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University M icrofilm s International 300 N. ZEEB ROAD. ANN ARBOR. Ml 48106 18 BEDFORD ROW. LONDON WC1 R 4EJ. ENGLAND PICKARD, Nathan Abraham SYSTEMIC MEMBRANE DISEASE IN THE PROXIMAL MUSCULAR DYSTROPHIES. The Ohio State University, Ph.D., 1978

University Microfilms International 300 n . z e e b r o a d, a n n a r b o r, m i 4b i o6

© 1978

NATHAN ABRAHAM PICKARD

ALL RIGHTS RESERVED SYSTEMIC MEMBRANE DISEASE IN

THE PROXIMAL MUSCULAR DYSTROPHIES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By Nathan A. Pickard, B.S.

*****

The Ohio State University

1978

Reading Committee: Approved By

Dr. ITobushisha Bab a Dr. Gerald Brierley Dr. Hanns-Pieter Gruemer Dr. Richard Matthe-ws user Dr. Keith Richardson Departmer/|t of Physiological Chemistry ACKNOWLEDGEMENTS

I would like to express my appreciation to Dr. Gruemer, my adviser, for his guidance, support and encouragement during my graduate studies at Ohio State University and the

Medical College of Virginia.

Dr. Edward Isaacs' support has been especially valuable in providing insight into the neuromuscular diseases and patients throughout this study. The cooperation of the

Departments of Pathology and Neurology, the Medical College of Virginia made this study possible. I would also like to acknowledge the contribution of patient samples by Dr.

Harland Verrill and Dr. Edwin Myers and the support of Drs.

Grisham and Owens.

I can only begin to thank my mother for her encourage­ ment and support of my education. This dissertation is dedicated to her and my father.

This work was supported by N. I. H. Training Grant

GM-01805, an intramural grant from the Department of

Pathology, Medical College of Virginia and a grant from the

Muscular Dystrophy Association. VITA

May 5, 1951 Born - Chicago, Illinois

1969 Graduated from Lane Technical High School, Chicago, Illinois

1973 B.S., University of Illinois, Circle Campus, Chicago, Illinois

1975-1978 N. I- H. Trainee, Division of Clinical Chemistry, Department of Physiological Chemistry, Ohio State University, Columbus, Ohio

PUBLICATIONS

Verrill, H. L., Pickard, N. A., and Gruemer, H.-D. Studies into the Mechanism of Cellular Enzyme Release: I. Alteration in Membrane Fluidity and Permeability, Clin. Chem. 23_, 2226 (1977) .

Verrill, H. L., Pickard, N. A., and Gruemer, H.-D. Diminished Cap Formation in Lymphocytes from Patients and Carriers of Duchenne Muscular Dystroohy, Clin. Chem. J23, 2341 (1977) .

FIELD OF STUDY

Major Field: Clinical Chemistry TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... ii

VITA ...... iii

LIST OF TABLES...... vi

LIST OF FIGURES ...... vii

INTRODUCTION AND HISTORICAL SURVEY...... 1

OBJECTIVES...... 9

REFERENCES...... 11

PART I. CLINICAL EVALUATION OF LYMPHOCYTE CAPPING IN MUSCULAR DYSTROPHY PATIENTS AND CARRIERS . . 13

INTRODUCTION...... 13

MATERIALS AND METHODS ...... 20

Isolation of human lymphocytes ...... 20 Labeling of B-lymphocyte surface immuno­ globulins ...... 21 Labeling of T-lymphocyte Concanavalin A (Con A) rec ep t o r s ...... 22 Patient Selection...... 22

RESULTS ...... 24

DISCUSSION...... 41

REFERENCES...... 53

iv TABLE OF CONTENTS

(Cont.)

Page

PART II. INVESTIGATIONS INTO THE MECHANISM OF ABNORMAL CAP FORMATION IN MUSCULAR DYSTROPHY PATIENTS AND CARRIERS...... 58

INTRODUCTION ...... 58

MATERIALS AND METHODS...... 61

Capping Experiments ...... 61 Incubation modifications...... 61 Electron spin resonance ...... 62 Lipid analysis...... 63 Patient selection ...... 65

RESULTS...... 66

DISCUSSION...... 78

REFERNCES...... 86

v LIST OF TABLES

PART I

Table Page

1.1 ENZYME AND CAPPING PATTERN IN MOTHERS AND IN SONS PRESUMED "SPONTANEOUS MUTATIONS". . . . 27

PART II

Table Page

1 1 . 1 SUMMARY OF RESULTS IN LIMB-GIRDLE MUSCULAR DYSTROPHY PATIENTS ...... 69

11.2 ELECTRON SPIN RESONANCE RESULTS IN LYMPHO­ CYTES FROM NORMALS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS...... 72

11.3 BIOCHEMICAL ANALYSIS OF LYMPHOCYTE LIPIDS IN NORMALS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS ...... 74

11.4 EFFECT ON CAPPING OF COLCHICINE AND CYTO- CHALASIN B IN LYMPHOCYTES FROM HEALTHY INDI­ VIDUALS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS ...... 77 LIST OF FIGURES

Figure PART I Page

1.1 CLINICAL EVALUATION OF CAPPING - DISTRIBUTION OF CAPPING RESULTS IN B -LYMPHOCYTES...... 25

1.2 THE R. L. FAMILY - A PEDIGREE DEMONSTRATING THE INHERITANCE OF FACIOSCAPULOHUMERAL MUSCULAR D Y S T R O P H Y ...... 29

1.3 THE A. P. FAMILY - CAPPING RESULTS IN A FAMILY WITH A HISTORY OF DUCHENNE MUSCULAR DYSTROPHY...... 31

1.4 THE P. E. FAMILY - CAPPING RESULTS IN A FAMILY WITH A HISTORY OF BECKER'S MUSCULAR DYSTROPHY. 33

1.5 THE C. C. FAMILY - CAPPING RESULTS IN A FAMILY WITH LIMB-GIRDLE MUSCULAR DYSTROPHY...... 35

1.6 THE J. C. FAMILY - CAPPING RESULTS IN A FAMILY WITH CONGENITAL MUSCULAR DYSTROPHY ...... 37

1.7 DISTRIBUTION OF CONCANAVALIN A (CON A) RECEPTORS IN LYMPHOCYTES ISOLATED FROM CONTROLS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS . 40

1.8 THE H. P. FAMILY - LYONIZATION IN A HETEROZYGOTE FOR DUCHENNE MUSCULAR DYSTROPHY ...... 46 LIST OF FIGURES

Figure PART II Page

II. 1 NORMAL CAPPING WITH TIME AT 37°C...... 67

11.2 EFFECT OF INCUBATION TIME AT 37°C ON CAPPING ...... 68

11.3 TYPICAL ESR SPECTRUM WITH PARAMETERS USED IN SAMPLE EVALUATION INDICATED ...... 71

11.4 EFFECT ON CAPPING OF COLCHICINE AND CYTOCHALASIN B ON LYMPHOCYTES FROM NORMAL AND PATIENT SAMPLES ...... 76

viii INTRODUCTION AND HISTORICAL SURVEY

The muscular dystrophies are a group of genetically determined disorders -with progressive degeneration of skeletal muscle. The muscular dystrophies have been sub­ divided into various types on the basis of the clinical dis­ tribution, the severity of muscle weakness, and the pattern of inheritance (1,2) . Thus five major types of muscular dystrophy have been recognized: Duchenne's muscular dystro­ phy (pseudohypertrophic muscular dystrophy), Becker's dystrophy, limb-girdle muscular dystrophy, facioscapulo­ humeral dystrophy (Landouzy-Dejerine dystrophy), and con­ genital muscular dystrophy.

Three major theories have been proposed to explain the pathogenesis of muscular dystrophies; abnormal microvascular supply of muscle, abnormal neuronal influence on muscle, or a genetic membrane defect (3,4). The vascular and neuro­ genic theories, though still receiving support of some inves­ tigators, are generally no longer accepted today (3). Evi­ dence supporting a membrane defect in muscular dystrophy continues to expand. The identification of numerous bio­ chemical abnormalities, coupled with the knowledge that genetic diseases are ultimately due to the production of defective proteins or imbalances in the synthesis or 2

degradation of metabolic enzymes, strengthens the theory that implicates the muscle or muscle membrane. An inborn error of metabolism could result in the abnormal synthesis or degradation of carbohydrates or lipids and be expressed at the level of the cell membrane.

Increased permeability of muscle membrane is suggested by abnormal serum enzyme pattern in patients and carriers and the presence of fibrin by-products in sera of patients.

Lactate dehydrogenase (LDH; EC 1.1.1.27), creatine kinase

(CK; EC 2.7.3.2) and both aspartate aminotransferase and alanine aminotransferase (AST; EC 2.6.1.1 and ALT; EC

2.6.1.2) are released into the blood stream in increased amounts in patients -with Duchenne muscular dystrophy (5,6) and to a lesser degree in limb-girdle and facioscapulo­ humeral muscular dystrophy (7). Silverman has demonstrated that proportionally more MB isoenzyme of CK is found in serum than is present in muscle (8).

CK is found primarily in skeletal muscle, smooth muscle, thyroid tissue and brain. There is little or no CK activity in liver or red blood cells. CK activity deter­ minations have aided the differential diagnosis of neuro­ genic and myopathic processes even though CK activity occa­ sionally is increased in the presence of neurogenic muscle disease (9). 3

Muscle biopsy is advantageously utilized for confirming

the diagnosis of muscular dystrophy- A diagnosis on clini­

cal grounds alone is usually not sufficient, as many other

conditions can present themselves in a similar way. The

results obtained from a biopsy depend on the biopsy site; a

severely involved muscle site may show mainly fibrosis, and

the diagnosis will be obscured, while a muscle site that is not involved may show no or little abnormalities. It is best to choose a muscle which is mildly weak, so that the

typical changes of the disease can be best discerned (6,10).

Electromyography also aids diagnosis. Typically a myo­ pathy results in numerous low voltage, polyphasic potentials with a normal interference pattern and muscle action poten­ tials of reduced amplitude and duration. Nerve conduction

studies are normal (6).

The Duchenne type muscular dystrophy is characterized by (a) expression in the male and rarely in the female;

(b) transmission in a sex-linked recessive mode with an assumed high mutation rate (as high as 33%) ; (c) recogniz­ able muscle weakness during the first three years of life;

(d) symmetrical involvement first of pelvic girdle muscle and later the shoulder girdles; (e) pseudohypertrophy, par­ ticularly of the calf muscle; (f) steady and usually rapid progression without periods of apparent arrest, inability to 4

walk usually occurs by the age of ten years but sometimes much later; (g) frequent deterioration that increases after periods of bed rest; (h) progressive skeletal distortion through muscular contractures and atrophy; (i) death from inanition or respiratory infection usually in the second decade of life.

The limb-girdle form shows (a) expression in either male or female; (b) transmission usually as an autosomal recessive mode of inheritance; (c) onset anytime from first decade to middle age; (d) primary involvement of either the shoulder girdle or pelvic girdle muscles with frequent asym­ metrical wasting; (e) spread from the upper to lower limbs or vice versa, within twenty years of onset; (f) abortive cases are uncommon; (g) muscular pseudohypertrophy is uncommon; (h) variable rate of disease progression; (i) severe disability after 20 to 30 years of onset; (j) muscular contractures are not common;, (k) survival to middle age occurs.

The characteristic features of facioscapulohumeral dystrophy are; (a) expression in either sex; (b) autosomal dominant inheritance; (c) variable age of onset; (d) occurence of abortive cases; (e) involvement, sometimes asymmetrical, of facial and scapulohumeral muscles with spread within twenty to thirty years to the pelvic girdle; 5

(f) muscular pseudohypertrophy is very rare; (g) slow insi­

dious progression with periods of prolonged arrest of the

disease; (h) severe disability does not usually occur; (i)

most patients survive and remain active to a normal age.

Congenital muscular dystrophy is a specific form of muscular dystrophy where (a) the infant is "floppy" due to weakness and associated hypotonia at birth, (b) there are

contractures of various muscle groups (arthrogryposis), (c) on muscle biopsy a nonspecific myopathic pattern is observed

(d) there is a tendency towards improvement rather than deterioration with time and (e) muscle enzyme activities in

serum are elevated from birth (21).

