Science and clinical myology
The Stephen Paget Memorial Lecture, 1990
Lord Walton of Detchant, TD, MA, MD, DSc, FRCP
Honorary Fellow and past Warden, Green College, Oxford; former Professor of Neurology, University of Newcastle upon Tyne.
I regard it as an exceptional honour and privilege to have been invited to deliver this prestigious annual lecture to honour Stephen Paget who, as you all know, was born in 1855, the fourth son of the distinguished surgeon, Sir James Paget, whose brother, Sir George, was one of my predecessors as President of the General Medical Council. James, of course, will always be remembered by the fact that his name has been attached to two specific diseases which are totally different in their course and significance, namely Paget's disease of bone and Paget's disease of the nipple . It may be that Stephen originally considered the possibility of entering the Church like two of his brothers, both of whom became bishops. However, after being educated at Shrewsbury and after reading classical honours at Christ Church, he eventually decided to follow his father into medicine, which he studied at St. Bartholomew's Hospital, obtaining his FRCS in 1885, before practising at the Middlesex, Metropolitan and West London Hospitals, first in aural surgery and later in thoracic surgery. He was also a most able writer and well acquainted, it appears, with many of the great men and women in literature, science and politics in Great Britain in the latter part of the 19th century, including Tennyson, Browning, George Eliot, Gladstone, Huxley, Matthew Arnold and Darwin. He published over 20 books on surgery and in addition many essays and biographies, as well as innumerable booklets and pamphlets. While he was particularly interested in medical education and also prepared much teaching material for medical students, his retirement in 1910, forced upon him by ill-health, meant that he had to give up surgery and he therefore devoted himself to literary work and the defence of medical research. His father was in fact a founder member of the Association for the Advancement of Medicine by Research (AAMR) which had been founded in 1882 following the passage in 1876 of the Cruelty to Animals Act but the influence of that body seemed to decline . Stephen became a member of the committee formed by Professor Ernest Starling (President of the AAMR) which prepared evidence for the Royal Commission on Vivisection in 1906 and which concluded that a new body was needed to defend research. He was elected founding Secretary of the Research Defence Society which was then established and devoted much of his time, energy and activities in the succeeding 20 years to its affairs. The society plainly owes much of its subsequent success and vitality to Stephen Paget's early work and spirited support of its aims.
My gratitude for your invitation was somewhat eroded by a glance at the exceptionally distinguished list of those who have given this annual lecture in each of the last 63 years. Their names read like a veritable "Who's Who" of British medicine and science of the 20th century, and I found the range of topics which they had chosen to be so varied but yet all-embracing as to cause me considerable concern about whether anything important or even of interest to your society could possibly be left to say.
I decided, however, to choose the topic "Science and clinical myology" if only because much of my medical life and such research as 1 have been able to undertake have been largely concerned with human neuromuscular disease, but with substantial reliance, whenever the need arose, upon complementary experimental research in appropriate animals, along with studies of those naturally-occurring genetically-determined diseases of muscle which have been discovered in many species in the animal kingdom. But before attempting to summarise some of the ways in which scientific discovery has illumined our understanding of human neuromuscular disease, it might be appropriate to begin by explaining my use of the term 'clinical myology' in the title of my lecture. I trust that you will forgive me if the succeeding comments are at first unashamedly autobiographical.
