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Recent Progress in the Molecular Genetics of the Muscular Dystrophies
Neuromusc. Disord.. Vol. 2, No. 4, pp. 245-248. 1992 Pnnted in Grea~ Britain
0960-8966/92 $5.00 ÷ 0.00 ~A992 Pergamon Press Ltd
WORKSHOP REPORT RECENT PROGRESS IN THE MOLECULAR GENETICS OF THE M U S C U L A R DYSTROPHIES A report of a conference held at the Australian Neuromuscular Institute (ANRI), Perth, on 14 and 15 May 1992 on the occasion of the 25th Anniversary of the Muscular Dystrophy Research Association of Western Australia (MDRA).
The molecular theme selected to commemorate the Silver Jubilee of the MDRA was considered to be appropriate for the occasion since it is in the use of recombinant DNA technology of the muscular dystrophies, that much recent progress has occurred. The spectacular discovery of the gene for Duchenne (DMD) and Becker (BMD) muscular dystrophyand its product (dystrophin) by Dr Lou Kunkel and his group in Boston in 1988 has led to many advances both fundamental and applied. Firstly, there is the search for a curative treatment for DMD using gene complementation (myoblast transfer) techniques and more recently direct gene therapy. These topics were the subject of two International Workshops held at the ANRI and published by Raven Press in 1990 [1] and 1992 [2]. The second avenue of progress has been in the use of the DNA methods in the clinical context. One particular outcome has been the unexpected revelation that a great variation exists in the phenotypic manifestations of abnormalities of the dystrophin gene, a group of myopathies now termed "the human dystrophinopathies'. The term "dystrophinopathy" was originally coined to describe the animal equivalents of DMD. These are the (mdx) mouse, the dog (the golden retriever and rottweiler) and the cat. However since the variable phenotypic expression with many "'atypical BMDs'" becoming evident, the term "human dystrophinopathy" is readily applied to the group. It is also to be noted that the term "DMD gene'" has been used incorrectly in the past and is now designated by the better title, the "dystrophin gene". The Conference was opened by Dr Judyth Watson, MLA, Minister in the Western Australian (WA) government for Aboriginal, M ulticultural Affairs and Seniors. In addition to her complimentary remarks she presented the Greg Coldham 245
Memorial Award for outstanding achievement to the Chief of the Neurogenetics Laboratory at the ANRI, Nigel Laing Ph.D. The award recognizes Dr Laing's contributions to the local community and more widely his publications since 1989. Dr Laing was introduced to DNA technology in the highly acclaimed laboratory of Dr Allen D. Roses at Duke University. Dr Laing and his group provide carrier and antenatal diagnosis for the dystrophinopathies in WA with consequent benefits for the community [3, 4]. His original work is also outstanding as reflected by his recent publications [5, 6]. Among his and his collaborators' discoveries is the assignment of nemaline myopathy to a locus on Chr 1 [7]. The search for the gene product is continuing. The Workshop sessions were divided into sections on dystrophin, muscle development, the human dystrophinopathies, myotonic dystrophy and other topics in myology. Overseas participants were Professor Victor Dubowitz of the Hammersmith Hospital, London, U.K., Dr Allen D. Roses of Duke University Medical Centre, Durham, NC, U.S.A., Dr Lefkos T. Middleton of the Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, Dr Olav Sola of the University of Washington, Seattle, U.S.A. and Professor John B. Harris, Director of the Muscular Dystrophy Group Research Laboratories, Newcastle Hospital, U.K. Highlights of the workshop are summarized below. The section on dystrophin began with a paper by Professor Harris who said that although it has become clear that dystrophin is an integral part of the cytoskeleton, its detailed function remains unknown. The pattern of expression of dystrophin is also controversial, but data from Newcastle derived from immunogold labelling suggest that dystrophin is distributed in a regular square lattice with an interparticle distance of 120 nm in each plane.
