Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from Postgrad Med J (1992) 68, 500- 506 © The Fellowship of Postgraduate Medicine, 1992

The muscular dystrophies Victor Dubowitz Department ofPaediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, UK

Introduction In 1879 William Gowers, the eminent British The main breakthrough has been in Duchenne neurologist, painted a remarkably lucid word pic- and Becker muscular dystrophy, or the Xp2l ture of Duchenne muscular dystrophy in his series dystrophies as some would now have us call them. of lectures on pseudohypertrophic muscular The location of the for the X-linked paralysis, published in the Lancet.' This disease, he Emery-Dreifuss muscular dystrophy on the long said, is one of the most interesting, and at the same arm of the at Xq28 was discovered time most sad, of all those with which we have to a few years back3 but there has as yet been no deal; interesting because ofits peculiar features and further progress in its resolution. The locus for the mysterious nature; sad on account of our dominantly inherited facioscapulohumeral dyst- powerlessness to influence its course. rophy on the long arm of chromosome 4 has only Almost exactly a century later Duchenne dys- recently been found4 and other dystrophies such as trophy found itself at the centre of one of the most the autosomal recessive congenital dystrophy and exciting breakthroughs in the modern science of limb girdle dystrophy will undoubtedly follow suit molecular genetics. It was the first once sufficient collaborative clinical and laboratory

in which a previously unknown biochemical abnor- effort is concentrated on them. copyright. mality was resolved by the process of reversed genetics, with initial location, isolation and cloning of the gene and then identifying the it The Duchenne dystrophy story encodes. Moreover, we now seem to be on the verge of treating the disease by one or other of the Locating the gene potential routes of gene therapy. This would cer- tainly have delighted Gowers. At the same time The discovery of the gene locus for Duchenne molecular genetics has generated a most complex dystrophy at Xp2l came, almost simultaneously, http://pmj.bmj.com/ and almost totally incomprehensible newjargon of from three sources. Through the application of the its own, which must surely have turned Gowers in new techniques of molecular genetics it became his grave. possible to isolate DNA sequences from cloned The muscular dystrophies, a term first coined by fragments derived from the human X chromosome Erb in 1891, are a group of genetically determined and to determine their exact location. Duchenne disorders characterized by degeneration of skeletal dystrophy was the first disease to be localized in this muscle and no associated structural abnormality in way by linkage with these restriction fragment on September 30, 2021 by guest. Protected the central or peripheral nervous system. They have length polymorphisms (RFLPs).5 Secondly, a been subdivided into various clinical types on the number of documented females with Duchenne basis of the clinical distribution and severity of dystrophy associated with an X:autosomal trans- muscle weakness, and the mode ofinheritance. The location all had the breakpoint on the X rate of progression of the disease is also variable chromosome at this locus. Thirdly, a number of and some, such as congenital muscular dystrophy, cases of Duchenne dystrophy with associated X- may remain relatively static or even show func- linked disorders, such as chronic granulomatous tional improvement over time. Some of the animal disease, and McLeod's synd- models of the dystrophies may be devoid of any rome were found to have a cytogenetically visible clinical weakness as may some of the milder deletion at the same site.6 Linkage studies in cases variants of, for example, Becker dystrophy, which of Becker dystrophy pointed to the same locus, can present solely with cramps on exercise.1'2 confirming that the two conditions are allelic and due to abnormalities in the same gene. This fits in with the clinical observation that Becker dystrophy has a similar clinical pattern to Duchenne apart Correspondence: Professor V. Dubowitz, M.D., Ph.D., from its later onset and milder course and that there F.R.C.P., D.C.H. is an overlap of cases between the two.' Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from THE MUSCULAR DYSTROPHIES 501