Having graduated in medicine in the Newcastle Medical School of the University of Durham in 1945, and having subsequently completed my military service, I was as a student and house officer so impressed and stimulated by the incomparable clinical skills and electrifying personality of the late Sir James Spence, the Professor of Paediatrics in Newcastle, that I was determined to become a paediatrician. Nevertheless, I was advised that before embarking upon such a career it would be necessary to have further experience in general internal medicine and to obtain the MRCP qualification before returning to the paediatric fold. I therefore obtained a registrarship in medicine in Newcastle where I came under the influence of an amiable and experienced senior physician, Dr Alan Ogilvie, and his newly-appointed assistant physician, Dr Henry Miller, whose principal interest was neurology. Having previously worked as house physician to the Professor of Medicine, Professor F.J. Nattrass, also a fine neurologist, I had begun to be greatly impressed by the intellectual challenge of that clinical discipline, which seemed to me as much as any specialty in medicine to depend upon a precise pathophysiological interpretation of clinical phenomena on the basis of fundamental knowledge of anatomy, physiology, biochemistry and genetics. Gradually, therefore, under the lively tutelage of Henry Miller and under the almost hypnotic influence of his extraordinary personality, my thoughts turned more and more towards neurology and away from paediatrics. Hence when, towards the end of my registrarship, Fred Nattrass approached me with the offer of a research assistantship in the Department of Medicine in order to investigate neuromuscular disease, I was finally persuaded.
The reason why this research assistantship had become available, with funds provided partly by the Ministry of Health and partly by the University of Durham, was because attempts by a man in the north east of England to establish a society to bring together the families of patients with muscular dystrophy had given rise to some national press publicity; this had in turn brought to the attention of the Ministry of Health a number of reports of patients who had allegedly been suffering from muscular dystrophy but who had recovered. Senior London neurologists when approached by Dr Albertine Winner (subsequently Dame Albertine) from the Ministry had, with a single voice, concluded that if these patients had been diagnosed as suffering from muscular dystrophy and had recovered, then the diagnosis must certainly have been wrong. But when Dr Winner approached Professor Nattrass in Newcastle, he felt that this was an interesting finding which ought to be further investigated; his motivation may not have been totally unrelated to the fact that the diagnosis in one of the patients alleged to have recovered had been made by himself, and in another by Sir James Spence. On being appointed, my first task was to spend three months at the National Hospital, Queen Square, in London and at St. Thomas' Hospital on leave of absence from my Newcastle appointment in order to acquire experience in the technique of electro-myography, as both Professor Nattrass and I felt that to be able to investigate fully the patients with neuromuscular disease whom I would subsequently be studying, a reasonable level of competence and diagnostic skill in the application of that method would be essential.
On returning to Newcastle, I first established an electromyographic service in the Department of Medicine and secondly set out to try to identify all of the patients with neuromuscular disease then living in the northern region. My objectives were first to define the clinical features and course of the muscular dystrophies and other related conditions; secondly to try to explain the recovery which had occurred in the patients who had been brought to the attention of the Ministry of Health and who had been thought to be suffering from a progressive muscular dystrophy; and thirdly to try to establish a firm revised classification of the muscular dystrophies based upon clearly-defined clinical and genetic criteria, in view of the fact that the purely descriptive criteria upon which such classification had largely been based seemed to me at that time to be in a state of some confusion. They were certainly unsatisfactory as a guide to prognosis and management. In parallel with these endeavours, I also set out to try to improve the precision of the electromyographic diagnosis of myopathy and to see whether, through a study of muscle pathology in man, accompanied by experimental studies in animals, I could cast some light upon the pathogenesis of human muscular dystrophy.