246
Workshop Report
He noted that studies of many patients with He also described a young female with slightly "Xp21.2 disease" supported the "in frame" and elevated serum creatine kinase who had central "out of frame" deletion hypothesis of Monaco. cores in her muscle fibres and mild abnormalities Professor Harris' data demonstrated a strong in dystrophin. The clinical picture was that of a correlation between the quantity of dystrophin congenital myopathy but the changes in dysexpressed and the loss of mobility in dystro- trophin were considered secondary. He phinopathy patients. Together with others, mentioned that this resembled data his group Professor Harris believes that the C-terminal of have published recently on patients with polydystrophin is crucial. Among other comments he myositis who often show abnormalities in stated that there were at least six other dystro- dystrophin expression. phin-like proteins. One of these utrophin is Professor Dubowitz said that it is possible to located on Chr 6 and has been well categorized. distinguish a female DMD carrier from an He mentioned that detailed analysis of autosomal muscular dystrophy by the presence dystrophin in unusual situations may reveal of dystrophin negative fibres. However, the much useful information concerning the func- proportion of such fibres does not correlate with tion of the protein, e.g. such as in brothers who the degree of weakness. It may however relate to have similar DNA deletions but one of whom age, the number of dystrophin negative fibres manifests as BMD and the other appears entirely decreasing with advancing years. normal. Patients with "out of frame" deletions The section on muscle development comwho produce little or no dystrophin and lack the menced with a paper by Miranda Grounds Ph.D. C-terminal, express a severe DMD phenotype. In and her group of the University of WA, on the about 40% of DMD patients there are a few effect of fibroblast growth factor (FGF) on fibres which express dystrophin. These are myogenesis. This was analysed in three animal sometimes referred to as "'revertant fibres". They dystrophinopathies, being the mdx mouse, dog usually occur in small numbers and less than 1% and human. She found a strikingly lower amount of fl FGF in the adult human and dog compared of the total. He also said that it seems that patients who with the mouse. Foetal muscle had stronger have lost the rod area of the dystrophin molecule labelling than adult. Regenerating muscles in the are able to splice the N- and C-terminal provided mdx and normal "crush injured" mouse showed no more than 50% of the dystrophin is lost. The much stronger fl FGF labelling than the human molecule seems to be sufficientlyelastic to stretch or dog. She suggested that this finding correlated from N to C, thus functioning to some extent and with the greater ability for regeneration and the producing the benign BMD phenotype. This symptom-free status of the mdx mouse. In the same session, S. Bao Ph.D. of the observation has therapeutic implications since minigenes with sufficient elasticity to stretch from University of Sydney spoke of his work which N to C may prove to be a method of treating the showed that MHCI expression was increased eight fold in human embryo myoblasts when disease. Professor Victor Dubowitz drew attention to cultured with interferon-?'. On 19 week embryos exceptions of the "'out of frame" rule. He IFN-y stimulated a 22-fold increase in MHCI described five patients who have deletions of antigen expression. J. Bower Ph.D. of Monash University, Melexons 3-7 which would be expected to cause a shift in the reading frame. However, they un- bourne reported the effects of several cytokines expectedly show mild or intermediate phenotype. on murine muscle precursor cells including TGFHe pointed out that about 6% of the dystro- ~, interleukin (IL-6) and leukaemia inhibitory phinopathy patients do not conform to factor (LIF). All were shown to be potent growth factors for muscle. Monaco's "in frame/out of frame" rule. Concerning the great clinical variability in The correlation between the amount of dystrophin and severity of disease (Hoffman's phenotype for the dystrophinopathies, Dr Byron hypothesisl also had exceptions. Immunoblots Kakulas of the ANRI described the preliminary show that in BMD patients there are variable results of investigations using DNA deletion amounts and variable sizes of the dystrophin analysis and immunoblots on patients with molecule. Professor Dubowitz said that patients spinal muscular atrophy (SMA), limb girdle with deletions of exons 45-47 were usually "fin muscular dystrophy (LGMD), autosomal recesframe" and showed the expected BMD pheno- sive muscular dystrophy (ARMD) and atypical facioscapulohumeral muscular dystrophy (FSH). type.
