J. med. Genet. (I965). 2, I.

Muscle Histology in Carriers of Duchenne MuscularDystrophy

ALAN E. H. EMERY*t

From the Division of Medical Genetics, Department of Medicine, Johns Hopkins University School of Medicine,Baltimore, Maryland, U.S.A.

There have been many studies with the lightmicroscope of histological changes in dystrophicmuscle. Recent reviews include those of Green-field, Shy, Alvord, and Berg (I957), Adams,Denny-Brown, and Pearson (i962), and Pearson(I963). Pearson (I963) has pointed out that themajority of investigations have been of moderatelyadvanced cases and that it is still not known inwhich portion of the muscle fibre the initialdystrophic process is manifest: sarcoplasm, sarco-lemmal sheath, contractile fibres, mitochondria, ornuclei. In order to investigate this problem,Pearson has studied preclinical cases of musculardystrophy where it would be expected that earlychanges would not be obscured by secondarychanges that occur later in the disease. Thoughstudies of preclinical cases have demonstratedthat definite pathological changes in the muscleare present even before the onset of clinical weak-ness, they have not been very helpful in determiningthe initial manifestation of the dystrophic process,for even in preclinical cases histological changesare fairly well advanced. In a 4-month-old infantwith preclinical muscular dystrophy, Pearson(I962) found widespread hyalinization of musclefibres and marked variation in fibre size.As an alternative to investigating preclinical

cases, it was considered that information con-cerning early structural changes in dystrophicmuscle might be obtained by studying musclebiopsies from carriers of muscular dystrophy:the rationale being that in carriers of musculardystrophy, according to the Lyon hypothesis

Received September 28, I964.* Supported by Grant No. G.M. IO, I89 of the U.S. PublicHealth Service and by a Grant from the Muscular DystrophyAssociation of America. The author was in receipt of a Dickin-son travelling fellowship of Manchester University, England.

t Present address: Division of Medical Genetics, Department ofMedicine, Manchester Royal Infirmary, Manchester I3,England.

concerning gene action in the X chromosome(Lyon, i96i, i962), all gradations in histologicalappearance from perfect normality to gross abnor-mality might be expected. The problem has beeninvestigated by recording subjective impressionsof apparent histological abnormalities and bymaking measurements of fibre size and the numberof central and peripheral (sarcolemmal) nuclei.

Another reason for studying muscle specimensfrom carriers was to determine whether there wasany correlation between histological appearancesand serum levels of creatine kinase, since thisenzyme has been found to be significantly raisedin a proportion of carriers of Duchenne musculardystrophy (Demos, Dreyfus, Schapira, and Schapira,I962; Hughes, I963; Pearce, Pennington, andWalton, I964; Pearson, Fowler, and Wright,I963; Richterich, Rosin, Aebi, and Rossi, I963;Sugita and Tyler, I963).

MethodA carrier of X-linked Duchenne muscular dystrophy

has been defined as a woman with at least two affectedsons or one affected son but a family history of othermale relatives having been similarly affected, i.e. abrother or maternal uncle

All the carriers were carefully examined for evidenceof any muscle weakness, and creatine kinase deter-minations were made on fresh specimens of serumby the method of Tanzer and Gilvarg (I959). Theresults are expressed in International units (InternationalUnion of Biochemistry, Report of the Commission onEnzymes, i96i), one unit being that amount of enzymewhich will catalyse the transformation of i jM creatine/minute/i,ooo ml. serum. In this laboratory the upperlimit for normal healthy women is I-5 units (Emeryand Pascasio, I965).The microscopic structure of gastrocnemius muscle

was studied in three healthy women with no history ofany neuromuscular disease and nine carriers ofDuchennemuscular dystrophy. The ages of the carriers rangedfrom 29 to 55 (mean 37.5) and the three controls were