While it would be difficult to summarise in a few paragraphs the wide range of experience I gained and the observations that I made during the five years that I spent as a research assistant to Professor Nattrass (interrupted by two fruitful years on travelling fellowships from the Nuffield Foundation and from King's College, Newcastle, the first spent at the Massachusetts General Hospital, Boston, and the second in the MRC's Neurological Research Unit at the National Hospital, Queen Square) several findings which I believe were important emerged from this work. First, I soon recognised that whereas the syndrome of dermatomyositis, with severe acute inflammation of both skin and muscle, had been well recognised for some years, it was very much less well known that polymyositis with inflammation, often sub-acute, restricted to skeletal muscle, was not uncommon and could often mimic the muscular dystrophies. So too could that variety of dermatomyositis, occurring especially in childhood, in which the skin changes were minimal, often consisting of no more than shiny, tight skin over the tips of the fingers and toes, with injection of the nail-beds, perhaps some scattered calcification around bony prominences, and often with an associated Raynaud phenomenon and/or dysphagia. I soon became convinced that this condition, which, before the widespread use of corticosteroid drugs, could sometimes remit spontaneously or more often burned itself out, leaving the patient with a greater or lesser degree of disability, had accounted for the so-called recovery from muscular dystrophy in some of the patients whose existence had initiated the survey. The results of this work were eventually published in Brain, having been used by Professor Nattrass for his Presidential Address to the Section of Neurology of the Royal Society of Medicine in 1952
These observations led me, with R. D. Adams, to study an extended series of cases of polymyositis and dermatomyositis and, in later years, to initiate some preliminary immunological studies, in collaboration with colleagues, attempting to define the role of cell-mediated disordered immunity and of humoral factors in this group of diseases . I believe it is now generally accepted that polymyositis and dermatomyositis are unquestionably autoimmune, that the former is due to T-cell mediated damage to skeletal muscle, while the latter results primarily from disordered humoral immunity with damage affecting not only the muscle cells themselves but also the intrmuscular blood vessels . And studies of experimental allergic myositis induced in animals by the injection of suspensions of skeletal muscle with adjuvant have helped in elucidating the pathogenesis of the human disease.
It was, however, clinical myology which stimulated and motivated some of these developments, since I and others became increasingly convinced that the major clinical differences between muscular dystrophy on the one hand and that group of inflammatory myopathies on the other was related not just to evidence of the importance of genetic factors so clearly demonstrated by the familial occurrence of many of the muscular dystrophies, but also by the clinical pattern of muscle involvement with which the patients presented . Thus in muscular dystrophy the disease process was invariably more indolent, but in addition each of the dystrophies demonstrated a curious and relatively consistent selectivity in the way in which individual skeletal muscles in the human subject were involved. Thus in the severe Duchenne type of dystrophy (to be mentioned below) enlargement of the calf muscles and of the deltoids was commonly seen, while the biceps brachii, pectorals and brachioradiales in the upper limbs were the first to become weak, and in the lower limbs the medial vasti became atrophic before the lateral and the anterior tibials long before the muscles of the calves. In polymyositis and dermatomyositis, by contrast, the weakness was much more often global, affecting all proximal limb muscles equally, without the clear-cut selectivity, and in addition the tendon reflexes were commonly preserved, whereas these disappeared in the weakened muscles early in the muscular dystrophies. Thus it was characteristic of Duchenne dystrophy to find absent knee jerks and brisk ankle jerks, even in the relatively early stages. And curiously, despite the inflammatory nature of the disease process in polymyositis and dermatomyositis, pain and muscle tenderness were a feature of the clinical course in only a relatively small proportion of patients.
Perhaps our next most significant observation related to the classification of the muscular dystrophies into three principal varieties, namely the Duchenne, limb-girdle and facioscapulohumeral types
It was clear that this severe Duchenne form usually led to difficulty in walking at about the age of 3, followed by a waddling gait, frequent falling and difficulty in rising from the floor, with the affected boys being confined to a wheelchair by the time they were about 10 years of age. At that stage, as most were left at home to become severely and grossly deformed, many died from respiratory or cardiac failure in the mid-teens. And when one saw, as I sometimes did, not just one but two or even three affected boys within a single family becoming progressively weakened by that disorder and recognised the devastating and tragic effects which the disease had not only upon the patients but also on family life, I soon realised that any improvement in their lot would be well worth while. Now, with improved management, the prevention of skeletal deformity and the early treatment of complications, many such patients are living into their twenties and some, despite their severe disability, have even achieved university degrees.
