ethnic group), and, when... R., Akiyama, J., and Marie, S K N (1996) Autosomal recessive limb-girdle muscular dystrophies, in Handbook of Muscle Disease (Lane, R J M.) Marcel Dekker, New York, pp 245–255 10 Banker, B Q (1986) Congenital muscular dystrophy, in Myology (Engel, A G and Banker, B Q.) McGraw-Hill, New York, pp 1367–1382 11 Lane, R J M., Shelbourne, P., and Johnson, K J (1996) Myotonic dystrophy, in Handbook of Muscle... suitable for HC and immunolabeling studies for periods of more than 20 yr Maintaining an archive of frozen material can prevent the need for rebiopsy, and, as new probes become available, retrospective diagnostic surveys are facilitated 2 Histological and HC Methods In most neuromuscular disease laboratories, a routine histopathological screening protocol of 10–15 histological and HC methods is in general... common in facioscapulohumeral muscular dystrophy (FSHD) and autosomal dominant (AD) limb-girdle muscular dystrophy (LGMD) (see histopathological profiles, Subheadings 3.4 and 3.5.) 2 Moth-eaten fibers show a disordered, patchy distribution of mitochondria, seen in many chronic LGMDs and adult Becker muscular dystrophy (BMD) 3 Central core disease, multicore (minicore) myopathy, and myotubular (centronuclear)... which the news is broken will be remembered forever, and the content and atmosphere of the consultation will, to a great extent, determine their subsequent attitude to the disease and to medical care The news must be broken with sensitivity and without haste, by a physician who knows the patient personally and has a full understanding of the disease and its many implications Enough information must be... immunoblotting or for DNA analysis However, many histopathology laboratories, particularly those in long-established institutions, may have access 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 15 16 Johnson to archives of formalin-fixed, paraffin-embedded material, which is proving to be a valuable... 19.0 ± 8.0 19.0 ± 6.5 19.0 ± 7.5 38 Zatz, Vainzof, and Passos-Bueno Fig 2 Mean CK Distribution according to age in DMD vs BMD 3.2.3 Limb-Girdle Muscular Dystrophies The limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited neuromuscular disorders characterized by proximal muscular weakness of the pelvic and shoulder girdles, and by a variable progression, with symptoms ranging... Borgers, M., Firth, J A., Stoward, P J., and Verheyen, A (1991) Phosphatases, in Histochemistry, Theoretical and Applied, vol 2 (Stoward, P J and Pearse, A G E.) 4th ed Churchill-Livingstone, Edinburgh, pp 187–218 5 Stoward, P J., Meijer, A E F H., Seidler, E., and Wohlrab, F (1991) Dehydrogenases, in Histochemistry, Theoretical and Applied, vol 3 (Stoward, P J and Pearse, A G E.) 4th ed Churchill-Livingstone,... 27–71 6 Bancroft, J D and Stevens, A (1990) Theory and Practice of Histological Techniques, 3rd ed Churchill-Livingstone, Edinburgh 7 Schmalbruch, H (1992) The muscular dystrophies, in Skeletal Muscle Pathology, 2nd ed (Mastaglia, F L and Lord Walton of Detchant Churchill-Livingstone, Edinburgh, pp 283–318 8 Emery, A E H and Dreifuss, F E (1966) Unusual type of benign X-linked muscular dystrophy J Neurol... Nervenheilkunde 1, 13 2 Bell, J (1943) On pseudohypertrophic and allied types of progressive muscular dystrophy, in Treasury of Human Inheritance, vol 4, Cambridge University Press, London, pp 3 Walton, J N and Nattrass, F J (1954) On the classification, natural history and treatment of the myopathies Brain 77, 169 4 Walton, J., Karpati, G., and Hilton-Jones, D (eds.) (1994) Disorders of Voluntary Muscle . Press Methods and Protocols Muscular Dystrophy Edited by Katherine M. D. Bushby Louise V. B. Anderson Methods and Protocols Muscular Dystrophy Edited by Katherine M. D. Bushby Louise V. B. Anderson Molecular. Methodologies in MDs 3 1 3 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,. vols. 1 and 2 Churchill, London. Histopathological Diagnosis of MDs 15 3 15 From: Methods in Molecular Medicine, vol. 43: Muscular Dystrophy: Methods and Protocols Edited by: K. M. D. Bushby and