Several diseases are clinically so similar to some

forms of muscular dystrophy as to present difficulties in the differential diagnosis. For example, polymositis is an inflammatory myopathy in which the muscle enzyme activities are frequently, but not invariably elevated in blood plasma as in patients with limb-girdle dystrophy. In polymyositis, the EMG shows a combination of spontaneous fibrillation potentials similar to those seen in denervation and poly- phasic, short duration potentials on voluntary contraction as in the myopathies, while the muscle biopsy shows the pre­ sence of an inflammatory response and the absence of hyper­

trophied fibers (11). Fortunately most polymyositis 6

patients respond to steroid therapy so that the differential diagnosis may be made on this basis, although recovery may be slow and incomplete.

The congenital myopathies (e.g. central core disease, nemaline myopathy, myotubular myopathy, congenital fiber type disproportion) may also present with pelvic girdle weakness. Enzyme activities and EMG results are often but not invariably normal. Therefore, muscle biopsies are help­ ful in differentiating the dystrophies from the congenital myopathies (12).

Anterior horn cell disease, such as that in spinal mus­ cular atrophy (Kugelberg-welander) can present itself in a similar fashion to Duchenne muscular dystrophy and other dystrophies. EMG and muscle biopsy aid the differential diagnosis, it is important to the diagnosis as early .as possible since prognosis and possible therapy vary widely among these similarly presenting neuromuscular diseases.

The myotonic syndromes are a group of diseases with an abnormal prolongation of induced or voluntary muscular con­ traction (13). Myotonia must be considered distinct from the dystrophies, as on biopsy the predominant feature is atrophy of muscle fibers and not dystrophy (14).

Myotonic atrophy is characterized by: (a) expression in males and females; (b) either autosomal dominant or 7

autosomal recessive inheritance; (c) variable age of onset;

(d) onset with myotonia and facial or peripheral weakness;

(e) simultaneous symmetrical wasting and weakness of forearm and of leg muscles which spread to proximal muscles; (f) mus­ cular hypertrophy occurs rarely; (g) cataract, testicular atrophy, frontal baldness and mental defect are common;

(h) the pituitary fossa is small and the skull vault thick;

(i) the disease is steadily progressive without periods of clinical arrest, leading to severe disability generally within 20 years; (j) death usually occurs in middle life.

In contrast to myotonia atrophy the following is observed in myotonic congenita: (a) onset usually at birth or shortly thereafter; (b) myotonia is the first symptom and is generalized throughout the skeletal musculature and is the sole cause of disability for many years; (c) generalized mus­ cular hypertrophy is common; (d) atrophic signs develop in many cases but often not for many years and, even then, they remain minimal; (e) the disease is generally compatible with normal survival without serious disability (15).

Initial interest in our laboratory concentrated on the application of the Parker and Mendell model for Duchenne dystrophy as a means of understanding enhanced enzyme release in disease (16). Silverman and Gruemer's demonstra­ tion of sarcolemmal membrane changes related to enzyme 8

release in the imipramine/serotonin animal model including cytoplasmic enzyme release/ diminished uptake of o(.-amino isobutyrate, and ribosuria supported the theory of a mem­ brane abnormality in the pathogenesis of Duchenne muscular dystrophy (8). Further investigation in this laboratory by

Verrill and co-workers on the effect of imipramine on sar- colemma-bound enzyme systems through inhibition studies showed a mixed type of inhibition which is consistent with an imipramine-induced interference at the enzyme sites and a disruption of lipid-protein interrelations. Such membrane conformational changes might contribute to the leakage of macromolecules such as enzymes from the cell interior (17).

Imipramine but not serotonin alone, caused release of enzymes from rat diaphragms and human lymphocytes and release of hemoglobin from erythrocytes in _in vitro experi­ ments. Imipramine also inhibited the capping phenomenon of human B-lymphocytes labeled with fluorescein-conjugated anti­ human immunoglobulins. Serotonin alone had no such effect, but administered together with imipramine potentiated the inhibition of capping by imipramine (18). This finding led to the observation of a diminished cap formation in the

B-lymphocytes of patients and carriers of DMD as compared to controls. OBJECTIVES

On the basis of earlier work, which indicated that in the animal model for Duchenne muscular dystrophy the defect was expressed at the level of the cell membrane and research by others identifying a number of defects in the structure and function of the erythrocyte membrane in both muscular dystrophy patients and carriers, it was of importance to evaluate and to test the validity of capping of human B- lymphocytes for the identification and differential diagnosis of the muscular dystrophies. Therefore, a study was under­ taken to study the membrane protein mobility in lymphocytes isolated from patients with Duchenne's, Becker's, limb-girdle, facioscapulohumeral, and congenital muscular dystrophy and patients with myotonia, polymyositis, Kugelberg-Welander's disease, and amyotrophic lateral sclerosis. The investiga­ tion was also extended to include cells isolated from carriers of the muscular dystrophies and to determine the sensitivity and specificity of the capping phenomenom as an indicator of carrier status for genetic counseling.

In the second part of this investigation studies were designed for the purpose of understanding the mechanism behind the defective capping ability of lymphocytes in

9 10

muscular dystrophy patients and carriers through investiga­ tions in membrane structure and function. First, the compo­ sition of the lipid portion of the cell membrane will be determined. Second, the integrity and functional ability of the cytoskeletal system will be determined through the study of drugs known to effect capping such as colchicine and cytochalasin B in muscular dystrophy patients and controls.

Third, electron spin resonance will be applied to investigate the fluidity of the bulk membrane lipids. REFERENCES

1. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach, in Major Problems in Neurology, Vol. 2, W. B. Saunders Company, Philadelphia, 1973, p. 1973.

2. Swaiman, K. F., Progressive muscular dystrophies, in The practice of Pediatric Neurology, K. F. Swaiman, Ed., C. V. Mosby Company, St. Louis, 1975, p. 990.

3. Roland, L. P., Pathogenesis of Muscular Dystrophies, Arch. Neurol. _33:315 (1976).

4. Appenzeller, o., and Ogin, G., Pathogenesis of Muscular Dystrophies, Arch. Neurol. _32:2 (1975).

5. Pennington, R. J. T., Biochemical aspects of muscle disease, in Disorders of Voluntary Muscle, H. N. Walton, Ed., Churchill Livingston, Edinburgh and London, 1974, p. 497.

6. Slater, G. E. and Swaiman, K. F., Muscular Dystrophies of Childhood, Pediatric Annals, _6;169 (1977).

7. Swaiman, K. F. and Sandler, B., The use of serum enzymes in the diagnosis of progressive muscular dystro­ phy, J. Pedia. _63:116 (1963).

8. Silverman, L. M. and Gruemer, H.-D, Sarcolemmal membrane changes related to enzyme release in the imipramine/ serotonin experimental animal model. Clin. Chem. 22:1710 (1976).

9. Dawson, D. M., and Fine, I. H., Creatine Kinase in human tissue, Arch, Neurol_1§*175 (1967).

10. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach, in Major problems in Neurology, Vol. 2, W. B. Saunders Company, Philadelphia, 1973, 1. 5-17.

11. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach in Major Problems in Neurology, Vol. 2, W. B. Saunders Company, Philadelphia, 1973, p. 316.

11 12

12. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach, in Major problems in Neurology, Vol. 2, w. Br. Saunders Company, Philadelphia, 1973, p. 253.

13. Walton, J. N. and Nattras, F. J. , On the Classification, Natural History and Treatment of the Myopathies, Brain 22:592 (1954).

14. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach, in Major Problems in Neurology, Vol. 2, W. B. Saunders Company, Philadelphia, 1973, p. 217.

15. Walton, J. N. and Nattras, F. J. , On the Classification, Natural History and Treatment of the Myopathies, Brain 77:12 (1954).

16. Parker, J. M. and Mendell, J. R., proximal Myopathy induced by 5-HT-Imipramine simulates Duchenne Dystrophy, Nature 247:103 (1974).

17. Verrill, H. L., Gruemer, H.-D and Baba, N., Mechanisms of Cellular Enzyme Release. II. Inhibition of Sarco- lemmal Enzymes by Myopathy-Inducing Agents, Clin. Chem. _23:2226 (1977).

18. Verrill, H. L., Pickard, N. A. and Gruemer, H.-D., Mechanisms of Cellular Enzyme Release. I.Alteration in Membrane Fluidity and Permeability, Clin. Chem., 23; 2219, (1977).

19. Roses, A. D. and Appel, S. H . , Erythrocyte Spectrin Peak II phosphorylation in Duchenne Muscular Dystrophy, J. of the Neurol. Sci. ^9:185 (1976).

20. Matheson, D. W. and Howland, J. L., Erythrocyte Deforma­ tion in Human Muscular Dystrophy, Sci. 184;165 (1973).

21. Dubowitz, V. and Brooke, M., Muscle Biopsy: A Modern Approach, in Major Problems in Neurology, Vol. 2, W. B. Saunders Company, Philadelphia, 1973, p. 251. PART I

CLINICAL EVALUATION OF LYMPHOCYTE CAPPING IN MUSCULAR DYSTROPHY PATIENTS AND CARRIERS Introduction

Increased serum enzyme activities are the biochemical

hallmark for the diagnosis of muscular dystrophy and for the

detection of carriers of X-linked Duchenne muscular dystro­

phy. Some investigators have postulated that muscle mem­ brane permeability changes are responsible for the abnormal

release of the cytoplasmic enzymes creatine kinase

(E.C. 2.7.3.2.), aldolase (E.C. 4.1.2.13.), lactate dehydro­

genase (E.C. 1.1.1.27.), and aspartate aminotransferase I (E.C. 2.6.1.1.) from the cell interior into serum (1,2,3).

There is an increasing amount of evidence that Duchenne muscular dystrophy is characterized by a generalized

systemic membrane defect not limited to muscle tissue (5-15).

The structure and function of erythrocyte membranes in

Duchenne muscular dystrophy patients and, in some cases,

Duchenne muscular dystrophy carriers have been the subjects of intensive studies. The observed biochemical abnormalities

appear to be secondary to the basic defect.

A substrate specific endogenous protein kinase altera­

tion has been reported. The protein kinase in the red cell

13 14

membrane is phosphorylated to a greater degree in patients

and carriers than in normals. The fact that the Duchenne

carriers showed the same abnormality as the affected

children, yet had no clinical abnormalities, suggests that

one gene dose may be sufficient to reveal alterations in the

protein kinase activity but may not be sufficient to produce

clinical dystrophy (5,6).

Adenosine triphosphatase (ATPase) activity of red cell

membranes in Duchenne muscular dystrophy patients was inhi­

bited by ouabain to a lesser degree than in normal indivi­

duals in assay systems continaing high or low salt content.

Epinephrine and cyclic adenosine monophosphate increased

total ATPase activity in all samples, and epinephrine

restored ouabain sensitivity in previously insensitive

Duchenne muscular dystrophy red cell membranes (7,8).

Another membrane phenomenon which has been demonstrated

is altered ion transport. Here the results showed a signi­

ficantly greater potassium influx in both patients and

carriers of Duchenne muscular dystrophy, relative to control, while ouabain inhibited the potassium influx and the sodium

efflux in the erythrocytes of all three groups, indicating an

imbalance between the active pump and passive leak ion move­ ments in Duchenne muscular dystrophy (9,10). 15

Electron spin resonance studies of erythrocyte membranes in patients with muscular dystrophy point to increased erythrocyte membrane fluidity (11). Electrophoretic mobility measurements made of red cells from patients with Duchenne dystrophy and myotonic atrophy showed altered mobilities as compared to controls (12) . Erythrocytes from patients with congenital muscular dystrophy (Duchenne, limb-girdle and facioscapulohumeral) exhibited dramatic surface deformation when observed with a scanning electron microscope. A similar alteration, but one affecting a smaller proportion of cells occurs in the case of the female carriers of Duchenne mus­ cular dystrophy (13,14). other studies of red cell membranes from Duchenne muscular dystrophy patients have revealed increased fragility (reduced deformability), as measured by a negative pressure required to aspirate an erythrocyte into a micropipette with an internal diameter of 1 - 2 pSA (15) .