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Alan E. H. Emery

30, 41, and 48 years old. All specimens were takenfrom the belly of the muscle and were obtained fromhealthy controls during the operation of varicose veinligation. Biopsy specimens were obtained from thecarriers under local anaesthesia, care being taken notto inject any of the anaesthetic into the part of themuscle to be excised. A block of tissue about 2 cm.long and I cm. wide, with the muscle fibres runningparallel to the long axis, was excised. In order tominimize the longitudinal shrinkage of muscle fibresthat occurs during fixation (Greenfield et al., I957,p. 97; Adams et al., i962, p. 710), the block of tissuewas placed on a thin piece of card and gently pressed.After about 5 minutes, when the tissue had becomewell adhered to the card, it was immersed in fixative.All the muscle specimens in this investigation werefixed in Formalin-Zenker's solution (5 parts of 40%Formalin to 95 parts of Zenker's fixative) for I5 toi8 hours. Transverse and longitudinal sections werecut at 6 j and stained with haematoxylin and eosin.All the slides were studied without any knowledge as tothe identity of the subject.

ResultsTwo carriers (Nos. i and 2 in the Table) who

were found to have muscle weakness have beenreported previously, and their status as carriershas been discussed (Emery, I963). Enlargedcalves were found not only in these two carrierswith muscle weakness but also in a third carrier(No. I2 in the Table) with no clinical evidenceof any muscle weakness. It may be that the en-larged calves in this woman represent normalhypertrophy. However, her serum creatine kinasewas I9 8 units which, in this series, is high forcarriers. Calf enlargement may therefore be amicrosymptom of muscle disease in carriers(Dubowitz, I963; Walton, I964).With regard to the histological findings, all the

controls and two of the carriers (No. 9 and No. I i)appeared to be perfectly normal. Sections fromcarriers No. IO and No. I3 were classified aspossibly abnormal because many of the fibres

TABLESUMMARY OF THE HISTOLOGICAL FINDINGS IN 9 CARRIERS OF DUCHENNE MUSCULAR DYSTROPHY

Histological Changes Fibre Size (,a)

Carrier Swollen Hyaline Fibre Size Increase in C.T. and Fat SerumGrade Fibres Change Variation Nuclei Necrosis Regeneration Infiltration Mean S.D.

9 0o - - 50 45 756 -5I I 0 - - _ =_ _ - 43.57 8-4I 0-553 I + - 52 20 1001 o-6IO I + - _ _ _ - 6384 I3-06o 2-I24 2 + + - - - - _ 4970 7-89 3.912 3 ± 1 + - --- 7I.37 137I 19-820 4 ±330.+ ± - - 8I30267I 2 2

I 5 t + + + - 72 69 2 i-8i 3-22 6 + + ± + + 78-03 i8 48 25 4

Degrees of abnormality have been arbitrarily classified from o (normal) to 6 (most abnormal).C.T. is connective tissue.

Sections were projected on to drawing paper at amagnification of x 300 and tracings made of musclefibres and all central and peripheral (sarcolemmal)nuclei were noted. Care was taken to choose onlythose parts of the slide where the muscle fibres hadbeen sectioned transversely and where there was nomarked shrinkage of fibres away from the endomysium.No attempt was made at this stage to determine ifthere were any structural abnormalities. In each caseioo fibres were traced and their diameters recorded.The slides were now restudied for evidence of

apparent structural abnorm.alities. They were dividedinto two groups: those that appeared to be perfectlynormal and those that did not. The slides in theabnormal group were then arranged according to thedegree of abnormality.

appeared to be swollen and, because of rounding-off, to have lost the normal polygonal outlineSwelling of muscle fibres was the only apparentabnormality in these two carriers. In carrier No. 24,not only did many of the fibres seem to be swollen,but several were stained deep red (eosinophilic)and cross-striations and the outline of myofibrilswere less clearly defined ('hyaline fibres'). Hyalinefibres were also seen in the sections of musclefrom the controls, but here they were very fewand always located on the periphery of sectionsas though due to fixation or staining artefact.However, in carrier No. 24 and the remainder ofthe carriers in whom hyaline fibres were considered