The conclusion that this disorder, like its more benign variant, the Becker type, which gives a similar clinical picture but does not often begin until the mid-teens and then produces severe disability only in middle life, were each due to an X-linked recessive gene, was based largely in early work upon the inspection of family pedigrees. In my own early studies , I was able to demonstrate first, through blood grouping studies carried out in an attempt to identify linkage, that carrier women could have affected children by more than one male, secondly that in very occasional families there was crossing over with red-green (deutan) colour blindness , and thirdly (most conclusive of all) the fact that in a single family I identified a young woman with the Duchenne type dystrophy who also had Turner's syndrome (ovarian agenesis) with an XO chromosome constitution , .
But as my work on the much more benign and slowly progressive autosomal recessive limb-girdle variety continued, so did that on the dominantly inherited facioscapulohumeral type. This, like so many dominant disorders, demonstrated extraordinary variability in clinical expression so that some patients were unaware that they were suffering from the condition, as its manifestations were so mild. I also began to recognise, with the aid of electromyographic (EMG) examination, that spinal muscular atrophy was a common clinical mimic of muscular dystrophy. The acute and severe form of spinal atrophy, usually called Werdnig-Hoffmann disease, which causes an infant to be extremely limp and floppy at birth and which usually leads to death from wide-spread muscular paralysis before the end of the first year of life, had been recognised for many years but it was not generally known that a more benign variety could occur in later childhood, adolescence and adult life. This was called the pseudomyopathic type of Kugelberg and Welander
My own EMG studies, using automatic frequency analysis with an audiofrequency spectrometer , seemed to add precision to the differential diagnosis between primary muscle disease on the one hand and muscular weakness secondary to neuropathic disorders on the other. It therefore helped in separating off from the autosomal limb-girdle syndrome many patients with spinal muscular atrophy in whom the disease sometimes arrested but who had previously been thought to be suffering from progressive muscular dystrophy.
Differential diagnosis was also greatly helped by the introduction of methods of serum enzyme estimation, first of aldolase and subsequently of creatine kinase activity. It was clearly demonstrated that in muscular dystrophy, especially in the severe Duchenne type, these enzymes leaked out of the diseased muscle cells into the blood in consider¬able quantity, particularly in the early stages, but that this leakage diminished as the disease progressed and muscle bulk lessened . But as we began to recognise that long¬standing denervation of skeletal muscle in the spinal muscular atrophies could result in secondary myopathic change and that, in consequence, raised serum creatine kinase activity could be observed in such cases too, it became increasingly clear that no single diagnostic method was totally precise; and the same proved to apply to examination of muscle biopsy sections first by conventional histological techniques, subsequently with the use of histochemical enzyme stains, and thirdly with the electron microscope - methods which I began to use after receiving an extensive grounding in muscle pathology with the notable Raymond Adams of the Massachusetts General Hospital in Boston, with whom I spent a year from 1953-1954 on a Nuffield Foundation Fellowship.
On arriving in Boston, I found that Ray's former teacher and mentor, Dr Derek Denny-Brown, had spoken to a meeting of the American Neurological Association and had claimed that the primary abnormality in muscular dystrophy was a failure in such cases of the skeletal muscle cells to regenerate. In consequence, I set out with Ray Adams to study skeletal muscle regeneration in traumatised muscles of the rabbit and then to compare my findings in those experiments with those obtained in patients with muscular dystrophy who had volunteered to have two muscle biopsies performed, one before and one after the production of a focal lesion in muscle produced by the injection of alcohol tagged with a visible marker . Our studies in the rabbit enabled us to define more precisely the sequential steps of muscle fibre regeneration, though we were not as successful in defining the role of the muscle satellite cell in this process as were subsequent workers; nevertheless we clearly demonstrated the formation of muscle buds and their distinctive basophilia, with large vesicular nuclei with prominent nucleoli which were invariably present in regenerating fibres. Appropriate stains confirmed the presence of large quantities of RNA in such fibres; subsequently those histological criteria of regenerative activity seem to have stood the test of time. In fact in the patients with muscular dystrophy, and especially in boys with the Duchenne type, we found that such regeneration did occur both spontaneously and in response to trauma, but nevertheless appeared to be abortive and ineffective, so that Denny-Brown was partly right. Subsequently on my return to Newcastle I was able, with G.W. Pearce and others, to confirm that in preclinical cases of Duchenne muscular dystrophy (diagnosed by creatine kinase activity early in life in families in which a previous affected boy had been diagnosed) the histological criteria of Duchenne dystrophy were, first, dense hyaline fibres seen to stain a bright glassy red in transverse sections stained with haemalum and eosin, secondly necrosis and phagocytosis of skeletal muscle, and thirdly clear-cut areas of focal muscle regeneration . I shall mention the significance of these findings later.