Erythrocyte membranes from Duchenne muscular dystrophy carriers and patients were shown to have a diminished amount of palmitoleic acid when compared to normal subjects.

Patients afflicted with this disorder show a similar, but more variable, diminution of palmitoleic acid. The change in fatty acid composition appeared related to a low membrane triglyceride content (16). A recent study identified the presence of increased amounts of serum hemopexin, a B]_ 16

globulin and the elevation was attributed to an alteration in the red blood cell membrane resulting in an abnormal release of haemin into the blood inducing an increase in hemopexin concentration (17) .

Abnormalities in muscle biochemistry include a marked increase in the capability for polyribosomal collagen forma­ tion (18) and an _in vivo increase in polyribosomal radio­ active amino acid incorporation (19). The oxidation of palmitate by muscle mitochondria is markedly reduced in

Duchenne muscular dystrophy patients and carriers (20)-

Freeze fracture studies of skeletal muscle from patients with Duchenne muscular dystrophy showed nonuniform distribu­ tion and depletion of intramembranous particles on both protoplasmic and extracellular faces of the muscle plasma membrane, indicating a muscle surface membrane abnormality in Duchenne muscular dystrophy, and that the alterations are present in the internal molecular architecture of the mem­ brane (21). Calcium uptake and adenosine triphosphatase

(ATPase) activity studies performed on fragmented sarco­ plasmic reticulum of Duchenne muscular dystrophy patients demonstrated decreased initial and total calcium uptake (4).

In x-linked recessive Duchenne muscular dystrophy, 50% of the carriers' daughters will be carriers and 5 0 % of her sons will be affected. 17

The incidence of Duchenne muscular dystrophy is one in

3,000 to 8,000 male births (22). In the absence of any effective therapy, genetic counseling remains a major tech­ nique in preventing the disease occurrence. Serum CK as well as muscle biopsy and EMG are the most helpful diagnostic tests. In at least 20% of known carriers, the CK values are however, normal. A suspected carrier with a normal CK pre­ sents an important problem in genetic counseling (23) . The situation becomes even more complicated due to the poorly defined normal limits for total CK (24).

Recently, two methods have been advocated as being at least as sensitive as CK activity measurements for carrier detection. The first, measurement of endogenous phosphory­ lation of peak II of the erythrocyte membrane, although having the advantage of being noninvasive, is time consuming and not readily applicable to the clinical laboratory (25).

The second, lactate dehydrogenase isoenzyme-5 determinations identified several mothers who had normal creatine kinase levels. In this study by Roses et al., the combination of creatine kinase and lactate dehydrogenase isoenzyme-5 deter­ minations identified 28 of 30 mothers as probable heterozy­ gotes (26,27). Both of these new studies suggest that cases of Duchenne muscular dystrophy previously considered to be 18

new (spontaneous) mutations are much less common than esti­ mated (28,29).

The conformational changes observed in muscle fibers and erythrocytes described earlier are, in all likelihood, secondary responses to an earlier, more common defect in all membranes and are likely to express themselves in altered membrane fluidity. The fluid mosaic model of membranes is at this time the generally accepted theory of membrane structure

(30). Membrane fluidity and conformational changes in mem­ brane proteins may be assessed by electron spin resonance

(ESR) (11). Additionally, the binding of specific membrane receptors with fluorescein-labeled ligands and subsequent redistribution of the receptor-ligand complex (capping phenomenon) may be used to study the mobility of integral membrane proteins in the plane of the cell membrane (31,32).

Decreased membrane protein mobility was observed by the capping technique in 10 patients and 8 carriers of Duchenne muscular dystrophy (33). The general availability of fluo­ rescent microscopes and the relative ease with which B- lymphocyte studies may be carried out, led us to investigate capping phenomenon for the clinical, differential diagnostic and biochemical investigation of the various forms of muscu­ lar dystrophy. Refinements in the recognition of conforma­ tional membrane changes should be useful in overcoming 19

& frequently encountered difficulties in carrier detection for genetic counseling. MATERIALS AND METHODS

Isolation of Human Lymphocytes (31)

Fifteen ml of heparinized blood were collected and the plasma separated by centrifugation for 10 minutes at 450 x g for later biochemical analysis. The cells were resuspended in 23 ml of Seligmann's balanced salt solution (SBSS, Grand

Island Biological Company, Grand island, NY 14072) and transferred into a 50 ml polycarbonate tube. Ten ml of a

Ficoll-Paque ® solution (Pharmacia Fine Chemicals,

Piscataway, NJ 08854) was layered beneath the cell suspension with a syringe fitted with a small bore tube. centrifugation of the samples (450 x g, 25 minutes at 25°C) resulted in four layers -erythrocytes, Ficoll-Paque, lymphocytes and SBSS.

The lymphocyte layer was removed and placed into a 50 ml polycarbonate tube. The cells were washed with SBSS, centri­ fuged, resuspended and any erythrocytes lysed by adding 9 ml of distilled water followed by the addition of 2.9 ml of

0.6 mol/liter NaCl within 10 seconds. The cells were washed twice in SBSS (250 x g, 10 minutes) and then resuspended in

5 ml of SBSS and counted in a Coulter Model F counter. This suspension of B and T lymphocytes constituted the starting material for the B and T cell labeling procedure. 21

Labeling- of B-Lymphocyte Surface Immunoglobulins (Slg)

Approximately 4 x 10^ lymphocytes were mixed with 0.1 ml of a 10 fold dilution of fluorescein labeled polyvalent anti­ human immunoglobulin (Meloy, Springfield, VA 22151) and incu­ bated in the dark at 4°C for 30 to 60 minutes. Dilution of the conjugated antiserum with 0.15 moles/liter NaCl was per­ formed immediately prior to use. The optimum dilution was determined by a comparison of serial dilutions of the anti­ serum against the percentage of positively labeled cells.

The largest dilution yielding maximum labeling was used. Low temperature labeling was utilized to inhibit mobility of the membrane proteins. The cells were centrifuged (200 x g, 10 minutes at 4°C), washed three times in ice cold SBSS and resuspended in a few drop^ of cold SBSS. The labeled cells were then removed from the low temperature environment and placed in a 37°C water bath for 30 minutes. A wet mount slide was prepared and observed under visible and fluorescent illumination at 1250x, utilizing a standard Zeiss microscope fitted with a VI Fl Epifluorescent condenser. A minimum of

50 labeled cells were classified in the following categories: uniform, clustered, patched or capped distribution. Only those cells that reach the capping state were reported in percent of total number of labeled cells because their dis­ tinct pattern, makes a misidentification very unlikely. The 22

microscopist was, in most cases, not aware of the identity of patient or control samples at the time of counting.

Labeling of T-Lvmphocyte Concanavalin A (Con A) Receptors(32)

Approximately 4 x 10^ lymphocytes were incubated with fluorescein labeled Con A (F-Con A, 100 jag/ml, Miles Labora­ tory, Inc., Research Division, Elkhart, IN 46514) in the dark at 4°C on a rotator for 15 minutes. The cells were centri­ fuged (700 x g, 3 minutes, 4°C), washed twice in cold SBSS and resuspended in 1 ml of SBSS. The cells were then incu­ bated at 37°C for 30 minutes, a wet mount slide prepared, and a minimum of 100 labeled cells classified into the following categories: uniform or capped. Only single cells and those in very small clumps (2 to 4 cells) were counted for percentage of caps.

Patient Selection

Only patients who in our judgement had an unequivocal diagnosis were selected for this study. The diagnoses were based on personal and family history, a neurological examina­ tion, serum enzyme determinations, an electromyography with nerve conduction studies, and a muscle biopsy with fresh frozen sections stained for hematoxalin and eosin, Gomori trichrome, NADH tetrazolium reductase, and acid (pH 4.5) and alkaline, (pH 10.0) ATPase. In cases with a positive family 23

history, some of the not affected members also underwent a neurological examination and, in some instances, an electro­ myography and a muscle biopsy as well, on this basis, patients were diagnosed as having one of the following neuromuscular disorders: Duchenne's muscular dystrophy,

Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, congenital muscu­ lar dystrophy, myotonic atrophy, Kugelberg-Welander1s disease, amyotrophic lateral sclerosis and polymyositis. RESULTS

The clinical evaluation of capping as a test for the

diagnosis of the muscular dystrophies has been based on a

study of 63 patients afflicted with Duchenne muscular dystro­

phy, Becker muscular dystrophy, limb-girdle muscular dystro­

phy, facioscapulohumeral muscular dystrophy and congenital

muscular dystrophy. All of these patients demonstrated without exception a quantitative reduction in the number of

fluorescent-labeled cells that reached the capping stage, while showing no change in the percentage of cells that were

labeled, that is they had normal B-lymphocyte counts. The

reduction in the number of capped cells is shown in

Figure 1.1 and is compared with controls for whom a range of

48 to 68 percent capping was obtained. In patients with

Duchenne muscular dystrophy the range of cap formation was between 0 and 22 percent for 30 patients, representing 24

families, while seven patients in one family with Becker

type x-linked dystrophy the range was from 20 to 40 percent.

Seven patients with facioscapulohumeral dystrophy had cap

values between 4 and 14 percent. B-lymphocytes from 16

patients with limb-girdle muscular dystrophy in nine families

demonstrated a rather wide spread from 2 to 44 percent.

Unfortunately, the relatives of the afflicted individuals 24 25

DMD BMD 100 CONTROL 1 0 0 Potients Corners 1 0 0 fPcrt*ents Carriers 90 90 90 80 80 80 I 70 70 70 I 60 60 O0 60 Uo 50 50 50 ^ 40 40 40 30 30 30 20 20 20

ALS/ LGMD FMD MA K-W/PM 100 100 100 (00 90 90 90 90 80 80 80 80 70 70 70 70 60 60 60 60 50 50 50 50 40 40 40 40 30 30 30 30 20 20 20 20

□ = carriers "with normal capping, 0 = carriers with normal capping and evidence for lyonization, DMD = Duchenne muscular dystrophy, BMD = Becker muscular dystrophy, LGMD = limb- girdle muscular dystrophy, FMD = facioscapulohumeral muscular dystrophy, MA = myotonia, ALS = amyotrophic lateral sclerosis K-W = Kugelberg-Welander's disease, PM = polymyositis.

FIGURE 1.1. Distribution of capping results in B-lymphocytes 26

were not always available for laboratory studies to establish the mode of inheritance, whether autosomal recessive or dominant, as such knowledge may be helpful to explain the variability of expression at the membrane level. Normal capping was observed in six patients with myotonic atrophy

(MA), two patients with amyotrophic lateral sclerosis (ALS), five with polymyositis (PM) , and two individuals with

Kugelberg-Welander disease (K-W) (Figure 1.1).

Out of the twenty-four Duchenne muscular dystrophy families where information on the family history was avail­ able (and presumed accurate), sixteen unrelated kindreds were observed where a spontaneous mutation according to tradi­ tional criteria might have occured. The criteria used were that there was no family history of muscular dystrophy, the maternal serum enzyme activities were within normal limits and there was only one affected son. In nine of the sixteen kindreds, the capping of B-lymphocytes in the mothers was abnormally low and fell into the range typical for carriers and patients of Duchenne muscular dystrophy (Table 1.1, lines 1 - 9), while in the other seven mothers, capping was normal (Table 1.1, lines 10 — 16) . Two of these five mothers had two children each with low capping (Table i.l, lines 15 and 16) . TABLE 1.1 ENZYME AND CAPPING PATTERN IN MOTHERS AND IN SONS PRESUMED "SPONTANEOUS MUTATIONS"

Serum Enzymes in mother Percent of labeled cells capped

Family AST1. CK2, LD3 Mother Affected son Others

1. M.B. Normal 4 4 2. C.R. Normal 4 2

3. D.A. Normal 6 0 r 4. V.H. Normal 10 NT§ 5. L.R. Normal 2 NT 6 • L • A • Normal 6 0 7. J.A. Normal 12 NT3 8. L.C. Normal 16 NTc 9. M.H. Normal 6 NT5 10. D.K. Normal 60 18 11. S.M. Normal 54 12 12. A.S. Normal 58 8 13. R.K. Normal 60 NT5 14. C.D. LD HB4 60 10 14 mother

A 8 grandmother 15. H.P. LD HB 54 8 10 daughter 54 daughter 56 husband 16. R.A. Normal 52 18 12 daughter 54 husband

1) AST = Aspartate aminotransferase 3) LD = Lactate dehydrogenase 2) CK = Creatine kinase 4) HB = High borderline 5) NT = Not tested 28

The R. L. Family (Figure 1.2) demonstrates the useful­ ness of identifying afflicted heterozygotes for genetic counseling in facioscapulohumeral muscular dystrophy and the reliability of the capping procedure in this autosomal domi­ nant disease. As may be seen from the pedigree, non-afflicted members in the second generation had only healthy offsprings and showed normal capping, while those afflicted with the disorder had, as expected if mated with normal individuals, a 50% chance of having afflicted children who in all cases demonstrated low capping. Two sons in the second generation and one son in the third had normal serum AST, CK and LD activities but all three had low capping values.