The clinical phenotype of cDNA probes covering the whole dystrophin gene. This enables one (in a sample) to detect Although it can be argued that all cases ofmuscular exon deletions within the gene, which occur in dystrophy associated with the Xp2l gene form a about 50-60% of all Duchenne as well as Becker continuous clinical spectrum of a single disease, it cases. This has considerably increased the diagnos- would be helpful to have some degree of interna- potential in carrier detection and antenatal tional consensus on the definition of the different diagnosis (with chorionic villus biopsy in early grades of severity of Xp21 disease, in order to at pregnancy), and also the accurate diagnosis of least be able to compare data from one laboratory isolated cases particularly of Becker dystrophy with those from another. It is of course also which may have been misdiagnosed as spinal important from the point ofview ofthe patient and muscular atrophy on the misinterpretation of his family to be able to give some form ofprognosis atrophic fibres in the muscle biopsy, or as and appropriate supportive therapy. autosomal limb girdle dystrophy. It has also helped The vast majority of boys with Duchenne mus- to confirm the diagnosis of Becker dystrophy in cular dystrophy lose the ability to walk cases presenting with cramps on exercise, a com- independently by the age of 12 years. If one defines mon and well-recognized presenting symptom.' Becker dystrophy as maintaining independent Some ofthese cases have no detectable weakness or ambulation beyond the 16th birthday, this clearly disability but have a grossly elevated creatine separates the two phenotypes and also recognizes kinase and a dystrophic muscle biopsy. cases of intermediate severity which bridge the gap Contrary to early speculations, neither the size of between the two and lose ambulation between 13 the deletion nor the location-bears any consistent and 16 years. One can then dispense with ill-defined relation to the severity ofthe clinical condition. In a terms such as 'outliers' or 'mild Duchenne' or review of 218 of our patients with Duchenne, 'severe Becker'. There is of course considerable Becker or intermediate phenotypes, 124 had dele- variability within the Becker group with some cases tions with the cDNA probes.' Seventy-four remaining ambulant and practically free of separate deletions were found,with 55 being unique disability into late adult life and others having to one patient and the remaining 19 occurring in at copyright. considerable disability already in their late teens. least two unrelated patients. Some deletions such as Similarly within the Duchenne group some cases of exons 33-34 and 33-35 occurred only with may already be unable to walk by 6 or 7 years of Becker patients and of exons 3-7 in four patients age. with intermediate severity and one Becker. We also found no correlation of associated mental retarda- tion with any selective deletions. Isolating, cloning and characterizing the gene In order to explain the varying clinical severity, Monaco et al." postulated that in Duchenne http://pmj.bmj.com/ The isolation of the gene was achieved by Kunkel dystrophy the deletion, irrespective of size, leads to and his colleagues by an ingenious approach of a frame shift of the triplet codons for amino acids preparing a library ofcloned sequences correspond- resulting in a severely truncated non-functional ing to the DNA deleted in a patient with Duchenne protein, whereas in Becker dystrophy the dystrophy, chronic granulomatous disease, nucleotides remain in frame, and can produce a retinitis pigmentosa and McLeod's syndrome and a functional protein, although reduced in size. Over visible deletion in the Xp2l region.7 This was 90% of cases with deletions conform to this on September 30, 2021 by guest. Protected followed within a comparatively short period of hypothesis. There have, however, been some excep- time by the complete cloning of the gene,8 and the tions. Thus Malhotra et al.'2 demonstrated a 3-7 discovery of the protein product of the gene, which exon deletion, with disruption of the translational they named dystrophin.9 The gene is a gigantic size, frame, in six Becker, five intermediate and two encompassing over 2,000 kilobases (2 million base Duchenne cases, and postulated mechanisms that pairs), with over 60 exons spanning the gene. might compensate for the effects of the frame shift Dystrophin is a correspondingly large protein of in the milder cases. over 400 kilodalton in size, but with very low abundance, comprising only about 0.002% oftotal Dystrophin in muscle biopsies striated muscle protein. In their initial biochemical studies of dystrophin in relation to Duchenne and Becker muscular dyst- Clinical applications rophies and other neuromuscular disorders, Hoffman et al.'3 concluded that dystrophin was Gene deletions absent in severe Duchenne dystrophy, was present Kunkel's group have produced, and made but of abnormal molecular size in mild Becker available to genetic laboratories worldwide, a series muscular dystrophy, and was present in normal Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from 502 V. DUBOWITZ amounts and size in other forms of muscular In addition to the original Hoffman antibodies, dystrophy. It was thus a valuable tool for excluding a number oflaboratories have produced antibodies Duchenne,Becker muscular dystrophy in the case to different parts ofthe dystrophin molecule which of other 'forms of dystrophy such as congenital has allowed a more comprehensive appraisal ofthe muscular dystrophy or autosomal limb girdle dyst- changes in dystrophin in the muscle in parallel with rophy that might have a similar clinical presenta- the deletion studies in the gene. Nicholson et al.'6 tion. Hoffman also claimed that one might be able recently reported their experience in a comparative to prognosticate in Duchenne or Becker dystrophy study of immunoblotting and immunocytochemis- on the basis of the dystrophin in the muscle. It try in a large series of 226 muscle biopsies, includ- almost looked as if the clinician had become ing 85 Duchenne and 55 Becker dystrophies, using redundant and all that was needed was a competent a monoclonal antibody to the rod domain of surgeon to take the muscle biopsy and a well- dystrophin, which was specific for dystrophin and informed biochemist, working in the splendid did not crossreact with spectrin or alpha-actinin. isolation ofhis laboratory, to do the rest! However, They found isolated positive fibres in 40% of the not all their cases fitted into this neat compartmen- Duchenne biopsies and a further 20% had weak talization. Thus one case of Becker dystrophy had labelling of a large number of fibres. Of the 54 absent dystrophin, which they ascribed to a dele- Duchenne and 52 Becker biopsies showing a tion corresponding to the same DNA region as the positive band on immunolabelling, 85% had a fusion used in preparing the original protein of abnormal size, whereas the remaining antibodies. By extrapolation this would imply that 15% had a protein of normal size but reduced one would need to know the deletion status of the abundance. They found that overall there was a gene, based on the DNA studies of the patient's good correlation between the abundance of dys- blood, before one could draw any conclusions in trophin and clinical severity. relation to prognosis from the absence of dyst- A lot more cumulative experience is still required rophin, particulary in a clinically milder case. comparing the severity of the clinical disease with Conversely, although there was little or no the deletion data in relation to the gene and the