Workshop Report
Two "SMA" patients were shown to have deletions of the dystrophin gene and abnormalities ofdystrophin on Western blots. One patient considered clinically to have LGMD was also shown to have a deletion of the dystrophin gene [8]. A girl in a family thought to have ARMD was also shown to have dystrophin abnormalities by Western blotting. Of six patients with a provisional diagnosis of atypical FSH, three were shown to have reduced quantities of dystrophin by Western blotting. This experience confirms the findings of others, particularly the group of Dr Sugita of Tokyo who has shown that the dystrophin gene and/or immunoblot abnormalities may be present in the Fukuyama type of congenital muscular dystrophy [9], and patients with quadriceps myopathy [10] or LGMD [11]. There are several other reports of patients, previously believed to have LGMD or SMA, being truly dystrophinopathies by DNA deletion analysis [12], some of whom were manifesting female carriers [11]. It is of interest that even patients with myalgia and cramps with no weakness may also have deletions of the dystrophin gene [13]. Others have massive cardiomegaly [14]. Even more surprising is a patient with a deletion of exons 31-44 has been shown to be asymptomatic [15]. Sporadic manifesting female carriers are easily misdiagnosed as having limb girdle muscular dystrophy or autosomal recessive muscular dystrophy [16]. Even these preliminary observations are sufficient to conclude that the current classification for the muscular dystrophies which is based on clinical criteria is inadequate. It may thus be foreshadowed that a classification will emerge based upon molecular criteria in the future. Concerning his experience of DNA mutations in DMD, Dr Lefkos Middleton reported 5% duplications, 65% deletions and 30% point mutations in his dystrophinopathy population. Steve Wilton Ph.D. of the ANRI described the methods involved in the examination of the dystrophin gene transcript. Total RNA extracted from BMD/DMD patients using the reverse transcriptase-polymerase chain reaction (RTPCR) is used to demonstrate the effect of gene deletion on the mature gene transcript. He believes that this approach will allow accurate determination of a woman's carrier state. Mutations within the gene affecting mRNA processing would be detected as abnormal sized PCR products. In one such study, combined with restriction fragment length polymorphisms
247
(RFLP) and single strand conformational po!ymorphisms (SSCP), analysis did not reveal an~¢ further base changes in dystrophin mRNA within the region ofexons 43-51 although several alternatively spliced gene transcripts were shown to be intact. SSCP provides information on the shape of the molecule when it is frozen, according to its amino acid sequences. RT-PCR can detect dystrophin gene rearrangements and denaturing gradient gel electrophoresis (DDGE) is used to identify alternative splicing. Nigel Laing Ph.D. of the ANRI reviewed the experience of his Neurogenetic Laboratory. Among his observations was that oogenesis seemed to be more mutagenic than spermatogenesis. There are 160 families in Western Australia with dystrophinopathy. Dr Allen Roses reported the preliminary results of his observations in dystrophia myotonica (DM) following the discovery of the CTG expansion of the gene on Chr 19 [17]. Recombinant events in DM families defined a 200 kb region that contains the DM gene. Among other observations, Dr Roses showed preliminary data on the variable expansion of the gene in different parts of the body. This may explain the differences in the severity of the lesions in the different tissues, an exciting observation which remains to be confirmed. This may also explain the greater involvement of skeletal muscle and lesser involvement of other tissues in DM. It is now known that the gene codes for a enzyme named myotonin protein kinase. Dr Roses suggested that this enzyme may be involved in the potassium channel disorder of the muscle membrane in DM. Concerning DM, Nigel Laing Ph.D. also reported his preliminary observations using the cDNA25 probe kindly provided by Dr Keith Johnson of London. DNA was analysed from nine DM patients without prior knowledge of the clinical severity in each. When this was revealed a striking correlation was found between the degree of expansion of the gene and the clinical severity of the disease. It is thus noteworthy that with the event of these advances it may now be possible to diagnose DM at any age and may predict its severity in any individual. This development is of great clinical significance and is especially useful for antenatal diagnosis. Angus Stewart Ph.D of Edith Cowan University in Perth, described his preliminary results of calcium activated force generation in skinned fast-twitched muscle fibres from C57 mdx mice. He found that differences may exist in strontium