Similarly, the capping test's effectiveness in the other forms of muscular dystrophy can be shown. The capping results for families with histories of Duchenne dystrophy

(Figure 1.3), Becker type dystrophy (Figure 1.4), limb-girdle

(Figure 1.5) and congenital muscular dystrophy (Figure 1.6) indicate that capping is a sensitive indicator of the carrier state, and is specific for the proximal muscular dystrophies.

In order to demonstrate that the decreased capping of

B-lymphocytes in muscular dystrophy patients was not due to any abnormality of either the surface immunoglobulins or B- lymphocytes, we have applied a technique that labels membrane glycoproteins and involves predominantly T-lymphocytes Figure 1.2. The proband was a 21-year-old male with a pro­ gressive weakness beginning with the shoulder girdle at age 13 and more recently involving the pelvic girdle and inability to exhibit facial expression which was noted prior to the age of 14. On examination there was marked weakness of the facial muscles, neck flexors, trapezius, scapula muscles, biceps and triceps and pelvic girdle muscles with noted sparing of the deltoids. There was no sensory loss, but deep tendon reflexes were absent. Exami­ nation of the patient's father showed a similar distribu­ tion of involvement except that the weakness was more severe. Laboratory findings: creatinine kinase 336 U/l (normal up to 195 U/l), EMG normal. Needle electrode studies showed short duration, very polyphasic, low ampli­ tude motor unit potentials. Only 4 percent of the patient's lymphocytes exhibited capping. In all affected individuals in this family, less than 14 percent of the lymphocytes demonstrated capping. The numbers in figure refer to age in years.

29 N = normal capping D=diminished capping

IT OrH O tB CM D t® Q ® Or 45 43 59 49 50 ur ® N N N N 10 20 17 22 29 21 13 29 6 23 12 15 17

FIGURE 1.2. Facioscapulohumeral muscular dystrophy The R.L. family Figure 1.3. Proband has been weak since age of 5. His shoulder girdles are somewhat atrophied, while his calves are hypertrophied and thighs atrophic. He can't touch his toes and arise or sit down and get up without climbing up his legs (Gower's sign). A maternal uncle had been afflicted with muscular dystrophy and a male sib also is effected. Serum enzymes activities were grossly elevated for CPK, with moderately elevated LDH, AST and ALT. Maternal muscle enzyme activities in serum were normal. No muscle biopsy was performed.

31 N = normal capping D = diminished capping

n 6 6 0 0 0

III

26 25 J 23 20 18 14 II

FIGURE 1.3. Duchenne muscular dystrophy The A.p. family co 10 Figure 1.4. Proband has had a life long problem of muscle ache and fatigue upon exertion. Earlier enzyme studies were moderately elevated. He has a son and a daughter, aged 7 and 4 respectively. His children are well. His parents are well, while one male sib is similarly effected. Five of his maternal uncles have muscular dystrophy. Two of his maternal female cousins previously have had abnormal muscle enzyme levels. One maternal aunt also has a son with muscular dystrophy. Upon examination the proband had normal upper limb reflexes while lower limbs showed evident wasting of the thigh muscles. In contrast, the calves are quite large. The patient has some degree of weakness in his quadriceps and hip flexors. A muscle biopsy showed sections of skeletal muscle which were markedly abnormal, with marked variability of muscle fibers within groups. The majority of fibers were large. The scattered small fibers were associated with proliferation of sarcolemma nuclei. Some histiocytic reaction and necrosis within fibers was apparent. Special stains for phosphatase and NADH showed the predominance of type 2 fibers.

33 N=normal capping D= diminished capping

60' 62|

67 4 0

[20 23 59

m

2mos

FIGURE 1.4. Becker muscular dystrophy The P.E. family u> Figure 1.5. Patient is a 45-year-old male with a three year history of progressive weakness and falling. There was no family history of a similar disorder. On examination he was noted to have mild weakness of the deltoid, biceps and tri­ ceps muscles and wrist extensors with sparing of the scapula and neck muscles. There was marked weakness of the ilio- soleus, glutenus maximus, quadriceps and tibialis anterior. CK is 1,800 U/l. Electromyography showed normal nerve con­ duction studies and low amplitude short duration, polyphasic motor unit potentials in the deltoid, biceps, quadriceps with more severe involvement of the tibialis anterior and quadriceps muscles. Biopsy demonstrated primarily myopathic changes with no evidence of inflammation, with numerous fibers showing regeneration. Histochemical analysis failed to demonstrate selective fiber type involvement of any fiber type grouping. B-lymphocytes capping was 18 percent. Two of his sons, ages 10 and 12, showed less than 22 percent capping; the older one having occasional stumbling and clumsiness but on examination appeared at least for the time being to be clinically normal. CK was 118 and 142 U/l respectively.

35 N = normal capping o D = diminished capping

n 0 0 t O t O 43 4 5------y ur II 20 20 17 19 25 21

FIGURE 1.5. Limb-girdle muscular dystrophy u> The C.C. family o\ Figure 1.6. The patient was a 14-month-old female noted to be weak and floppy since birth with apparent progression in weakness. On examination there was generalized weakness of all muscle groups. Deep tendon reflexes were absent. The patient’s 3-year-old sister had an identical history and similar results on examination. The CK was 1,737 U/l and B-lymphocyte capping 10 percent. An EMG showed normal nerve conduction and low amplitude, polyphasic, short duration motor unit potentials seen in all muscles. There was elec­ trical silence at rest with no evidence of denervation. The EMG performed in the younger sister was identical. Muscle biopsy showed severe, diffuse myopathic changes with a marked increase in endomysial, perimysial and epimysial fibrous tissue. Histochemical anaylsis demonstrated the presence of both fiber types, but there was a mild irregularity in the distribution of enzymatic activity within the fibers. The patient's mother had a serum CK of 24 U/l with only 6 per­ cent capping. She was clinically normal. The patient's father's lymphocytes showed 52 percent capping and he was also clinically normal.

37 N= normal capping N o D=diminished capping

FIGURE 1.6. Congenital muscular dystrophy The J.C. family 39

instead of B-lymphocytes. Using Concanavalin A as the ligand, a decrease of capping in twelve patients and ten carriers of Duchenne muscular dystrophy with a range of 2 to

14 percent was observed as compared to control values of 14 to 32 percent in fourteen health individuals (Figure 1.7).

The possibility existed that the decrease in capping seen in our patients could be due to a factor present only in dystrophic serum. To check this possibility, lymphocytes from healthy individuals which had previously shown normal capping were incubated in fresh dystrophic serum for one hour prior to labeling and then washed, labeled and then observed for capping. Under these conditions no reduction in capping by the normal lymphocytes was observed. 40

4 0

3 0 -

o z o o CL o o oo % 2 0 - o CJ oo

o

10- □ a

o DMD ■ DMD carriers a FMD a LGMD o controls

FIGURE 1.7. Distribution of Concanavalin A (Con A) - receptor capping results in controls and muscular dystrophy patients and carriers. DISCUSSION

The results of this study are consistent with previous suggestions of a systemic membrane defect in red blood cells and in muscle tissue of patients with Duchenne, limb-girdle, and facioscapulohumeral muscular dystrophy and extends the identification of the defect to include B and T-lymphocytes.

These forms of muscular dystrophy as well as congenital dystrophy and Becker dystrophy may then reflect a syndrome that, from a pathogenic point of view, could be designated as an inherited, systemic membrane disorder. It can now be con­ cluded, because all the dystrophies show the same functional abnormality in lymphocytes that the conformation change expressed in the membrane is similar even though the primary defect in these patients may be different.

From a clinical standpoint, the capping tests' ability to reveal primary membrane abnormalities in cells other than those undergoing degenerative changes makes this method use­ ful in distinguishing the muscular dystrophies from the neurogenic diseases which can present themselves clinically, biochemically, and histologically like muscular dystrophy.

Of greatest concern in differential diagnosis is the identi­ fication of patients with inflammatory myopathies such as polymyositis or dermatomyositis and patients with spinal

41 42

muscular atrophy such as Kugelberg-Welander disease. These

diseases often present with muscle weakness similar in dis­

tributions to those seen in the dystrophies and with abnor­

mally high serum creatine kinase activity. In fact the

differential diagnosis between limb-girdle muscular dystro­

phy and polymyositis is often based on the patient's respon­

siveness to steroid therapy. As can be seen from Figure 1.1,

patients with either polymyositis or Kugelberg-Welander have

normal capping and are easily differentiated from the dystro­

phies despite the aforementioned biochemical ambiguities.

Moreover, the capping test confirms that myotonia atrophica,

often called myotonia dystrophica is not a dystrophy since

all proximal muscular dystrophies appear to be characterized by primary membrane abnormalities. The capping test differentiates to only a limited extent among the various

forms of muscular dystrophy. Differentiation is aided if

additional family members are available to establish the mode of inheritance, where helpful, such as determining sex

linkage in Duchenne muscular dystrophy and Becker muscular dystrophy, or autosomal dominance or recessiveness in limb- girdle muscular dystrophy.

If the intermediate capping values observed in our one kindred with Becker muscular dystrophy is further substan­

tiated, capping will be helpful in the differential diagnosis 43

of a mild form of Duchenne dystrophy and Becker dystrophy,

where survival to middle age is common. The data here are

not meant to imply the capping tests ability to reflect on

the degree of severity of the cellular defect or of dystro­

phy itself. For example, patients with both facioscapulo­

humeral and limb-girdle dystrophy show capping between 0 and

20 percent as do the afflicted sons and nonafflicted carriers

in Duchenne muscular dystrophy. Moreover, there was no

apparent correlation between the severity of the disease and

capping within a particular form of dystrophy, one can state

that the diminished ability of a patients' lymphocytes to cap

is not a characteristic of the particular form of muscular

dystrophy but more importantly capping reflects only one

aspect of cellular activity that may be modified to varying

degree by an unknown number of possible genetic defects at or

near the membrane level and the cytoskeletal system and it must be assumed that there are factors that effect the

eventual expression of the original genetic defect.