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Muscle Histology in Carriers of Duchenne Muscular Dystrophyf s <.~~~~~~~~~~~~'

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FIG. i. Transverse sections of gastrocnemius muscle. (a) Normal muscle (control). (b) Swelling of muscle fibres(carrier No. I2). (c) A vacuolated necrotic fibre, and phagocytosis of a necrotic fibre (carrier No. 2). (d) Regeneratingmuscle fibres (carrier No. 2). ( x 250).

to be an abnormal finding, these fibres were muchmore frequent and were situated within fasciculi.In some of the deeply staining eosinophilic fibres,the sarcoplasm appeared to have been convertedto floccular masses. In carrier No. I2 there appearedto be increased variation in fibre size (Fig. i).In carrier No. 20 swelling of muscle fibres, hya-linization and increased variation in fibre size werepresent as well as an apparent increase in thenumber of sarcolemmal nuclei. A few fibres werenecrotic. Necrotic fibres differed from normalfibres in that they stained grey-blue and not pink,there were no clearly defined myofibrils, andassociated nuclei were shrunken and pyknotic.This appearance of the nuclei is characteristic offibres undergoing necrosis (Pearce and Walton,1962). Some necrotic fibres were vacuolated and

others were being phagocytosed by histiocytes(Fig. i and 2). In carriers No. i and No. 2 somenecrotic fibres had been completely phagocytosed,leaving only the endomysial tube within whichregeneration of muscle fibres presumably occurs(Kitiyakara and Angevine, I960). In carrierNo. 20 no regenerating fibres were found and veryfew were present in sections from carriers No. iand No. 2. The fibres believed to be regeneratingwere rounded, smaller than adjacent fibres, baso-philic, and associated with vesicular nuclei withprominent nucleoli (Fig. i). These appearanceshave been accepted by others as evidence offibre regeneration (Pearce and Walton, I962;Walker and Drager, I962; Gilbert and Hawk,I963). The final stage was proliferation of con-nective tissue and fatty infiltration, but this was

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4 Alan E. H. Emery

seen only in carrier No. 2 and was not extensive.The histological findings in the nine carriershave been summarized in the Table.Measurements of fibre size and number of

nuclei are given in the Table and representedgraphically in Fig. 3. The mean fibre diameter

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defined, and cross striations are indistinct. The lower fibre appearsto be normal with well-defined myofibrils and distinct cross-striations. Between the two fibres are the remains of a necroticfibre which is undergoing phagocytosis. (x 25o.) (b) Higher powermagnification. (.x 500.)

these values, perhaps as a result of different fixationmethods. Greenfield et al. (I957) considered thatin the vastus lateralis, muscle fibres exceedingIOO [L in diameter were abnormal. They alsoconsidered abnormal more than 8 nuclei perfibre in cross-section or more than 3 internally

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FIG. 3. Histograms of fibre diameter (It) and number of peripheral(sarcolemmal) and central nuclei per fibre. Control (A), and carriers9 (B), II (C), 13 (D), Io (E), 24 (F), 12 (G), 20 (H), I (C), 2 (J).The carriers have been arranged, from above downwards in orderof increasing histological abnormalities.

for the three controls was 45.3 ,L (S.D. 9.5),49-8 ,U (S.D. 8-o), and 52-5 [± (S.D. io-6). Theseresults compare well with those obtained bySchwalbe and Mayenda (I8gO) of 47-5 V. and byFeinstein, Lindegard, Nyman, and Wohlfart (i955)of 54-I t± for the gastrocnemius muscle. Otherinvestigators have obtained values greater (62-5 ,.u,Halban, I894) and less than (3I6 ,u, Sissons, I963)

placed nuclei per IOO fibres. From the results ofthis study (Fig. 3) it seems that, in the gastrocnemiusmuscle, fibres exceeding 8o ,u in diameter or morethan IO nuclei per fibre are abnormal. Centralnuclei were found in as many as 6% of fibres inthe three controls.With the exception of carrier No. 24, where

sampling of muscle fibres may not have beencompletely random, the increase in fibre size in