significant dystrophin present in 35 oftheir 38 cases changes in the dystrophin in the muscle. Mean- copyright. of Duchenne dystrophy, there were three samples while it would be prudent (for biochemists as well which had detectable levels above 3% of normal. as physicians) not to lose sight ofthe importance of Similarly in Becker muscular dystrophy the sugges- careful clinical assessment ofthe patient in relation tion of altered size or molecular weight of dyst- both to the extent of weakness and the rate of rophin excluded the possibility of a normal-sized progression of the disease, rather than trying to protein of reduced amount. predict these from the laboratory data in isolation. The application of immunocytochemical techni- ques to the study of dystrophin in muscle sections Animal models ofX-linked (Xp2l) dystrophies http://pmj.bmj.com/ added a new dimension to the interpretation. In contrast to the early suggestion based on the A number ofanimal models ofthe Xp2l dystrophy biochemical fractionation studies that dystrophin have been identified in recent years and have might be localized to the triads, the cytochemical already attracted a lot of research interest both studies showed a clear-cut localization to the from the investigative and from the therapeutic sarcolemmal membrane.14"15 point of view. While confirming the general absence of any staining for dystrophin in Duchenne biopsies, it has The mdx mouse on September 30, 2021 by guest. Protected also consistently revealed small numbers, usually less than 1%, of positive staining fibres in some An X-linked dystrophy in the mouse was picked up Duchenne biopsies. It has been suggested that these purely by chance during a mutagenesis screen of may represent a somatic , with reversion serum enzymes.'8 Following a period ofnecrosis of to normal, in individual fibres. In Becker biopsies the muscle at about 2-3 weeks ofage there is active there may be a variation in the intensity of stain in regeneration and recovery and the mice then the positive fibres and also the presence of patchy remain essentially normal and have a normal rather than continuous staining of the membrane lifespan.'9 The gene for the mouse dystrophy is in individual fibres. In female carriers the results homologous to the human dystrophin gene,20 and are also interesting, but somewhat complex. dystrophin is also absent in the mdx mouse mus- Although manifesting carriers may show a mosaic cle.9 It has recently been shown that the genetic pattern of positive and negative fibres,'6 the abnormality is a point mutation at nucleotide majority of non-manifesting carriers that we have position 3185, with the replacement of the reviewed have shown an essentially normal dyst- nucleotide cytosine by a thymine, resulting in a rophin pattern with practically all fibres showing a termination codon (TAA) in place of a glutamine normal staining pattern.'7 codon (CAA).2' This results in premature termina- Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from THE MUSCULAR DYSTROPHIES 503 tion of translation at 27% of the length of the a feature of selected muscles in the human disease, dystrophin polypeptide and the production of a the so-called pseudohypertrophy already noted by truncated protein. Presumably this residual protein Duchenne, who coined the term. is sufficiently stable and functional to sustain regeneration and fibre integrity in the mouse dystrophy. Possibly some of the human Duchenne Therapeutic possibilities and Becker dystrophies cases that do not have a detectable deletion in the gene may have a similar Gene therapy change in a single base pair among the more than 2 million base pairs that make up the gene. The dramatic advances in the past few years in relation to the dystrophy gene naturally raised the The XMD dog hopes of sufferers and their families that a cure might be around the corner. It also sparked An X-linked muscular dystrophy (XMD) has been speculation in the scientific community on the discovered in a Golden Retriever strain of dogs possibility of gene therapy. Techniques already which is also genetically homologous to the human exist for the introduction of DNA into somatic or disease and lacks dystrophin in the muscle.2223 germline cells, either directly or with viral or other Clinically the dog manifests a severe weakness with vectors. One major hurdle was the tremendous size an early onset and steady progression, comparable of the gene which would be beyond the capacity of to the Duchenne type. From an early stage there is retroviral vectors. Possible ways around this were also marked wasting ofthe muscles, but none ofthe the use of alternative vectors such as yeast artificial prominence and apparent enlargement of the chromosomes or the construction of a 'mini' gene muscles one sees in the human disease. The his- from the DNA of a patient with mild Becker type tological picture is identical to that in Duchenne dystrophy and the deletion of about half of the dystrophy, including the early proliferation of dystrophin, yet still capable of producing a func- endomysial connective tissue. Selective involve- tional protein about half the normal size.27 ment ofsome muscles at an earlier stage than others Moreover it has recently been shown that DNA copyright. has been demonstrated in the neonatal period,24 injected directly into muscle remains functionally comparable to the selective involvement from an viable, thus bypassing the need for a vector.28 To early stage in Duchenne dystrophy. crown it all, Caskey's laboratory have just suc- The exact genetic mutation has not yet been ceeded in producing a construct of the whole identified, but no deletions have been found. As in dystrophin gene, and shown that the recombinant the case of the mdx mouse, all the descendants dystrophin that it produced was indistinguishable within the same strain will presumably have the from mouse muscle dystrophin by Western blot same mutation. Cooper has recently diagnosed a analysis with anti-dystrophin antibodies, and was http://pmj.bmj.com/ dystrophin-deficient dystrophy in a pair of sibling localized to the cell membrane with fluorescent male Rottweiler pups, aged 5 months.25 Clinically antibody techniques.29 This adds further credence they were more severely affected than the previous and hope to the possibilities ofdirect gene therapy, Retriever progeny. The canine X-linked muscular but there are still major hurdles to be overcome dystrophy is thus likely to affect many different such as the targeting of the replacement gene to the strains of dog, and a number of case reports of affected muscles. in dogs in the earlier veterinary literature may well have been cases of abnor- Myoblast transfer on September 30, 2021 by guest. Protected malities in the dystrophin gene. An attractive, feasible and potentially practical The XMD cat short cut, which could provide an alternative approach to the replacement of either the gene or A dystrophin-deficient muscular dystrophy has its protein product, has been the introduction of been documented in a single neutered male cat, normal muscle cells, in the form ofactively dividing which was not clinically weak and had marked cultured myoblasts, directly into the dystrophic prominence of the muscles.26 Cooper has recently muscle. These myoblasts can then fuse with the investigated a similar male cat with a dystrophin- regenerating mononucleate satellite cells of the deficient dystrophy, which had a remarkable degree host, which lie dormant under the sarcolemmal of muscular hypertrophy and no obvious clinical membrane of normal as well as dystrophic muscle, weakness. This feline model ofmuscular dystrophy until activated. The multinucleate muscle cells is of special interest in parallel with the mouse formed are thus a combination of normal and dystrophy for its absence of clinical weakness and dystrophic cells, comparable to the female carrier its marked muscle hypertrophy, which probably of the Duchenne gene, and hopefully sufficiently also occurs in the mdx mouse, and is, ofcourse, also functional to provide reasonable muscle power. 504 V. DUBOWITZ Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from