In 1935, Haldane (28) estimated the rate of spontaneous mutation in hemophilia on the assumption that the number of

genes lost by individuals unable to reproduce must equal the

number of new mutations, if the defective gene pool is to

remain stable. Since Duchenne dystrophy is a genetic lethal

disease, one-third of the males afflicted with this disorder 44

were thought to be the result of new mutations, that is their mothers were not carriers. This view was supported by accounts of a stable gene pool in the general population

(33,36). Contradicting the assumption of a high proportion of new mutations in the afflicted male population are the recent results using methods such as lactate dehydrogenase isoenzyme-5 determinations (26), endogenous phosphorylation of peak II in red cell membranes (25) , and measurement of muscle protein synthesis (19) . The observation by Roses et al. (26) that there are more carriers of the Duchenne trait than the theory permits has not been reconciled with the principles of the Haldane theory. It may, however, be concluded from this debate that estimates of spontaneous mutation can only be as accurate as the means for detecting carriers, and as newer, more sensitive methods are developed, estimates of new mutations in the hemizygotic sons become lower than earlier thought, our experience tends to confirm these conclusions. The afflicted sons in Table 1.1 were spontaneous mutations according to traditional criteria; there was no previous history of muscular dystrophy in any of these families, each mother had only one afflicted son, and the mothers had normal serum enzyme activities. The mothers in lines 1 through 9 demonstrated a severe reduction in cap formation indicating that the mothers were indeed 45

carriers for the Duchenne trait. The last seven mothers of

Table 1.1, lines 10 through 16 on the other hand had normal capping and normal serum enzyme activities, findings that could have been interpreted as the result of spontaneous mutation were it not for the observations in the H. P., R. A. and C. D. families. The mother in the H. P. family, for instance, consistently demonstrated normal serum enzyme acti­ vities and normal cap formation while two of her children, the proband and one daughter had capping values below 10 per­ cent on two different occasions (Figure 1.8). That the capping test had not reached the limits of sensitivity in disclosing a membrane abnormality in the mother's somatic cells, is supported by the failure to observe intermediate capping values in our patient and carrier population of

Duchenne muscular dystrophy, that is the defect is either present or absent. Since the probability of two spontaneous mutations occurring in one family is remote, unequal somatic inactivation would seem to be the more likely explanation for these two mothers. Similarly, our observations in the

C. D. family (Table 1.1, line 14) of a mother who has normal serum enzyme activities and normal capping yet has a son who is afflicted and whose mother and grandmother have low capping support favorable Lyonization in the mother's somatic cells such that phenotypically she is normal yet Figure 1.8. The proband was 12-year-old boy in whom pro­ gressive muscle weakness was noticed at age 6 and who has been confined to a wheelchair for approximately two years. There was no other family history of muscle disease. He had diffuse muscle weakness with proximal pelvic and shoulder weakness and contractures of knees, hips and elbows. Creatine kinase was 815 U/l and an EMG showed low voltage, polyphasic motor units with no evidence of denervation. Nerve conduction studies were normal. Muscle biopsy showed severe myopathic changes with no histochemical abnormality. The B-lymphocyte capping was 16 percent in the patient's 20-year-old sister with a normal creatine kinase of 15 U/l. The patient's mother showed 58 percent capping with a creatine kinase of 90 U/l. The numbers in the figure refer to age in years.

46 N = normal capping D=diminished capping 42

n 20 16 / 12 10 16__ 7 Normal, not tested

FIGURE 1.8. Duchenne muscular dystrophy The H.P. family 48

genetically a carrier. Lyonization may also apply in the

daughter of the proband in our Becker muscular dystrophy

pedigree (Figure 1.4) . As the daughter of an afflicted

male, she is an obligate heterozygote (she must have inheri­

ted her father's defective X-chromosome), yet her enzyme

levels and lymphocyte capping are both normal. Lyonization

has been described in X-linked hemophilia A, where three

sisters who were obligatory carriers exhibited the three

phenotypes possible for heterozygous females; clinically

affected, clinically normal but phenotypically abnormal as

determined by laboratory tests, and clinically and pheno­

typically normal (37). Lyonization was also implicated in a

family carrying Christmas disease, a rare X-linked recessive

character seldom found in females. Here a case was reported

of a young girl who suffered from Christmas disease but whose monozygotic twin sister did not (38). The postulated high mutation rate has been questioned on the basis of the

occurrence of Lyonization in carriers of the X-linked Lesch-

Nyhan disease (39). An extreme phenotype resulting from

Lyonization in Duchenne dystrophy has been previously des­

cribed in one of female identical twins (44). The assumption of Lyonization may also apply to the other four mothers of

the afflicted children of Table 1.1, lines 10 through 13.

However, unless carriers or afflicted males can be located 49

in these kindreds the possibility of a mutation in the mothers or their ancestry cannot be ruled out. Thus, using lymphocyte capping to detect carriers, at most only 4 out of

30 probands appear to present new mutations. The apparent discrepancy between the predicted and observed proportion of new mutations can easily be reconciled if the mutation rate in males is assumed to be higher than that in females. This hypothesis could be tested by observations on the maternal grandmothers of affected probands. If the defective gene is passed from the grandfather to the mother, she would be likely to demonstrate defective capping and abnormal serum enzyme activities while the grandmother would not.

Two clinical and laboratory patterns may occur as a con­ sequence of mosaicism in carriers of Duchenne muscular dystro­ phy. One pattern with diminished capping and increased serum enzyme activities is the most typically seen with the defective X-chromosome being expressed in both somatocytes and oocytes. The mothers of the H. P., R. A. and C. D. families are assumed to belong to the second pattern, since they are phenotypically normal on the basis of presently available methodology, which can only infer carrier status.

Females belonging to this pattern are by definition carriers, yet have normal serum activities and normal capping of B-lymphocytes and are normal by any procedure 50

that evaluates functional responses of the somatic cells.

The absence of mass screening programs for Duchenne muscular dystrophy carriers and the relative insensitivity of existing methods for carrier detection have hindered widespread popu­ lation studies and pedigree analysis. For purpose of genetic counseling and contrary to present practice, all mothers of patients with Duchenne muscular dystrophy, even those with only one afflicted son should be considered con­ firmed carriers as should their female relatives or offspring who have defective capping ability or who have on three separate occasions abnormally high serum CK or LD-5 activity.

The occurrence of seven mothers with normal serum enzyme activities and normal capping in this study demonstrates that even phenotypically normal relatives of confirmed carriers can be at risk for giving birth to sons hemizygotic for

Duchenne dystrophy. The incidence of "false negatives" resulting from lyonization cannot be readily estimated from this population since this investigation selectively included cases presumed to be "spontaneous mutations". Refinements in prenatal diagnosis, as recently reported, requires methods with improved sensitivity and specificity at the membrane level for the evaluation of the pregnant woman's carrier status before an intrauterine procedure with all its risks is attempted (40-43). 51

The membrane abnormalities found in red cells of dystro­

phic patients and carriers, along with our results extending

the identification of a membrane defect to B- and T-lympho­

cytes, indicates that the defect is already present in the

stem cell. The identification of a stem cell defect and

reports of abnormalities in the muscle plasma membrane (3,4)

support the hypothesis of a systemic membrane disorder in

the muscular dystrophies.

In summary, the evaluation of lymphocyte capping,

through the use of fluorescent microscopy is a valuable aid

in the diagnosis of muscular dystrophy, the differentiation between polymyositis and limb-girdle muscular dystrophy, as well as between Kugelberg-Welander and Duchenne muscular dystrophy. Defective capping has proven to be a highly

sensitive indicator of carrier status and will be a valuable

tool in cases where the serum enzyme activities are normal or borderline. Capping has the advantage of being essentially noninvasive, capable of providing same day results, and of

sufficient ease to allow most hospitals to develop its use.

Prom a research standpoint, it will be of value to demo-

strate a multitude of intracellular and cell membrane abnor­ malities. The-identification of a membrane defect in lympho­

cytes from patients with proximal muscular dystrophy supports previous suggestions of a systemic membrane disease in 52

Duchenne's, limb-girdle and facioscapulohumeral dystrophy and extends it to include Becker's and congenital muscular dystrophy.

Our investigation of carriers for the Duchenne type of muscular dystrophy demonstrated that the occurrence of new mutations is much lower at the hemizygote level than earlier thought and, that in fact, mothers of afflicted boys should be assumed to be carriers. Accepting the limitation that our carrier population was biased by our selectively including mothers whose afflicted sons were presumed spontaneous mutations, by currently popular estimates of a high mutation rate (as high as 33%) we should have expected to observe sixteen afflicted sons born to mothers who were normal by all carrier detection methods. In fact, at most, we saw only four such individuals.

Also, the defective capping observed in the lympho­ cytes of patients with musculary dystrophy is the first description of its occurrence in cells other than in cancer cells (45,46). REFERENCES

1. Roses, A. D., Appel, S. H.: Muscular dystrophies. Lancet 1:1400 (1974).

2. Tyler, F. H.: Muscular dystrophies, the Metabolic Basis of Inherited Disease. Third edition. Edited by J. B. Stanbury, J. B. Syngarden, D. S. Fredrickson. New York, McGraw-Hill Book Company, 1972.

3. Mokri, B., Engel, A. G.: Duchenne dystrophy: Electron microscopic findings pointing to a basic or early abnor­ mality in the plasma membrane of the muscle fiber. Neurology 25:1111 (1975).

4. Samaha, F. J. and Gergly, J., Biochemical abnormalities of the sarcoplasmic reticulum in muscular dystrophy, N. Eng. J. Med. 280:184 (1969).

5. Roses, A. D., Herbstreith, M. H., and Appel, S. H . , Membrane protein kinase alteration in Duchenne muscular dystrophy, Nature 254:350 (197 5).

6. Roses, A. D. and Appel, S. H., Erythrocyte Spectrin Peak II Phosphorylation in Duchenne Muscular Dystrophy, J. of the Neurol. Sci. _29:185 (1976) .

7. Mawatari, S., Schonberg, M. and Olarte, M., Biochemical Abnormalities of Erythrocyte Membranes in Duchenne Dystrophy, Arch. Neurol. _33:489 (1976).

8. Araki, S. and Mawatari, S., Ouabain and Erythrocyte- Ghost Adenosine Triphosphatase, Arch. Neurol. 24:187 (1971).

9. Sha'afi, R. I., Rodan, S. B., Hintz, R. L., Fernandez, S. M. and Rodan, G. A., Abnormalities in membrane micro­ viscosity and ion transport in genetic muscular dystro­ phy, Nature 254;525 (1975).

10. Howland, J. L., Abnormal potassium conductance associated with genetic muscular dystrophy, Nature 251:724 (1974).

11. Butterfield, D. A., Electron spin resonance studies of erythrocyte membranes in muscular dystrophy, Arch, of Chem. Res. 10:111 (1977). 53 54

12. Bosmann, H. B., Gersten, D. M. Griggs, R. C., Howland, J. L., Hudecki, M. S., Katyare, S., and McLaughlin, J., Erythrocyte Surface Membrane Alterations, Arch Neurol. 23:135 (1976).

13. Matheson, D. W. and Howland, J. L., Erythrocyte Deforma­ tion in Human Muscular Dystrophy, Sci. 184;165 (1973).

14. Korczyn, A. D., Fishman, P., Djaldetti, M. and Berginer, V. M., Scanning Electron Microscope Study of Erythro­ cytes from Patients with Myopathy, Israel Journal of Medical Sciences, 1J:131 (1977).

15. Percy, A. K. and Miller, M. E., Reduced deformability of erythrocyte membranes from patients with Duchenne muscular dystrophy, Nature 258:147 (1975).

16. Howland, J. L. and- Iyer, S. L. Erythrocyte Lipids in Heterozygous Carriers of Duchenne Muscular Dystrophy, Sci. 19§:309 (1977).

17. Danieli, G. A. and Angelini, C., Duchenne Carrier Detec­ tion, Lancet 2^:90 (1976) .

18. Ionasescu, V., Distinction between Duchenne and other muscular dystrophies by ribosomal protein synthesis, J. Med. Genet. _12:49 (1975) .

19. Ionasescu, V., Zellweger, H., Shirk, P., Conway, T. W. , Identification of carriers of Duchenne muscular dystro­ phy by muscle protein synthesis. Neurol. 23t497 (1973).

20. Lin, C. H., Hudson, A. J. and Strickland, K. P., Fatty acid oxidation by skeletal muscle mitochondria in Duchenne muscular dystrophy, Life Sci. _11:355 (1 9 7 2 ).

21. Schotland, D. L., Bonilla, E., and Van Meter, M., Duchenne Dystrophy: Alteration in Muscle Plasma Membrane Structure, Sci. 196:1005 (1977).

22. Zellweger, H. and Antonik, A., Newborn screening for Duchenne muscular dystrophy. Pediatrics _5=>:30 (1975).

23. Emergy, A. E. H. and Holloway, S., Use of Normal Daugh­ ter's and Sister's Creatine Kinase Levels in Estimating Heterozygosity in Duchenne Muscular Dystrophy. Hum. Hered. 27:118 (1977). 55

24. Galen, R. S. and Gambino, S. R., What Upper Limit of Creatine Kinase Activity Defines Disease, Clin. Chem. 21:272 (1975).