248
Workshop Report
inactivation in the dystrophic vs normal. Control data produce two clear curves corresponding to fast and slow, using this technique, whereas mdx fibres produce three curves corresponding to fast and slow fibres and intermediate fibres. Sue Fletcher Ph.D. of the ANRI has shown that the trophic factors MyoD and myogenin are expressed during necrosis and regeneration of muscle in old mdx mice. She believes that this increase in the expression of MyoD/myogenin indicates that regeneration and, by inference, necrosis, of the skeletal muscles of adult, mdx mice is continuous and not transient or nonprogressive as suggested in some reports. Dr Olav Sola of the University of Washington, Seattle, U.S.A. reviewed his pioneering work on the use of the latissimus dorsi muscle for myocardioplasty operations. Anatomically he has found that three separate segments of the human latissimus dorsi exist. He has defined the histochemical fibre type distribution in each, the highest total number of slow fibres being in the superior segment and in decreasing numbers in the oblique and lateral segments. In each segment the largest number of slow fibres is found in the central or perivascular regions. In conclusion, the human dystrophinopathies may be classified as: (1) DMD; (2) BMD, this group includes the atypical patients previously diagnosed as Kugelberg-Welander SMA and limb girdle MD patients; and (3) the clinically atypical dystrophinopathies. The atypical group includes patients with quadriceps myopathy, those with cardiomyopathy, the "'creatine kinase aemias'" and patients with just myalgia and cramps. As a preliminary observation there are also some patients with atypical FSH which show "'facioscapulohumeral distribution of weakness" who have been found to show abnormalities ofdystrophin by Western blotting.
BYRON A. KAKULAS Medical Director Australian Neuromuscular Research Institute Professor of Neuropathology University of Western Australia and Head of Neuropathology Royal Perth Hospital Perth. Western Australia
REFERENCES
1.
Kakulas B A, Mastaglia F L (Eds). Pathogenesis and
2.
New York: Raven Press, 1990. Kakulas B A, Howell J McC, Roses A D (Eds).
Therapy o f Duchenne and Becket Muscular Dystrophy.
Duchenne Muscular Dystrophy: Animal Models and Genetic Manipulation. New York: Raven Press,
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
1992. Laing N G, Mears M E, Chandler D C, et al. Two years experience in a comprehensive carrier screening and prenatal diagnostic laboratory of the diagnosis of Duchenne and Becker muscular dystrophies. Med J Aust 1991; 154: 14-18. Walker A P, Laing N G, Yamada T, et aL A Taql map of the dystrophin gene useful for deletion and carrier status analysis. J Med Genet 1992: 29: 14-19. Laing N G, Layton M G, Johnsen R D, et aL Two distinct mutations in a dystrophin gene: chance occurrence or pre-mutation? Am J Med Genet 1992; 42: 688~92. Laing N G, Akkari P A, Chandler D C, et al. Duchenne muscular dystrophy (DMD) gene cDNA8 Pstl and Taql polymorphisms involve exon 51 of the Hindlll map. Nucl Acids Res 1990; 18: 4284. Laing N G, Majda B T, Akkari P A, et al. Assignment of a gene (NEM1) for autosomal dominant nemaline myopathy to chromosome 1. A m J H u m Genet 1992: 50: 576-583. Laing N G, Mears M E, Thomas H E, et al. Differentiation of Becker muscular dystrophy from limb girdle muscular dystrophy and KugelbergWelander disease using a eDNA probe. Med J Aust 1990; 152: 270-271. Arikawa E, ishihara T, Nonaka I. Sugita H, Arahata K. Immunocytochemical analysis of dystrophin in congenital muscular dystrophy. J Neurol Sei 1991; 105: 79-87. Sunohara N, Arahata K, Hoffman E P, et al. Quadriceps myopathy: form fruste of Becker muscular