Perhaps, however, the most significant conclusions which I drew from these years of investigation were, first, that despite the increasing sophistication of ancillary diagnostic methods, clinical criteria were nevertheless all-important and, I believe, still are in the diagnosis of neuromuscular disease. Among these are the nature, distribution and rate of evolution of the patient's muscular weakness and of pain and tenderness, if any such are present; secondly, the presence of observed atrophy and, when noted, of fasciculation in muscle fibres or, by contrast, the presence of enlargement or hypertrophy; thirdly, contracture or shortening of muscles and tendons and skeletal deformity must be noted; fourthly, on manual muscle testing, it is crucially important to define the exact distribution of muscle weakness and to identify those muscles which are weak and those which are comparatively spared; fifthly, it is clearly important to examine the tendon reflexes, noting whether they are absent, depressed, normal or exaggerated, of normal speed or abnormally slow, and to look for myotonia both on contraction and relaxation of the grip and on percussion of a muscle such as the deltoid or tongue; and finally, of course, it is important to seek clinical evidence of disease in other tissues or organs such as, for example, the endocrine glands, the skin, connective tissue, heart, lungs, abdomen and lymph nodes, just as one must also look for sensory abnormalities which might indicate that the disease is not primarily a neuromuscular one but that it may be a consequence of dysfunction of mixed motor and sensory peripheral nerves or of the central nervous system.
One final clinical example underlining this thesis may suffice . Quite recently in Oxford I was invited by a general physician of considerable distinction to examine a 17-year-old boy whom he had seen as an out-patient, when he had wondered, because of the patient's complaint of muscle pain and stiffness on exertion and because of the clear-cut hypertrophy of many skeletal muscles which he showed, whether the patient might be suffering from a muscular dystrophy, possibly of the Becker type. By the time I saw the boy in consultation, he had had several investigations ordered by the senior registrar on the firm, a young man with extensive endocrinological experience. Those investigations had included a wide range of biochemical studies, including serum enzyme estimations, and electromyography, which demonstrated evidence of minimal myopathic change, as well as muscle biopsy which had failed to demonstrate any significant abnormality. When 1 saw the boy just as he was about to have topical magnetic resonance studies, I noted that the history of pain and stiffness on exertion was clear-cut, but in addition he seemed to me to have a slightly deep voice and generally slow movements. He did demonstrate diffuse muscle hypertrophy but there was no myotonia in the tongue or in the hands, and on percussion of the deltoid the muscle contracted and relaxed slowly. His tendon reflexes were also much reduced in tempo and I was not therefore greatly surprised to find that he had a diffuse enlargement of the thyroid gland, a pulse rate of 45 per minute, some loss of hair on the lateral third of the eyebrows and a rather dry skin; in other words, he had many of the clinical features of myxoedema and was presenting the manifestations of Hoffmann's syndrome - that is, the muscular manifestations of hypothyroidism. I simply mention this case not, I hope, in order to flaunt my own personal clinical expertise, but to underline the fact that in clinical medicine it is all too easy to be totally misled by one or two clinical manifestations (such as, in this case, the diffuse muscle hypertrophy) which may lead one in the wrong direction, unless one's examination of the patient as a whole is so assiduous as to make it possible to identify features of disease in other systems. Clearly the muscular symptoms in this case dominated the clinical picture to such an extent that the physicians under whose care he had been admitted to hospital had overlooked the signs of hypo-thyroidism which they would normally have recognised at once had they not been so impressed by the overt symptoms of muscle dysfunction.