25. Roses, A. D., Roses, M. J., Miller, S. E., Hull, K. L. and Appel, S. H., Carrier Detection in Duchenne Muscular Dystrophy, N. Eng. J. of Med. 294;193 (1976).

26. Roses, A. D., Roses, M. J., Nicholson, G. A. and Roe, C. R., Lactate dehydrogenase isoenzyme 5 in detecting carriers of Duchenne muscular dystrophy, Neurol. 27;414 (1977).

27. Roses, A. D., Nicholson, G. A. and Roe, C. R., Screening for Duchenne muscular dystrophy, Pediatrics 60:248 (1977).

28. Haldane, J. B. S., The rate of spontaneous mutation of a normal gene. J. Genet. _31;317 (1935).

29. Gardner-Medwin, D., Mutation rate in Duchenne type of muscular dystrophy. J. Med. Genet. J7:334 (1970).

30. Singer, S. J. and Nicholson, G. L., The Fluid Mosaic Model of the Structure of Cell Membranes, Sci. 175:720 (1972).

31. Verrill, H. L., Pickard, N. A., Gruemer, H.-D., Mechanism of cellular enzyme release. I. Alteration in membrane fluidity and permeability. Clin. Chem. 23:2219 (1977).

32. Ben-Basset, H. , pollick, A., Rosenbaum, S. M., Naparstek, E., Shouval, D. and inbar, M., Fluidity of Membrane Lipids and Lateral Mobility of Concanavalin A Receptors in the Cell Surface of Normal Lymphocytes and Lympho­ cytes from patients with Malignant Lymphomas and Leukemias. Can. Res. _37:1307 (1977).

33. Dubowitz, V., Carrier Detection and Genetic Counselling in Duchenne Dystrophy, Develop. Med. Child, Neurol 17: 352 (1975).

34. Gardner- Medwin, D., Mutation Rate in Duchenne Type of Muscular Dystrophy, J. Med. Genet. _7:334 (1970).

35. Zellweger, H. and Antonile, A., Newborn Screening for Duchenne Muscular Dystrophy, Pediat. _55:30 (1975). 56

36. Stephens, F. E. and Tyler, F. H., Studies in Disorders of Muscle, V. The inheritance of Childhood Progressive Muscular Dystrophy in 33 Kindreds. Amer. J. Hum. Genet. 1:111 (1951).

37. Graham, J. B., Miller, C. H., Reisner, H. M . , Elston, R. C. and Olive, J. A., The Phenotypic Range of Hemo­ philia A Carriers, Am. J. Hum. Genet. 18:482 (1976).

38. Revesz, T. , Schuler, D., Goldschmidt, B. and Elodi, S., Christmas Disease in One of a Pair of Monozygotic Twin Girls, Possibly the Effect of Lyonization. Am. J. Med. Genet. _9:396 (1972) .

39. Francke, U., Felsenstein, J., Gartler, S. M., Migeon, B. R., Dancis, J., Seegmiller, J. E., Bakay, F . and Nyhan, w. L., The Occurrence of New Mutants in the X-linked Recessive Lesch-Nyhan Disease. Am. J. Hum. Genet. 18:123 (1976).

40 Vassilopoulos, D., Emery, A. E., Muscle nuclear changes in fetuses at risk for Duchenne muscular dystrophy. J. Med. Genet. 14:13 (1977).

41. Wyatt, P. R., Cox, D. M.: Duchenne's Muscular Dystro­ phy, Studies in Cultured Fibroblasts. Lancet 1:172 (1977).

42. Mahoney, M. J., Haseltine, F. P., Hobbins, J. C., Banker, B. Q., Caskey, C. T. and Golbus, M. S., Prenatal Diagnosis of Duchenne1s muscular dystrophy. N. Eng. J. Med. 192:968 (1977).

43. Toop, J., and Emery, A. E., Muscle histology in fetuses at risk for Duchenne muscular dystrophy, Clin. Genet. _5:230 (1974).

44. Gomez, M. R., Engel, A. G., Dewald, G. and Peterson, H. H., Failure of inactivation of Duchenne dystrophy X- chromosome in one of female identical twins. Neurol. 12:537 (1977).

45. Cohen, H. J. and Gilbertson, B. B., Human Lymphocyte Surface Immunoglobulin Capping, Normal Characteristics and Anomalous Behavious of Chronic Lymphocyte Leukemia Lymphocytes, J. Clin. Invest. _55:84 (1975). 57

46. Ben-Basset, H., Polliack, A., Rosenbaum, S. M., Naparstek, E., Shouval, D. and Inbar, M., Fluidity of Membrane Lipids and Lateral Mobility of Concanavalin A Receptors in the Cell Surface of Normal Lymphocytes and Lymphocytes from Patients with Malignant Lymphomas and Leukemias. Can. Res. _37:1307 (1977).

t PART II. INVESTIGATION INTO THE MECHANISM OF DIMINISHED CAP FORMATION IN MUSCULAR DYSTROPHY PATIENTS AND CARRIERS Introduction

The mechanism of human lymphocyte capping is far from understood, though certain cellular and subcellular compo­ nents have been implicated in lymphocytes and fibroblasts

(1-4). In brief, capping permits one to observe the mobility of integral membrane proteins in the plane of the cell sur­ face. Normally, these proteins are found uniformly through­ out the plasma membrane and are anchored into place by microtubules (1). In the presence of certain drugs such as colchicine, vincristine or vinblastine, or at low temperature the microtubules depolymerize and, upon raising the tempera­ ture above 4°C, the proteins move from their dispersed or uniform state through various stages of aggregation (clus­ ters) to an asymmetric distribution of larger aggregates

(patches) into a state that appears under the microscope as a cap sitting on the trailing end of the cell (uropod). This same redistribution can be stimulated by the presence of polyvalent ligands (such as fluorescein labeled antiimmuno­ globulins) upon warming of the cells to 37°C. The role of

Ca++, of the membrane associated contractile elements, the microfilaments, and of the anchoring microtubules, in

58 59 pulling specific cell membrane proteins to one pole of the cell is under active investigation. It is not surprising, therefore, that a -wide number of factors can result in sur­ face Ig-receptor-antibody mobility changes including abnor­ malities in membrane conformation and composition and in the availability of energy in the form of ATP (5-11). Thus, measurement of membrane protein mobility appears to be a sensitive indicator of multitude of possible intracellular and associated cell surface changes.

Decreased lateral mobility of both Concanavalin A

(Con A) and surface immunoglobulin (Slg) receptors in lympho­ cytes from patients afflicted with chronic lymphocytic leukemia has recently been described (6,7). Cap formation in these cells could not be stimulated by supplementing the energy source or protein content. Using.a fluorescence polarization technique Shinitzky and Inbar observed in malignant lymphocytes an opposite correlation between the lipid fluidity and the mobility of Con A receptors? that is, the lower rotational mobility of the Con A receptors was associated with higher lipid fluidity (5). Lymphocytes isolated from chronic lymphocytic leukemia patients during remission stages of the diseases exhibited a higher cap forming ability than during the acute (progressive) phase.

The cap forming ability of cells from chronic lymphocytic leukemia patients was not affected by treatment with steroids. 60

The microviscosity of the lipid core of membranes is determined by three variables, the most important being the mole ratio of cholesterol to phospholipid, with the micro­

viscosity being directly proportional to the cholesterol/phos- pholipid ratio. The two other characteristics are the degree of unsaturation of the phospholipid acyl chains and

the relative amount of sphingomyelin in the membrane (5).

In an attempt to understand the mechanism behind the defective capping ability of lymphocytes from patients and

carriers of muscular dystrophy we have applied some tech­ niques that are commonly used for the evaluation of membrane

structure and function. Electron spin resonance will be used to evaluate the fluidity of the lipid portion of the cell membranes. The lipid composition, in particular, the

relative amounts of cholesterol and phospholipid and per­ centage composition of the different phospholipid fatty acids will be determined. The functional integrity of the microfilaments and microtubules will be studied by deter­ mining the effects of cytochalasin B and colchicine on

Con A-receptor capping. Colchicine has been shown to dis­ rupt the microtubules and cytochalasin B is thought to pre­ cipitate the microfilaments (8). MATERIALS AND METHODS

Capping Experiments

Only patients -who in our judgement had an unequivocal diagnosis were selected for these studies. The diagnoses were based on clinical, histological or electrophysiological evaluations. The collection of blood, isolation of lympho­ cytes, labeling of membrane proteins, fluorescent microscopy, and surface pattern classification were performed as pre­ viously described (7-13).

Incubation Modifications

Attempts to modify the distribution of the membrane receptor-ligand complexes in lymphocytes from controls and patients and carriers with muscular dystrophy were performed by washing the cells in Seligmann's balanced salt solution

(SBSS) containing the appropriate agent and performing the incubation procedure in the presence of the agent to be test­ ed. In each case, except that of cytochalasin B (Sigma

Chemical Co., St. Louis, Mo. 63178), the drug was dissolved directly in SBSS at the desired concentration. In this man­ ner the effects of colchicine (Eli Lilly and Co., Indianapolis

Ind.), at a final concentration of 10 pmoles/liter and pred­ nisolone phosphate (Merck, Sharp and Dohme) were tested.

61 62

Cytochalasin B was first dissolved in 100% ethanol

(ETOH) at a concentration of 833 mg/ml- A 22 fold dilution

was made with SBSS to give a final cytochalasin B concen­

tration of 38 mg/ml (79 pmoles/liter).

Electron spin resonance (ESR)

It has been shown that nitroxide spin label derivatives

of fatty acids and fatty acid methyl esters can be effective

probes of membrane structure and fluidity in a variety of

systems (14) . In this study the "12-doxyl1' derivative of

methyl stearate (12-doxyl methyl stearate =12-4, '4'-

dimethyl-oxazolidinyl-N-oxyl stearate) was used to examine

the fluidity of lymphocyte membrane isolated from normal

individuals and muscular dystrophy patients and carriers.

The 12-doxyl methyl stearate (12-DMS) was synthesized

according to the method of Waggoner et aJL. (15) and showed

one spot on thin layer chromatography.

In a typical experiment 2 ^il of a 10 mmoles/liter solu­

tion of 12-DMS in methanol was evaporated on the wall of a

small test tube. This concentration of label was shown to

result in sufficient label uptake into the cell membrane and decreased the likelihood of excessive label-label inter­

action. 100-200 jj.1 of a concentrated suspension of lympho­

cytes was added and the sample was agitated vigorously on a

i vortex mixer for 3-4 minutes. The sample was taken up in a 63

quartz aqueous sample cell and placed in the spectrometer

cavity at 22+l°C. The spectral data were obtained -with a

Varian E-109 EPR spectrometer (Varian Instrument Division,

Palo Alto, CA 94303) at a modulation amplitude of 1 gauss; and microwave power of 20-100 mW. The field was 25 gauss/ min., and a time constant of 1 second of 12.5 gauss/min. with a time constant of 2 seconds. The EPR spectrometer was interfaced to a DEC PDP 11 VO 3 computer. The spectra were stored on disk, and then replotted at constant center peak amplitude for comparison purposes.

For sample evaluation, the line widths (A H) and peak ratios of the spectra will be used (Figure II.3). The spectrum obtained represents a population of spin-labels oriented in a specific direction due to an applied magnetic field. Superimposed on the orientation of the molecules are the molecular motions. The molecules undergo rapid tumbling

(isotropic movement) and rotation around their long axis

(anisotropic movement) that tends to average out the orientations of the labels (14,20).

Lipid Analysis

Lymphocyte suspensions of approximately 10® cells iso­ lated from blood of normal individuals, muscular dystrophy patients and carriers were pelleted, then resuspended in

0.5 ml of 100 mmol/liter KCL (16,17). 3„ 16 ml of ice cold 64

redistilled methanol was added folio-wed by heneicosanoic

acid as fatty acid internal standard. Redistilled choloro-

form (6.33 ml) containing 0.5 mg/100 ml BHT (Butylated hydro-

xytoluene) was added and then subsequently sonicated for 30

seconds in ice to disrupt the cells. The solutions were

warmed for 5 minutes at 50^0. 2.0 ml of the solution of

KCl were added and then the solutions were centrifuged. The

lower phase was recovered and evaporated to dryness under

nitrogen and then the samples were redissolved in 0.5 ml

chloroform.