dystrophy. Ann Neurol 1990: 28: 634-639. Arikawa E, Hoffman E P, Kaido M, et al. The frequency of patients with dystrophin abnormalities in a limbgirdle patient population. Neurology 1991: 41: 14911496. Lunt P W, Cumming W J K. DNA probes in differential diagnosis of Becker muscular dystrophy and spinal muscular atrophy. Lancet 1989: 7: 46~,7. Gospe S M, Lazaro R P. Lava N S, et al. Familial Xlinked myalgia and cramps: a nonprogressive myopathy associated with a deletion in the dystrophin gene. Ann Neurol 1989; 26(3}: 466. Gold R, Kress W, Meurers B, et al. Brief communication: Becket muscular dystrophy of unusual disease courses by combined approach to dystrophin analysis. Museh" Nerve 1992: 15: 214-218. Koh J, Bartlett R J, Pericak-Vance M A, et al. Inherited deletion at Duchenne dystrophy locus in normal male. Lancet 1987;2:1154 1155. Norman A M, Hughes H E, Gardner-Medwin D, Nicholson L V B. Dystrophin analysis in the diagnosis of muscular dystrophy. Arch Dis ChiM 1989; 64:15011503. Harley H G, Rundle S A, Reardon W, et al. Unstable DNA sequence in myotonic dystrophy. Lancet 1992: 339:1125 II28.
0960-8966/92 $5.00 ÷ 0.00 ~A992 Pergamon Press Ltd
WORKSHOP REPORT RECENT PROGRESS IN THE MOLECULAR GENETICS OF THE M U S C U L A R DYSTROPHIES A report of a conference held at the Australian Neuromuscular Institute (ANRI), Perth, on 14 and 15 May 1992 on the occasion of the 25th Anniversary of the Muscular Dystrophy Research Association of Western Australia (MDRA).
The molecular theme selected to commemorate the Silver Jubilee of the MDRA was considered to be appropriate for the occasion since it is in the use of recombinant DNA technology of the muscular dystrophies, that much recent progress has occurred. The spectacular discovery of the gene for Duchenne (DMD) and Becker (BMD) muscular dystrophyand its product (dystrophin) by Dr Lou Kunkel and his group in Boston in 1988 has led to many advances both fundamental and applied. Firstly, there is the search for a curative treatment for DMD using gene complementation (myoblast transfer) techniques and more recently direct gene therapy. These topics were the subject of two International Workshops held at the ANRI and published by Raven Press in 1990 [1] and 1992 [2]. The second avenue of progress has been in the use of the DNA methods in the clinical context. One particular outcome has been the unexpected revelation that a great variation exists in the phenotypic manifestations of abnormalities of the dystrophin gene, a group of myopathies now termed "the human dystrophinopathies'. The term "dystrophinopathy" was originally coined to describe the animal equivalents of DMD. These are the (mdx) mouse, the dog (the golden retriever and rottweiler) and the cat. However since the variable phenotypic expression with many "'atypical BMDs'" becoming evident, the term "human dystrophinopathy" is readily applied to the group. It is also to be noted that the term "DMD gene'" has been used incorrectly in the past and is now designated by the better title, the "dystrophin gene". The Conference was opened by Dr Judyth Watson, MLA, Minister in the Western Australian (WA) government for Aboriginal, M ulticultural Affairs and Seniors. In addition to her complimentary remarks she presented the Greg Coldham 245
Memorial Award for outstanding achievement to the Chief of the Neurogenetics Laboratory at the ANRI, Nigel Laing Ph.D. The award recognizes Dr Laing's contributions to the local community and more widely his publications since 1989. Dr Laing was introduced to DNA technology in the highly acclaimed laboratory of Dr Allen D. Roses at Duke University. Dr Laing and his group provide carrier and antenatal diagnosis for the dystrophinopathies in WA with consequent benefits for the community [3, 4]. His original work is also outstanding as reflected by his recent publications [5, 6]. Among his and his collaborators' discoveries is the assignment of nemaline myopathy to a locus on Chr 1 [7]. The search for the gene product is continuing. The Workshop sessions were divided into sections on dystrophin, muscle development, the human dystrophinopathies, myotonic dystrophy and other topics in myology. Overseas participants were Professor Victor Dubowitz of the Hammersmith Hospital, London, U.K., Dr Allen D. Roses of Duke University