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Muscle Histology in Carriers of Duchenne Muscular Dystrophy

carriers closely paralleled the degree of histologicalabnormality determined independently. Theresults of the measurements of fibre size confirmedthe impression that swelling of muscle fibres isapparently the earliest change, and that increasein sarcolemmal and central nuclei is found onlywhen structural changes are moderately advanced(Fig. 3).

Since there can be no certainty that the dataare normally distributed, Kendall's non-parametricrank method (Siegel, I956, p. 2I3) was used incalculating correlation coefficients. The correlationcoefficient between apparent structural changesand serum creatine kinase levels was + o69 andhighly significant (p = o-oo6). The correlationcoefficient between fibre size and serum creatinekinase levels was + o044, but not significant.As might have been expected, muscle biopsies

appeared most abnormal in the 2 carriers withmuscle weakness.

DiscussionThe results of the present investigation suggest

that swelling ofmuscle fibres is the earliest structuralchange in dystrophic muscle. This has also beensuggested as the earliest change by Pearce andWalton (I962) from the results of their extensivestudies of biopsies from affected individuals.They were unable to determine whether thisenlargement represented a genuine hypertrophyof fibres; attempts to demonstrate that theseenlarged fibres contained an increased number ofmyofibrils were unsuccessful for technical reasons.Pearson (I963) has criticized the views of thoseinvestigators who have considered hyalinizationof muscle fibres as merely fixation or stainingartefact. From his studies of preclinical cases hehas concluded that hyalinization probably repre-sents one of the earliest forms of fibre alteration.The results of the present investigation supportPearson's opinion. There were several reasons forconsidering such fibres abnormal in this study.First, when similar fibres were seen in normalmuscle they were invariably situated at the peri-phery of sections and always few in number,whereas in carriers they were situated withinfasciculi and were much more numerous. Secondly,their presence was associated with other histologicalabnormalities that were significantly correlatedwith the level of serum creatine kinase.

According to the Lyon hypothesis concerninggene action in the X chromosome (Lyon, I96I,I962), it might have been expected that in carriersof X-linked Duchenne muscular dystrophy eachmuscle fasciculus would be a mosaic of necrotic

and normal fibres, the proportion of necroticfibres reflecting the proportion of cells in which theactive X chromosome was the one bearing themuscular dystrophy gene. In each of two femalecarriers, Pearson et al. (I963) observed onlytwo populations of muscle fibres, one normaland the other dystrophic, and concluded thatthis was evidence of X chromosome mosaicism.In the present investigation two populations ofcells were not found. Instead structural changesappear to have progressed to different extents indifferent carriers and all gradations from apparentnormality to quite marked abnormality wereobserved. These findings seem more in accordwith modern views regarding the development ofthe multinucleate muscle fibre: in vitro experiments(Stockdale and Holtzer, I96I) and electron-microscope studies (Bergman, I962; Hay, i963)suggest that multinucleate muscle fibres arise byfusion of uninucleate myoblasts. A possibleexplanation for the histological findings couldtherefore be as follows: If the myotomes aremosaics of nuclei, in some of which the activeX chromosome is the one bearing the musculardystrophy gene whereas in others the active Xchromosome is the one bearing the normal gene,then fusion of uninucleate myoblasts derivedfrom the myotomes would result in multinucleatemuscle fibres which would also possess two differenttypes of nuclei in differing proportions. Theproportion of nuclei of the two different types inany muscle fibre would depend on the proportionof the two types in the myotome and by chancein the myoblasts which fuse together to form afibre. Perhaps the proportion of nuclei in whichthe active X chromosome is the one bearing themuscular dystrophy gene within any fibre deter-mines whether it will be normal, swollen, hyalinized,or even necrotic (Fig. 4).