Phospholipid phosphorous was determined by Bartlett's

method after hydrolysis with perchloric acid for 2 hours at

170OC (18). The chloroform lipid extracts were separated by

one-dimensional thin layer chromatography (TLC) on silica

gel into phospholipid, triglyceride, cholesterol and chole­

sterol ester using petroleum ether: diethyl ether: glacial

acetic acid (80:20:1). The marker lipids were detected in

iodine vapor and the corresponding not exposed sample areas were removed and methylated using 0.7 ml boron trifluoride-

methanol. Methyl esters were recovered (17) and determined

on a Hewlett-Packard 5830A gas-liquid chromatograph using a

glass column packed with 10% EGSS-X on Gas chrom P at 170°C.

Cholesterol and cholesterol esters were eluted from TLC

scrapings with chloroform following the addition of 5 — 65

cholestane as internal standard. The chloroform was removed under a stream of nitrogen and the cholesterol esters saponi' fied in ethanol (0.5 ml ) and potassium hydroxide

(10 moles/liter, 0.05 ml ) for 2 hours at 50°C. After extraction into pentane, cholesterol was assayed by GLC on a column of 3% SE-30 on Gas-Chrom Q at 225°C. Values were determined by comparison with standard solutions containing cholestane and cholesterol.

Patient Selection

For _in vitro experiments no attempt was made to select patients with a particular form of muscular dystrophy, but rather as in initial investigation all forms of proximal muscular dystrophy were considered together, as long as low capping had been observed previously in the patient. RESULTS

Figure II.1 shows the progression towards capping of

normal lymphocytes with time. Most cells while kept below

4°C are in the uniform or clustered distributions, but within minutes upon warming to 37°C the ligand-receptor com­

plexes redistribute such that within 30 minutes at least 48%

of the cells are in the capped state and with continued

exposure at 37°C, there is no further increase in capping;

in fact, counting becomes more difficult to perform as endo-

cytosis and shedding result in fewer cells being labeled.

To determine whether the decreased capping seen in dystrophic lymphocytes was an "absolute" phenomenon or

simply a slowing of the normal capping process, lymphocytes

from patients and a carrier of muscular dystrophy were incu­ bated for an extended period of time. As can be seen in

Figure II.2 the same plateau is seen after 30 minutes at 37°C

as in normal lymphocytes and thus the low cap values in

lymphocytes from dystrophic patients do not approach the

values seen in normal individuals.

The great variability of the decreased capping ability of B-lymphocytes in limb-girdle muscular dystrophy patients

from values typical for a dystrophic individual to near nor­ mal capping is shown in Table II.1. Patients without a

family history of limb-girdle had intermediate- capping 66 Figure II.1- Normal capping with time at 37° C 37° at time with capping Normal II.1- Figure PERCENTAGE CELLS LABELLED 100 40 - 40 20 60 - 60 50 - 50 - 90 30 30 - 70 - 70 10 - -

-1 Uniform tm idctd inminutes) indicated (time ITIUIN F LABEL OF DISTRIBUTION Cluster M/60' 30 67 CapPatch FIGURE II.2. Effect of incubation time at 37° C on capping on C 37° at time incubation of Effect II.2. FIGURE

% % CAPPING 100 40 40 - 20 60 60 - 30 - 80 80 - 90 - 70- 50- 10 - - 0 0 0 0 0 0 70 60 50 40 30 20 10 MINUTES AT37*C DMO-C © - = « CONTROL « FMD.FMD DMD

68 TABLE II.1

Summary of Limb-Girdle Muscular Dystrophy

Capping Family Steroids Capping with steroid Patient History CK LDH in vivo Un/Cl/Pa/Ca in vitro

1 . HH 254 230 yes 0/34/22/44

- 599 265 no 4/22/34/4Q 4/6/38/52

2. DR - 286 328 no 0/56/24/20

3. YM — 2052 600 yes 0/52/12/36 6/34/28/32 4/20/36/40

4. AW + 1057 406 no 6/64/14/16 2/70/14/14

5. RH + 500 301 no 6/86/6/2 4/60/24/12 2/62/26/10

6. JB + 571 291 no 6/68/16/10 6/70/12/12

7. CC + 1875 395 no 4/60/18/18 0/66/18/16

8. MD + 5061 500 no 6/84/2/8 6/72/12/10 6/68/14/12

9. GL + 347 224 no 10/70/10/10 2/68/12/18 70

values while those with a family history all had values

below 20%. Since two patients without a family history were

on steroid (prednisolone) therapy, we undertook a study to

see whether steroids could effect capping of B-lymphocytes

in vivo and _in vitro. The lymphocytes were isolated as

earlier described, and labeling and incubating of the cells was done in the presence of prednisolone at concentrations

comparable to therapeutic levels in patients (1 mg/liter).

We found that we were able to increase capping slightly only

in the two patients with limb-girdle muscular dystrophy without a family history, while the others were not respon­

sive to prednisolone in vitro. Additionally, one of the

limb-girdle muscular dystrophy patients (H.H.) was taken off

drug therapy during the course of these experiments; after

termination of therapy no change in the patient's capping

results were seen.

Electron spin resonance

The parameters in the ESR spectrum that were used for

the comparison of the fluidity of lymphocyte membranes from

normal and dystrophic individuals, were the line width of

the low-field peak (A H) and the ratio of the center and

low-field peak amplitudes (B/A). These parameters are shown

for a typical spectrum in Figure II.3. The values of AH and

B/A for a series of normal and dystrophic lymphocyte samples FIGURE II.3. Typical ESR spectrum with parameters used in sample evaluation indicated x = 22.3 ± 2.8 1.73 + .16 + 1.73 2.8 22.3 ± x = Legend; DMD = Duchenne dystrophy patient dystrophy Duchenne = DMD Legend; ioo*^icr>ui4>UihO(-* 19.5 24.0 22.5 27.0 24.0 20.8 18.0 LGMD s*patient LGMD dystrophy Limb-girdle carrier dystrophy Duchenne = DMDC 2 FMD = Facioscapulohumeral dystrophy patient dystrophy Facioscapulohumeral = FMD ______. Controls ESR PARAMETERS IN LYMPHOCYTES ISOLATED FROM NORMALS, FROM ISOLATED LYMPHOCYTES IN PARAMETERS ESR B/A AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS AND PATIENTS DYSTROPHY MUSCULAR AND 1.63 1.85 2.06 1.72 1.64 1.55 1.70 1.68

TABLEII.2

11 10 12 4 2 X 8 7 6 5 3 1 9 = DMD M 301.74 23.0 DMD M 17.0 DMD DMD DMD MC20.5 LGMD DMDC GD19.0 FMD 18.0 LGMD LGMD DMDC DMDC 21.2 401.95 24.0 21.5 16.0 20.0 18.5 15.5 19.5 + 2.7 19.5+ ______H A ______Dystrophies B/A

_____ 1.94 1.33 1.68 1.65 1.66 1.49 1.85 1.71 1.72 1.22 1.66 73

are listed in Table II.2 along with the mean values for the two classes of cells. No significant or consistent differences were found between the two classes of cells as a group or individually by disease (P < .01).

Lipid Analysis

The lipid composition of lymphocytes from healthy indi­ viduals and patients and carriers was determined. Due to limitations in the amount of blood that can be obtained from human subjects, we were unable to obtain sufficient starting material to enable isolation of the lymphocyte membranes.

The results shown in Table II.3 are for the whole cell and therefore to only a limited extent reflect the composition of the lymphocyte membrane. There is no gross difference between controls and diseased individuals for any of the lipids analyzed. Results are expressed as the average number of moles per milliliter of lipid extract.

Cytoskeletal system

In order to assess the integrity and functional cap­ abilities of the microfilaments and microtubules lymphocytes isolated from ten healthy individuals and eleven muscular dystrophy patients and carriers were washed and incubated in the presence of colchicine or cytochalasin B. In normal T- lymphocytes, the exposure to colchicine significantly TABLE II.3 LYMPHOCYTE LIPIDS IN NORMALS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS

Fatty Acids (°L of total fatty acids) Cholesterol Phosphorous Triglyceride Arach- Controls (nmol/ml) (pmol/ml) (nmol/ml)_____ Palmitic Stearic Oleic Linoleic idonic

1 0.05 0.28 1.14 11.8 22.8 18.9 8.6 28.3 2 0.02 0.46 2.49 20.0 20.8 16.7 5.1 28.3 3 0.19 0.52 2.01 18.8 22.9 17.8 6.4 29.1 4 0.16 0.40 3.46 20.0 21.4 17.1 7.9 26.1 5 0.10 0.66 0.87 20.1 21.1 16.8 9.0 26.5 6 0.06 0.20 --- 20.7 21.5 17.1 10.3 23.1

X 0.098 0.419 2.0 18.6 21.8 17.4 7.9 26.9

Fatty Acids (7a of total fatty acids) Patients/ Cholesterol Phosphorous Triglyceride Arach- Carriers (pmol/ml) (^amol/ml) (nmol/ml) Palmitic Stearic Oleic Linoleic idonic

1 DMD 0.09 0.42 4.69 20.8 22.6 16.1 8.4 24.2 2 DMD 0.05 0.56 3.10 20 0 22.0 17.2 7.5 25.3 3 DMD 0.09 0.51 4.52 21.6 19.2 18.1 5.2 26.4 4 DMD 0.17 0.52 1.83 21.4 21.4 18.3 8.9 22.6 5 DMDC 0.16 0.38 1.42 21.5 21.7 17.8 8.2 23.2 6 LGMD 0.05 0.24 0.51 19.3 22.0 19.4 9.8 23.6 7 FMD 0.08 0.24 --- 26.8 19.7 16.5 7.4 21.9 8 FMD 0.05 0.30 1.19 21.4 21.5 17.7 8.0 24.4

X 0.093 0.396 2.5 21.6 21.3 17.6 7.9 24.0

4* 75

(P .001) increased capping from 23 + 5% to 35 + 8% (mean +

S.E.M.) labeled cells capped (Figure II.4 and Table II.4).

Cytochalasin B, which disrupts microfilaments caused a severe reduction in the ability of normal lymphocytes to undergo capping with only about 2 % of all labeled cells in the capped configuration. As had previously been shown, lymphocytes isolated from muscular dystrophy patients and carriers are also less able to redistribute their Con A receptors into caps with an average of 8% of all labeled cells capped. In contrast to what was seen in normal lympho­ cytes, no enhancement in the number of cells capped was observed upon exposure of lymphocytes from patients and carriers upon treatment with colchicine. Cytochalasin B further inhibited the already low capping seen in this group.

The bars in the figure indicate the range of results seen for each treatment. No difference was seen among the results obtained for the various types of muscular dystrophy. IUE I4 Efc ncpigo ociie n ctcaai i lymphocytes in B cytochalasin and colchicine of capping on Effect II.4. FIGURE average values with the ranges indicated by the bars). the by indicated ranges the with values average rmcnrl admsua ytoh ains n ares sonae the are (shown carriers and patients dystrophy muscular and controls from 3/o CAPPING .55 i 40- 45- 50- 0 2 25- 30- 35- 10 15- - 0 5- - -

NORMAL n=IO DYSTROPHIC □ Untreated □ = II n= Cytochalasin B-treated Colchicine-treated

TABLE II.4 EFFECT ON CAPPING OF COLCHICINE AND CYTOCHALASIN B IN LYMPHOCYTES FROM HEALTHY INDIVIDUALS AND MUSCULAR DYSTROPHY PATIENTS AND CARRIERS

Controls (% capping) Patients/Carriers (% capping) # Untreated Colchicine Cytochalasin B #______Untreated Colchicine Cytochalasin B

1. 16 34 4 1. DMD 14 12 2

2. 26 34 2 2. DMD 12 8 2

3. 19 28 1 3. DMD 8 9 1

4. 22 34 2 4. DMD 4 5 3

5. 26 44 0 5. DMD 4 4 0

6. 32 43 4 6. DMDC 14 14 4

7. 16 28 2 7. DMDC 6 6 2

8. 20 30 0 8. DMDC 2 3 0

9. 22 36 2 9. LGMD 6 8 1

10. 26 32 2 10. FMD 8 8 1

X = 23 35 2 11. FMD 5 5 1

X = 8 8 1

- j -j DISCUSSION

Capping has been shown to occur in lymphocytes, fibro­ blasts and polymorphonuclear leukocytes. The capping of specific membrane receptors is a well documented but inade­ quately understood phenomenon (1-11). This is due in part to lack of knowledge of the contribution of each of the many cellular, subcellular and extracellular components of the process. It is, however, generally recognized that the cytoskeletal system, the fluidity of the cell membrane and the energy supply (ATP) are of considerable importance in the control of capping (1).