In the earlier part of this talk, I have described how I became involved in research into neuromuscular disease and have mentioned some of my early investigations. Following my return to Newcastle and my eventual appointment as a consultant neurologist, with the aid of grants from the Medical Research Council, the Muscular Dystrophy Associations of America and Canada, and eventually the Muscular Dystrophy Group of Great Britain and Northern Ireland, of which I am now honoured to be Chairman, I was able to establish a block of Muscular Dystrophy Research Laboratories at the Newcastle General Hospital and to recruit a team of colleagues to work on the clinical, genetic, electrophysiological, histological, histochemical, electron microscopic, biochemical and cell biological aspects of these disorders.
Time would not allow me to summarise any but a few of the findings of the Newcastle team. I believe, however, that one fundamental observation was that of Cullen and Fulthorpe in 1975 , who used the electron microscope to examine the earliest structural changes occurring in the muscle fibres in patients with preclinical Duchenne dystrophy, With standard histological methods and light microscopy we had demonstrated, as 1 have mentioned, the waxy hyaline fibres seen so prominently in transverse sections which many previous authors, including my mentor, Raymond Adams, had concluded were the result of fixation artefact. However, Cullen and Fulthorpe went on to examine first, under phase-contrast illumination, unfixed muscle fibres in which they found multiple contraction bands which were clearly responsible for those hyaline changes. In plastic-embedded fibres stained with toluidine blue, it was evident that such areas of hypercontraction might be extremely focal, involving only a small segment of a transverse section of the fibre, usually directly beneath the sarcolemma; they also demonstrated subsequently with specific stains a marked excess of calcium within these areas. This work led them to conclude that in Duchenne dystrophy there might be a defect in the plasma membrane of the skeletal muscle fibres, allowing the ingress of calcium from the extracellular space and that this in turn might activate calcium-activated neutral proteases which were then, at least in part, responsible for the breakdown and digestion of the muscle fibres. My colleague Dr Pennington confirmed that such calcium-activated proteases were indeed present in excess . Almost simultaneously Mokri and Engel , working at the Mayo Clinic, also using electron microscopy, demonstrated focal defects in the plasma membrane which they called 'the delta lesion'; hence it seemed that the mechanism of fibre breakdown in muscular dystrophy was becoming more clearly understood.
But the greatest impetus to research in this field has, of course, resulted from work in molecular biology. In 1987, following upon work in many laboratories in different parts of the world, including Holland, Toronto and Oxford, to name but three, but ultimately through the extraordinary energy, industry and expertise of Dry Kunkel, Dry Hoffman and their colleagues at the Boston Children's Hospital, the gene responsible for muscular dystrophy of the Duchenne type was finally isolated, localised and characterised in the Xp 21 region of the female X chromosome and has proved to be one of the largest genes known in human genetics with a length of almost 2 megabases , . Of even greater importance is the fact that the genes for Duchenne and Becker dystrophy have been shown to be allelic and that their clinical expression appears to depend, at least in part, upon the number and extent of the deletions present within the gene. However, the relationship is somewhat imprecise and there are some patients with Duchenne dystrophy who do not have such deletions and in whom the disease is presumed to result from a point mutation. Identification of the gene led to isolation of the missing gene product, a protein now called dystrophin , which is totally absent in almost all cases of Duchenne dystrophy and much reduced in those of the Becker variety. Further work has demonstrated that dystrophin forms a vital structural component of the plasma membrane of the skeletal muscle fibre ; hence it appears that the earlier observations of Cullen, Fulthorpe, Mokri and Engel upon the pathogenesis of muscle fibre breakdown have been largely vindicated. Presumably it is the absence of dystrophin which renders the muscle fibre membrane incompetent and which leads on to the breakdown process which I have outlined .