Medical Centre, Durham, NC, U.S.A., Dr Lefkos T. Middleton of the Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, Dr Olav Sola of the University of Washington, Seattle, U.S.A. and Professor John B. Harris, Director of the Muscular Dystrophy Group Research Laboratories, Newcastle Hospital, U.K. Highlights of the workshop are summarized below. The section on dystrophin began with a paper by Professor Harris who said that although it has become clear that dystrophin is an integral part of the cytoskeleton, its detailed function remains unknown. The pattern of expression of dystrophin is also controversial, but data from Newcastle derived from immunogold labelling suggest that dystrophin is distributed in a regular square lattice with an interparticle distance of 120 nm in each plane.
246
Workshop Report
He noted that studies of many patients with He also described a young female with slightly "Xp21.2 disease" supported the "in frame" and elevated serum creatine kinase who had central "out of frame" deletion hypothesis of Monaco. cores in her muscle fibres and mild abnormalities Professor Harris' data demonstrated a strong in dystrophin. The clinical picture was that of a correlation between the quantity of dystrophin congenital myopathy but the changes in dysexpressed and the loss of mobility in dystro- trophin were considered secondary. He phinopathy patients. Together with others, mentioned that this resembled data his group Professor Harris believes that the C-terminal of have published recently on patients with polydystrophin is crucial. Among other comments he myositis who often show abnormalities in stated that there were at least six other dystro- dystrophin expression. phin-like proteins. One of these utrophin is Professor Dubowitz said that it is possible to located on Chr 6 and has been well categorized. distinguish a female DMD carrier from an He mentioned that detailed analysis of autosomal muscular dystrophy by the presence dystrophin in unusual situations may reveal of dystrophin negative fibres. However, the much useful information concerning the func- proportion of such fibres does not correlate with tion of the protein, e.g. such as in brothers who the degree of weakness. It may however relate to have similar DNA deletions but one of whom age, the number of dystrophin negative fibres manifests as BMD and the other appears entirely decreasing with advancing years. normal. Patients with "out of frame" deletions The section on muscle development comwho produce little or no dystrophin and lack the menced with a paper by Miranda Grounds Ph.D. C-terminal, express a severe DMD phenotype. In and her group of the University of WA, on the about 40% of DMD patients there are a few effect of fibroblast growth factor (FGF) on fibres which express dystrophin. These are myogenesis. This was analysed in three animal sometimes referred to as "'revertant fibres". They dystrophinopathies, being the mdx mouse, dog usually occur in small numbers and less than 1% and human. She found a strikingly lower amount of fl FGF in the adult human and dog compared of the total. He also said that it seems that patients who with the mouse. Foetal muscle had stronger have lost the rod area of the dystrophin molecule labelling than adult. Regenerating muscles in the are able to splice the N- and C-terminal provided mdx and normal "crush injured" mouse showed no more than 50% of the dystrophin is lost. The much stronger fl FGF labelling than the human molecule seems to be sufficientlyelastic to stretch or dog. She suggested that this finding correlated from N to C, thus functioning to some extent and with the greater ability for regeneration and the producing the benign BMD phenotype. This symptom-free status of the mdx mouse. In the same session, S. Bao Ph.D. of the observation has therapeutic implications since minigenes with sufficient elasticity to stretch from University of Sydney spoke of his work which N to C may prove to be a method of treating the showed that MHCI expression was increased eight fold in human embryo myoblasts when disease. Professor Victor Dubowitz drew attention to cultured with interferon-?'