Difficulties in attempting to demonstrate Xchromosome mosaicism in such conditions aschoroideraemia and ocular albinism (Beutler andBaluda, I964) might also be the result of morpho-genetic changes occurring during development intissues that originally exhibited mosaicism.Dubowitz (I963) recently reported his histo-

logical findings in muscle biopsy specimens fromfour carriers of Duchenne muscular dystrophy,but pointed out that one of the difficulties he hadin interpreting minimal pathological changes wasto know the limits of normality. Another importantfactor to be considered in such studies is thesomewhat subjective interpretation of musclehistology. By taking the precautions of usingcontrols and studying specimens without any

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Alan E. H. Emery

NORMA.

410 C. C

VARIOUS DEGREES OF

ABNORMAL!.r K

FIG. 4. Diagram illustrating a possible explanationlogical findings in carriers of Duchenne muscular d

knowledge as to the identity of the su

hoped that in this investigation thesehave been largely avoided.The results of the present investigati4

that histological studies of muscle biopsymay be a useful adjunct to serum e

detecting apparently healthy carriers ofmuscular dystrophy.

SummaryThe histology of gastrocnemius mus

specimens has been studied in 9

Duchenne muscular dystrophy andcontrols. The earliest structural chanto be swelling of the muscle fibres buttions from apparent normality in 2

quite marked abnormality were observedchanges correlated well with serum levelskinase. The observed structural changesinterpreted in terms of the Lyon hypcthe mode of development of multinucle

fibres. Histological studies of muscle biopsyspecimens would seem to be a useful adjunct to

Yt-!:!* the estimation of serum creatine kinase in detectingcarriers of Duchenne muscular dystrophy.

I should like to express my sincere gratitude toDr Victor A. McKusick for his encouragement andguidance throughout this investigation. My thanks

MYoBL ASV are also due to Mr E. P. Walker of the PathologyDepartment at Johns Hopkins Hospital for technicalassistance in the preparation of the material for histology.

REFERENCES

Adams, R. D., Denny-Brown, D., and Pearson, C. M. (i962).Diseases of Muscle-A Study in Pathology, 2nd ed. HoeberHarper, New York.

E' R,E5 Bergman, R. A. (5962). Observations on the morphogenesis of

rat skeletal muscle. Bull. Johns Hopk. Hosp., 0IO, I87.Beutler, E., and Baluda, M. C. (i964). The separation of glucose-6-

phosphate-dehydrogenase-deficient erythrocytes from the bloodof heterozygotes for glucose-6-phosphate-dehydrogenase de-ficiency. Lancet, I, 189.

Demos, J., Dreyfus, J. C., Schapira, F., and Schapira, G. (i962).Anomalies biologiques chez les transmetteurs apparemment sainsde la myopathie. Rev. canad. biol., 21, 587.

Dubowitz, V. (X963). Myopathic changes in muscular dystrophycarriers. Proc. roy. Soc. Med., 56, 8IO.

Emery, A. E. H. (1963). Clinical manifestations in two carriersof Duchenne muscular dystrophy. Lancet, I, 1126.

OF Mv i:L. -, and Pascasio, F. (1965). The effects of pregnancy on theconcentration of creatine kinase in serum, skeletal muscle andmyometrium. Amer. J. Obstet. Gynec., 9I, I8.

Feinstein, B., Lindegard, B., Nyman, E., and Wohlfart, G. (I955).Morphologic studies of motor units in normal human muscles.Acta anat. (Basel), 23, I27.

Gilbert, R. K., and Hawk, W. A. (I963). The incidence of necrosis

for the histo- of muscle fibres in Duchenne type muscular dystrophy. Amer.