Future research will clearly be directed towards methods of attempting to repair the defect in the muscle fibre membrane or of identifying affected children very early in life in the hope of offering them some form of treatment (perhaps, ultimately, gene replacement) which will prevent the membrane from becoming as defective as it would in the natural course of events. Of very great importance when one thinks of introducing experimental methods of treatment is the fact that a naturally-occurring muscular dystrophy of X-linked inheritance has been clearly identified in the mdx mouse and yet another has been detected in a strain of golden retrievers . Current evidence strongly suggests comparability of these afflictions with the human disease. In both of these species the Duchenne gene transcript is lacking and dystrophin has been found to be absent in the muscle cells of affected animals. Cardiomyopathy like that of Duchenne dystrophy occurs in the dogs , and in carriers of the X-linked canine disorder, mosaic expression of dystrophin comparable to that found in the muscle of human female carriers has been noted33 . Plainly experiments in gene replacement should not only become feasible ultimately in such animals but will surely become a reality once a method of reintroducing the defective gene or the missing product into the skeletal muscle cell has been devised.
In the meantime, through work being done in Great Britain by Dr Terry Partridge and his colleagues at Charing Cross Medical School and by Dr Peter Law in the United States and others, methods are being realised of harvesting large numbers of myoblasts grown in culture from muscle satellite cells derived from normal animals and of introducing these by injection into the affected muscles of dystrophic animals , . Current experimental evidence suggests that problems of rejection are being overcome and that such myoblasts introduced into diseased muscle are capable of producing dystrophin which can be demonstrated by immuno-histochemical techniques, using monoclonal antibodies which are available both in the United States from Dr Hoffman's laboratory and in the UK from Professor John Harris's muscular dystrophy laboratories in Newcastle, where I formerly worked. Early experiments have indeed been carried out using myoblast transfer in human subjects by Dr Law in the United States and it has been shown in a few boys with Duchenne muscular dystrophy that such myoblasts are capable of restoring dystrophin to some muscle fibres of the extensor digitorum brevis .
The question as to whether myoblast transfer therapy will ever become of practical benefit in human patients is still a very open one. The problems to be overcome are first those of producing a sufficient number of myoblasts of human origin for use in this work; secondly, the ever-present problem of rejection will clearly be important; and thirdly, and perhaps above all, we must recognise the enormous difficulty that will be encountered in trying to introduce a sufficient number of myoblasts at a sufficient number of sites in a large number of muscles in order to make the effects of the treatment clinically useful. Work will clearly be necessary on the question as to whether intra-arterial injection or some other route of administration may prove effective.
The final point in relation to Duchenne dystrophy which I wish to bring to your attention relates to embryo research. Identification of the gene and of its missing protein product has now led to 95-98% accuracy, through the application of DNA studies, of carrier detection in informative families. Such female carriers in the past could only be advised that if they fell pregnant, they should undergo amniocentesis at the fourteenth week and should then have an abortion if the fetus was shown to be male. Now, with the aid of chorionic cell biopsy at eight or nine weeks, it is possible not only to sex the unborn fetus but, using current molecular biological techniques, to demonstrate whether or not, if the fetus is male, the abnormal gene is present. Hence selective abortion of only affected males is now feasible in many such families. Of even greater importance is the fact that in vitro fertilisation and so-called pre-embryo biopsy is now making possible pre-implantation diagnosis. I do not, of course, need to remind this audience that were it not for the extensive animal research which has been carried out over the course of the last 20 and more years, in vitro fertilisation in human subjects would never have become possible. Indeed if the Human Fertilisation and Embryology Bill, which has now received the royal assent after its final passage through both Houses of Parliament, had not become law, allowing research on the human embryo up to 14 days after fertilisation, all research of vital importance to the infertile and to carriers of the gene responsible for Duchenne dystrophy and many other crippling inherited disorders would have been prevented by law.