. On 19 week embryos exceptions of the "'out of frame" rule. He IFN-y stimulated a 22-fold increase in MHCI described five patients who have deletions of antigen expression. J. Bower Ph.D. of Monash University, Melexons 3-7 which would be expected to cause a shift in the reading frame. However, they un- bourne reported the effects of several cytokines expectedly show mild or intermediate phenotype. on murine muscle precursor cells including TGFHe pointed out that about 6% of the dystro- ~, interleukin (IL-6) and leukaemia inhibitory phinopathy patients do not conform to factor (LIF). All were shown to be potent growth factors for muscle. Monaco's "in frame/out of frame" rule. Concerning the great clinical variability in The correlation between the amount of dystrophin and severity of disease (Hoffman's phenotype for the dystrophinopathies, Dr Byron hypothesisl also had exceptions. Immunoblots Kakulas of the ANRI described the preliminary show that in BMD patients there are variable results of investigations using DNA deletion amounts and variable sizes of the dystrophin analysis and immunoblots on patients with molecule. Professor Dubowitz said that patients spinal muscular atrophy (SMA), limb girdle with deletions of exons 45-47 were usually "fin muscular dystrophy (LGMD), autosomal recesframe" and showed the expected BMD pheno- sive muscular dystrophy (ARMD) and atypical facioscapulohumeral muscular dystrophy (FSH). type.
Workshop Report
Two "SMA" patients were shown to have deletions of the dystrophin gene and abnormalities ofdystrophin on Western blots. One patient considered clinically to have LGMD was also shown to have a deletion of the dystrophin gene [8]. A girl in a family thought to have ARMD was also shown to have dystrophin abnormalities by Western blotting. Of six patients with a provisional diagnosis of atypical FSH, three were shown to have reduced quantities of dystrophin by Western blotting. This experience confirms the findings of others, particularly the group of Dr Sugita of Tokyo who has shown that the dystrophin gene and/or immunoblot abnormalities may be present in the Fukuyama type of congenital muscular dystrophy [9], and patients with quadriceps myopathy [10] or LGMD [11]. There are several other reports of patients, previously believed to have LGMD or SMA, being truly dystrophinopathies by DNA deletion analysis [12], some of whom were manifesting female carriers [11]. It is of interest that even patients with myalgia and cramps with no weakness may also have deletions of the dystrophin gene [13]. Others have massive cardiomegaly [14]. Even more surprising is a patient with a deletion of exons 31-44 has been shown to be asymptomatic [15]. Sporadic manifesting female carriers are easily misdiagnosed as having limb girdle muscular dystrophy or autosomal recessive muscular dystrophy [16]. Even these preliminary observations are sufficient to conclude that the current classification for the muscular dystrophies which is based on clinical criteria is inadequate. It may thus be foreshadowed that a classification will emerge based upon molecular criteria in the future. Concerning his experience of DNA mutations in DMD, Dr Lefkos Middleton reported 5% duplications, 65% deletions and 30% point mutations in his dystrophinopathy population. Steve Wilton Ph.D. of the ANRI described the methods involved in the examination of the dystrophin gene transcript. Total RNA extracted from BMD/DMD patients using the reverse transcriptase-polymerase chain reaction (RTPCR) is used to demonstrate the effect of gene deletion on the mature gene transcript. He believes that this approach will allow accurate determination of a woman's carrier state. Mutations within the gene affecting mRNA processing would be detected as abnormal sized PCR products. In one such study, combined with restriction fragment length polymorphisms
247