Lystrophy. J. Path., 43, 107.Greenfield, J. G., Shy, G. M., Alvord, E. C., Jr., and Berg, L.

(1957). An Atlas of Muscle Pathology in Neuromuscular Diseases.Livingstone, Edinburgh.

Lbject, it is Halban, J. (I894). Die Dicke der quergestreiften Muskelfasernund ihre Bedeutung. Anat. Hefte, 3, 267.

difficulties Hay, E. D. (1963). The fine structure of differentiating muscle inthe salamander tail. Z. Zellforsch., 59, 6.

On indicate Hughes, (I963). enzyme special

to the Duchenne type dystrophy. In Research in Muscularspecimens Dystrophy (Muscular Dystrophy Group), p. I67. Pitman, London.

nzymes in International Union of Biochemistry (I96I). Report of the Com-mission on Enzymes. Pergamon Press, Oxford.

Duchenne Kitiyakara, A., and Angevine, D. M. (I960). Regeneration ofinjured and transplanted striated voluntary muscle in rats.Amer. 7. Path., 37, 613.

Lyon, M. F. (I96I). Gene action in the X-chromosome of themouse (Mus. musculus L.). Nature (Lond.), 9go, 372.

(1962). Sex chromatin and gene action in the mammalianX-chromosome. Amer. J. hum. Genet., 14, 135.

scle biopsy Pearce, G. W., and Walton, J. N. (I962). Progressive muscular

carriers of dystrophy: the histopathological changes in skeletal muscleobtained by biopsy. _. Path. Bact., 83, 535.

3 healthy Pearce, J. M. S., Pennington, R. J. T., and Walton, J. N. (x964)ge appears Serum enzyme studies in muscle disease. Part III Serum creatine

all grada- kinase activity in relatives of patients with the Duchenne typeof muscular dystrophy. Neurol. Neurosurg. Psychiat., 27, I8i.carriers to Pearson, C. M. (I962). Biochemical and histological features of

1, and these early muscular dystrophy. Rev. canad. Biol., 21, 533.(I963). Pathology of human muscular dystrophy. In Muscular

;of creatme Dystrophy in Man and Animals, ed. G. H. Bourne and M. N.have been Golarz, p. I. Karger, Basle and New York.

)thesis and -, Fowler, W. M., and Wright, S. W. (1963). X-chromosomemosaicism in females with muscular dystrophy. Proc. nat.

-ate muscle Acad. Sci. (Wash.), 50, 24.

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Richterich, R., Rosin, S., Aebi, U., and Rossi, E. (I963). Pro- Stockdale, F. E., and Holtzer, H. (I96I). DNA synthesis andgressive muscular dystrophy. V. The identification of the carrier myogenesis. Exp. Cell Res., 24, 508.state in the Duchenne type by serum creatine kinase determination. Sugita, H., and Tyler, F. H. (I963). Pathogenesis of muscularAmer. J. hum. Genet., 35, I33. dystrophy. Trans. Ass. Amer. Phycns, 76, 231.

Schwalbe, G., and Mayenda, R. (I890). Ueber die Kaliberver- Tanzer, M. L., and Gilvarg, C. (i959). Creatine and creatinehaltnisse der quergestreiften Muskelfasern des Menschen. kinase measurement. J. biol. CheM., 234, 3201.Z. Biol., 27, 482.

Siegel, S. (I956). Nonparametric Statistics for the Behavioral Walker, B. E., and Drager, G. A. (I962). Evidence of regenerationSciences. McGraw-Hill, New York. in repeat biopsies of dystrophic human muscle. Neurology

Sissons, H. A. (I963). Investigations of muscle fibre size. In (Minneap.), 12, 38I.Research in Muscular Dystrophy (Muscular Dystrophy Group), Walton, J. N. (I964). Muscular dystrophy: some recent advancesp. 89. Pitman, London. in knowledge. Brit. med. 7., I, 1344.

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