May I remind you that when the female ovum, released into the uterus at the time of ovulation, is fertilised by a sperm, the process of cell division begins and within the first few days floating free in the uterus are groups of undifferentiated but pluripotential cells, each forming what I prefer to call a conceptus or a pre-embryo rather than an embryo. The term 'pluripotential' means, of course, that it is impossible to identify which cells will form the membranes within which the fetus will eventually lie and which will later form an identifiable embryo from which a fetus will form. By about the fourth or fifth day, the conceptus becomes a blastocyst in which there is a nodule or cluster of cells called the inner cell mass from which the embryo later derives, and also an outer ring of cells capable of forming the membranes and the placenta. But no such blastocyst is yet attached to, or embedded in the wall of the uterus and about 80% of those formed are spontaneously aborted. About one in five begins to attach to the uterine wall at about the seventh day, subsequently receiving a blood supply and nourishment from the maternal circulation, and later, at about the fourteenth day, that specific linear arrangement of cells within the basal cell mass which constitutes the primitive streak appears.
Work done in the last year or two by Professor Robert Winston at Hammersmith and by others has clearly demonstrated that it is now feasible, without damage to the subsequent development of the embryo, to carry out biopsy by removal of a single cell from the blastocyst at about the fourth or fifth day; that single cell is removed from the part of the blastocyst which will ultimately form the membranes and the placenta. Sexing of the conceptus is now being commonly performed and I understand that within the last few months Professor Winston and his colleagues have not only found it possible to identify in such single cells the sex of the conceptus but are also close to being able to determine whether or not the dystrophic gene is present . This would make it feasible for such carrier women to have normal sons and non-carrier daughters, a prospect undreamed of a few short years ago.
I fully understand, of course, and appreciate the sincerity of those who believe that human life begins at conception and that any experiment on what they regard as a human life, or what can become one, is to them abhorrent. I am, however, personally satisfied, as are many eminent theologians including the Archbishop of York, Lord Soper, the Rev. Prof. Gordon Dunstan and the Rev. Dr Norman Ford, a distinguished Roman Catholic theologian , that individuation of the human embryo does not begin until the primitive streak appears at the fourteenth day. I therefore am in no doubt that Parliament was right to accept the Human Fertilisation and Embryology Bill which passed both Houses with very large majorities. The work it is making possible will, I believe, bring inestimable benefits to human health, not least to the families of patients with Duchenne dystrophy and to those in which many other serious crippling inherited diseases are present.
Mr Chairman, Ladies and Gentlemen - I appreciate that unlike many of the others who have delivered the Stephen Paget Lecture in previous years, I have concentrated in my discourse today particularly upon studies of the human animal, studies in which I have been personally involved. But 1 hope that that approach requires no apology, as I have also, I hope, paid due tribute to the contribution which research on animals has made to our understanding of human neuromuscular disease. Whenever it has been necessary to illumine my own work or to investigate mechanisms which it was not feasible to study in man, 1 have had no hesitation in having recourse to animal work, bearing in mind, of course, the very necessary constraints of humane management which it is essential to employ in all such experiments, just as is the case in volunteer human subjects. Much of the applied research to which I have referred has been made possible by fundamental observations in other members of the animal kingdom and it is a truism which I often quote that today's discovery of basic laboratory science brings tomorrow's practical development in patient care. In no field do I believe this to be more true than in research into neuromuscular disease. As I come to the end of my professional working lifetime, I cannot but acknowledge with gratitude the contribution made by many of the pioneers whose original observations, often derived from animal experimentation, have made possible those major developments in the care of patients with neuromuscular disease which are practical today.
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Last edited: 27 August 2014 05:49