Earlier experimental work in mice showed the the myoblasts in. The presence ofdystrophin in the fusion of donor and host myoblasts to produce injected muscles on both sides is being assessed hybrid multinucleate muscle cells,30 and to intro- after 3 months in open biopsy samples with both duce from the donor cells.3' This approach Western blotting and immunocytochemical techni- was subsequently used by Law and his colleagues32 ques. The muscle strength in the elbow flexors is to try to correct the abnormality in the autosomal also being measured sequentially. recessive muscular dystrophy in the mouse. In a Law and his colleagues in Memphis, USA, are recent application of the technique in the mdx conducting a similar series of experiments on the mouse, Partridge and his colleagues demonstrated extensor digitorum brevis muscle on the dorsum of the fusion of normal donor muscle cells with the the foot, using cultured myoblasts from the father, muscle cells of the host with the production of and comparing the results with the sham-injected hybrid multinucleate fibres. These fibres were also opposite side. The small size of the muscle has capable of expressing dystrophin, which was con- potential advantages in relation to the number of sistently absent in the host muscle.33 cells needed for the procedure, but limitations if While the mdx mouse is a good model for more than one biopsy were envisaged or in accurate establishing the experimental aspects of this measurement ofmuscle force. These studies are still therapeutic approach, and has the advantage ofthe very much in the experimental stage. It will not be small muscle size in relation to the number of feasible to proceed to the next phase of actual injected cells, it is not a good model for assessing therapeutic trials ofmyoblast transfer in appropri- potential clinical benefit in the absence of any ate patients to assess the potential impact on the clinical weakness. In this respect the XMD dog, disease itself until we have a clear-cut and un- with its clinical phenotype and pathological picture equivocal answer to the basic question as to more closely resembling Duchenne dystrophy, whether the technique produces significant and should provide a better model. adequate expression of dystrophin in the host muscle, and what beneficial effect this has on the Human myoblast transfer experiments muscle pathology and particularly on the clinical