(RFLP) and single strand conformational po!ymorphisms (SSCP), analysis did not reveal an~¢ further base changes in dystrophin mRNA within the region ofexons 43-51 although several alternatively spliced gene transcripts were shown to be intact. SSCP provides information on the shape of the molecule when it is frozen, according to its amino acid sequences. RT-PCR can detect dystrophin gene rearrangements and denaturing gradient gel electrophoresis (DDGE) is used to identify alternative splicing. Nigel Laing Ph.D. of the ANRI reviewed the experience of his Neurogenetic Laboratory. Among his observations was that oogenesis seemed to be more mutagenic than spermatogenesis. There are 160 families in Western Australia with dystrophinopathy. Dr Allen Roses reported the preliminary results of his observations in dystrophia myotonica (DM) following the discovery of the CTG expansion of the gene on Chr 19 [17]. Recombinant events in DM families defined a 200 kb region that contains the DM gene. Among other observations, Dr Roses showed preliminary data on the variable expansion of the gene in different parts of the body. This may explain the differences in the severity of the lesions in the different tissues, an exciting observation which remains to be confirmed. This may also explain the greater involvement of skeletal muscle and lesser involvement of other tissues in DM. It is now known that the gene codes for a enzyme named myotonin protein kinase. Dr Roses suggested that this enzyme may be involved in the potassium channel disorder of the muscle membrane in DM. Concerning DM, Nigel Laing Ph.D. also reported his preliminary observations using the cDNA25 probe kindly provided by Dr Keith Johnson of London. DNA was analysed from nine DM patients without prior knowledge of the clinical severity in each. When this was revealed a striking correlation was found between the degree of expansion of the gene and the clinical severity of the disease. It is thus noteworthy that with the event of these advances it may now be possible to diagnose DM at any age and may predict its severity in any individual. This development is of great clinical significance and is especially useful for antenatal diagnosis. Angus Stewart Ph.D of Edith Cowan University in Perth, described his preliminary results of calcium activated force generation in skinned fast-twitched muscle fibres from C57 mdx mice. He found that differences may exist in strontium
248
Workshop Report
inactivation in the dystrophic vs normal. Control data produce two clear curves corresponding to fast and slow, using this technique, whereas mdx fibres produce three curves corresponding to fast and slow fibres and intermediate fibres. Sue Fletcher Ph.D. of the ANRI has shown that the trophic factors MyoD and myogenin are expressed during necrosis and regeneration of muscle in old mdx mice. She believes that this increase in the expression of MyoD/myogenin indicates that regeneration and, by inference, necrosis, of the skeletal muscles of adult, mdx mice is continuous and not transient or nonprogressive as suggested in some reports. Dr Olav Sola of the University of Washington, Seattle, U.S.A. reviewed his pioneering work on the use of the latissimus dorsi muscle for myocardioplasty operations. Anatomically he has found that three separate segments of the human latissimus dorsi exist. He has defined the histochemical fibre type distribution in each, the highest total number of slow fibres being in the superior segment and in decreasing numbers in the oblique and lateral segments. In each segment the largest number of slow fibres is found in the central or perivascular regions. In conclusion, the human dystrophinopathies may be classified as: (1) DMD; (2) BMD, this group includes the atypical patients previously diagnosed as Kugelberg-Welander SMA and limb girdle MD patients; and (3) the clinically atypical dystrophinopathies. The atypical group includes patients with quadriceps myopathy, those with cardiomyopathy, the "'creatine kinase aemias'" and patients with just myalgia and cramps. As a preliminary observation there are also some patients with atypical FSH which show "'facioscapulohumeral distribution of weakness" who have been found to show abnormalities ofdystrophin by Western blotting.
BYRON A. KAKULAS Medical Director Australian Neuromuscular Research Institute Professor of Neuropathology University of Western Australia and Head of Neuropathology Royal Perth Hospital Perth. Western Australia
REFERENCES
1.
Kakulas B A, Mastaglia F L (Eds). Pathogenesis and
2.
New York: Raven Press, 1990. Kakulas B A, Howell J McC, Roses A D (Eds).
Therapy o f Duchenne and Becket Muscular Dystrophy.
Duchenne Muscular Dystrophy: Animal Models and Genetic Manipulation. New York: Raven Press,
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
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