function. It is also imperative for the wide publicity copyright. Fired by the apparent success of the technique in that this work has already had in the lay press to be the mdx mouse, a number of centres in North toned down in order to avoid unwarranted hope in America opted to plunge forthwith into the sufferers and their families together with the experiments in patients with Duchenne dystrophy frustration of thinking (misguidedly) that they are to evaluate the potential therapeutic value in the already missing out on actual beneficial treatment context of the human disease. They anticipated by not being where the action lies. that they would be able to resolve such basic Meanwhile, we may be able to get a further

questions as the minimum and optimal number of appraisal ofthe therapeutic potential ofthis techni- http://pmj.bmj.com/ cells needed to be effective, the method ofintroduc- que in the dystrophic dog, and I have established a tion and dispersion ofthe cells into the host muscle, collaborative programme with Dr Barry Cooper's and the short-term or long-term need for group at the New York State College ofVeterinary immunosuppression, as part of the experimental Medicine at Cornell University in Ithaca, USA. design. As in any therapeutic trial, it is absolutely There are a number of advantages in the dog essential to have objective and reliable methods to model. One can use siblings as donors and carefully assess the outcome of the experiment, both from control the timing ofthe injections in relation to the the point of view of the successful fusion of the stage of the pathological process and also compare on September 30, 2021 by guest. Protected donor with the host cells and the expression of different muscles which may be at different stages in dystrophin in a previously dystrophin-negative the same animal. There are also major hurdles to be subject, and also the effect this has on the overcome in the canine model such as the difficulty pathological state of the muscle, and its clinical in culturing cloned myoblasts in quantity with function. existing culture media which may be optimal for From an immunological compatibility point of human or mouse muscle, and the relatively view, the ideal donor would probably be a sibling, rudimentary status of knowledge in relation to the but in view of the ethical constraints on using dog's immunological systems, compared to man or minors in the context of such experiments, the mouse. father has usually been chosen. Karpati and his team in Montreal, Canada, are injecting cultured myoblasts from the father into Conclusions the biceps muscle of boys with proven Duchenne dystrophy and an associated deletion in the dyst- The muscular dystrophies are currently at a very rophin gene. This is done randomly and blindly, the exciting stage of active resolution of many of the other biceps having a comparable injection without fundamental questions that have puzzled and Postgrad Med J: first published as 10.1136/pgmj.68.801.500 on 1 July 1992. Downloaded from THE MUSCULAR DYSTROPHIES 505 eluded clinicians and scientists for the past century. remarkable technical advances we have witnessed The Duchenne gene has been cloned, its protein in relation to gene manipulation in the last few identified and its location accurately mapped to the years. inner surface of the sarcolemmal membrane. Its function has not yet been fully established but it is thought to have a stabilizing role in the integrity of Dedication the membrane in close association with linked glycoproteins. The absence or abnormality of I am delighted to contribute this paper on the dystrophin presumably triggers the process of muscular dystrophies to the festschrift for Lord progressive degeneration and disintegration of the Walton and to express my personal appreciation muscle cell, but the other factors, such as the influx for all the help and advice he has given me in so of calcium, which have an important role in many ways over the years, since my interest in perpetuating the process still await more detailed muscular dystrophy was first kindled during a study and clarification. residency post at Queen Mary's Hospital for The new era of potential treatment of the Children in 1957 and also my gratitude to the remorselessly progressive and disabling Duchenne Muscular Dystrophy Group of Great Britain for form of muscular dystrophy is just beginning, and their continuous support of my research things look set fair for its success, given the endeavours over the past 33 years.

References

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