As seen by Figures il.l and II.2, capping is both tem­ perature and time dependent. We have shown the extent of capping to be reduced to less than 20% in muscular dystrophy patients and carriers. increasing the length of time of incubation does not enhance capping, that is, a plateau is reached after 30 minutes. Also, raising the incubation tem­ perature to 46°C and 57°C produces no increase in capping in patients or carriers and therefore the capping process does not appear to simply be less in rate.

Patients with limb-girdle muscular dystrophy had a wide range in capping, and in only a few cases was capping increased by steroids in _in vitro experiments (Table II.1).

78 79

Our patient population is too small to determine whether our observations on the effect of steroids on capping _in vitro may be related to recent observations that steroids _in vivo reversibly decrease the release of muscle enzymes into serum in certain muscular dystrophy patients (23,24). In this connection one should recognize that it is not understood what the wide range of the capping results in limb-girdle patients reflects. Some patients with intermediate capping values had no relevant family history in previous generations yet often show more than one afflicted sibling each of whom has different symptoms. More patients and most importantly, more families of the patients, need to be evaluated in order that the impact of the mode of inheritance on the capping test be established, whether the mode is autosomal dominant or recessive. Testing of additional family members may also help establish whether incomplete penetrance of the dominant trait is a significant phenomenon in those cases where only one parent can be shown to carry the trait and more than one offspring in the succeeding generation is afflicted with the disease. The only disease in which defective capping has been previously reported is chronic lymphocytic leukemia (6,7).

Lymphocytes isolated from chronic lymphocytic leukemia patients demonstrated reduced membrane protein mobility. The 80

reduced membrane protein mobility which was directly propor­ tional to increased membrane fluidity was attributed to a lowering in the ratio of cholesterol to phospholipid in the lymphocyte membrane by a factor of two (7) . The lipid por­ tion of the lymphocyte membrane can be evaluated through the use of spin-labels (paramagnetic probes) and lipid analysis.

Spin-labels are stable paramagnetic molecules whose structure results in an attachment to or a physical relationship with biological macromolecules such as those found in membranes.

Nitroxide spin-labels can be incorporated into the system of interest to provide information concerning, among other things, structure, function, relative polarity, fluidity and conformational change.

The fluidity of the lymphocyte membrane was measured by the tumbling of the hydrophobic label, 12-doxy1 methyl stearate. The spectra obtained by this method is dependent upon the environment in which the probe is located in the membrane. The lipid environment effects the movement of the radical in response to an applied magnetic field. If the probe is located in a very fluid lipid environment, that is, it is more free to interact with other membrane lipids (and probes), both the line width ( A H) and peak ratio (B/A) should be low. Thus, if the decreased capping observed in lymphocytes of muscular dystrophy patients and carriers is 81

due to higher lipid fluidity than seen in lymphocytes from our control population than we would expect to see in our patients and carriers lower line widths (A H) and peak

ratios (B/A). The absence of statistically significant and predictable difference for either of the two parameters,A H or B/A, for the two types of cells (Table II.2) suggests

that the overall bulk lipid fluidity of the cell membrane is

similar for normal and dystrophic lymphocytes. The major

limitation of this procedure is that, if there are too few cells or the label is present in too high a concentration, exchange broadening due to excessive label-label interaction, will occur, and will falsely elevate the A H and B/A results.

This effect was avoided by determining the optimum label con­ centration relative to the number of cells used in these experiments. We cannot exclude the possibility of either a

localized abnormality in fluidity in the boundary lipids

(those in close association with membrane protein) or an

abnormality in fluidity that would only be expressed in the phase transitions occuring on either side of the membrane during the capping process itself (19).

Membrane fluidity in part is dependent upon the lipid

composition of the cell membrane. The mole ratio of chole­

sterol to phospholipid and the degree of unsaturation of the 82

phospholipid acyl chains are the two most important determi­ nants of membrane fluidity (5). In contrast to what is seen in chronic lymphocytic leukemia, analysis of cells from our patient population (Table II.3) demonstrated no difference in the cholesterol/phospholipid ratio nor any gross alteration in the relative percentage of the most common fatty acids.

These results are consistent with our observations on the normal fluidity of the bulk lipids in lymphocytes as deter­ mined by electron spin resonance. A 4% decrease in erythro­ cyte palmitoleic acid in Duchenne muscular dystrophy patients and carriers when compared with normal erythrocytes has been reported (25). Very little or no palmitoleic acid could be seen in any of our lymphocyte samples. It should be reiterated that the lipid analysis was done on the whole cell and not on isolated membranes because of insufficient start­ ing material, making this approach less sensitive an indi­ cator of small alterations in lipid composition than if the analysis had been done on isolated plasma membranes.

The integrity and functional abilities of the micro­ filaments and microtubules are best evaluated through the use of drugs thought to specifically disrupt each of these structural proteins. Capping of surface receptors can be inhibited or even reversed by drugs that act on the membrane associated cytoskeletal system such as cytochalasin B, a 83

fungal metabolite that disrupts microfilaments (8). Both

Con A and surface-Ig receptors are linked though a micro­ tubule system that anchors these receptors, in the presence of colchicine, which promotes microtubule depolymerization, capping was significantly enhanced by about 57% over untreated cells in the control population (Figure II.4).

Exposure of lymphocytes from muscular dystrophy patients and carriers to colchicine on the other hand failed to produce any detectable enhancement of capping. As expected cyto­ chalasin B reduced Con A capping in both normal and afflicted individuals lymphocytes to less than 2%. The inability to see enhanced mobility of membrane protein receptors after the

(assumed) depolymerization of the microtubules with colchicine indicates a defect in the membrane protein itself or its association with the underlying cytoskeletal system. As little is known concerning the association of the micro­ filaments and microtubules to the integral membrane proteins one cannot differentiate between a defect in cytoskeletal system itself or its point of attachment to the cell surface receptors.

Recently it has been reported (20) that electron spin resonance was abnormal in erythrocytes from muscular dystro­ phy patients suggesting that alterations in membrane protein conformation and/or organization are present in these 84

diseases. Increased phosphorylation of the major structural protein in erythrocytes (spectrin) from Duchenne muscular dystrophy patients has been demonstrated (26). Scanning electron microscopy of erythrocytes from muscular dystrophy patients showed dramatic surface deformations (22), support­

ing evidence of an abnormality arising in the membrane it­ self or the underlying cytoskeletal system. An alteration in the physical state of membrane proteins or membrane conforma­ tion may possibly explain the alterations in protein kinase activity (27), ATPase activity (28), electrophoretic mobility

(29), and membrane deformability (30) observed in erythro­ cytes from muscular dystrophy patients. An alteration in membrane protein conformation could be expected to effect those functions that require the binding of molecules on either side of the membrane. Thus, a change in the confor­ mation of the S-Ig receptor could effect the binding of the microfilaments and microtubules to the membrane proteins where proximity of the cytoskeletal system to the membrane is critical. Therefore, the receptors would be able to bind the fluorescein-labeled anti-immunoglobulins as indicated by the normal number of labeled cells in the lymphocytes of dystro­ phic patients but the microfilaments would not be able to bind to and pull the ligand-receptor complex into a cap. 85

Further investigations will determine whether the pri­

mary defect arises primarily in the boundary lipids {those

in close association with membrane proteins) resulting in a

change in protein conformation or in the proteins themselves.

The importance of specific boundary lipids to ATPase acti­

vity has already been shown (21) for erythrocyte ghosts, it was concluded in that study that the (Na++K+)-stimulated

ATPase activity was strongly dependent upon the presence of

phosphatidylserine, of which only a minor fraction (the boundary region) in the erythrocyte membrane was directly

involved. Any theory of the pathogenesis of the muscular dystrophies must address itself to the underlying reason why

different proteins in the erythrocyte membrane such as the

ATPases and protein kinase demonstrate abnormal activity, and why in lymphocytes, the mobility of surface immunoglobulin and

Con A receptors are abnormal. The variety of protein abnor­ malities identified in different blood cells and tissues may t be best understood if a defect is assumed in the insertion of membrane proteins into the membrane or if there is an abnor­ mality in a specific boundary lipid that alters the conforma­

tion of the membrane proteins. REFERENCES

1. Schreiner, G. F., and Unanue, E. R., Membrane and Cyto­ plasmic Changes in B-Lymphocytes Induced by Ligand- Surface Immunoglobulin Interaction, in Advances in Immunology, Vol. 24, F. J. Dixon and H. G. Kunkel, Eds., Academic Press, New York, N.Y., 1976, p.37.

2. Unanue, E. R. and Karnovsky, M. J., Ligand-induced Move­ ment of Lymphocyte Membrane Macromolecules; V. Capping, Cell Movement and Microtubular Function in Normal and Lectin-Treated Lymphocytes. J. Exp. Med. 140:1207 (1974).

3. Taylor, R. B., Duffus, P. H., Raff, M. C. and de Petris, S., Redistribution and Pinocytosis of Lymphocyte Surface Immunoglobulin Molecules induced by Anti-Immunoglobulin Antibody. Nature New Biology 233:225 (1971).

4. Raff, M. C. and de Petris, S., Movement of Lymphocyte surface antigens and receptors: the fluid nature of the lymphocyte plasma membrane and its immunological signi­ ficance. Fed. Proc. _32:48 (1973) .

5. Shinitzky, M. and inbar, M., Microviscosity Parameters and Protein Mobility in Biological Membranes, Biochem. Biophys. Acta 433:133 (1976).

6. Cohen, H. J. and Gilbertson, B. B., Human Lymphocyte Surface Immunoglobulin Capping, Normal Characteristics and Anomalous Behavious of Chronic Lymphocyte Leukemia Lymphocytes, J. Clin. Invest. _55:84 (1975).

7. Ben-Basset, H., Polliak, A., Rosenbaum, S. M., Naparstek, E., Shouval, D. And Inbar, M., Fluidity of Membrane Lipids and Lateral Mobility of Concanavalin A Receptors in the Cell Surface of Normal Lymphocytes and Lymphocytes from Patients with Malignant Lymphomas and Leukemias. Can. Res. ^7:1307 (1977).

8. Nicolson, G. L. and Poste, G., The cancer cell: Dynamic aspects and modifications in cell surface organization. N. Eng. J. Med. 295:253 (1976). 86 87

9. Poste, G . , Papahajopoulos, D., and Nicholson, G. L., Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution cell surface receptors. Proc. Natl. Acad. Sci, USA, _72:4430 (1952).

10. Nicholson, G. L., Transmembrane Control of the receptors on normal and tumor cells, I. Cytoplasmic influence over cell surface components. Biochem. Biophys. Acta 457:57 (1976).

11. Edelman, G. M., Yahara, I. and Wang, J. L., Receptor mobility and receptor cytoplasmic interactions in lymphocytes. Proc. Natl. Acad. Sci. USA 70:1442 (1973).

12. Verrill, H. L., Pickard, N. A. and Gruemer, H.-D, Dimi­ nished Cap Formation in Lymphocyte Membranes from Patients and Carriers of Duchenne Muscular Dystrophy. Clin. Chem. _23;2341 (1977).

13. Verrill, H. L., Pickard, N. A., and Gruemer, H.-D, Mechanism of cellular enzyme release. 1. Alteration in membrane fluidity and permeability. Clin. Chem. 23:2219 (1977).

14. Griffith, 0. H. and Jost, P. C., Lipid Spin Labels in Biological Membranes in Spin Labeling: Theory and Applications, Edited by L. J. Berliner, Academic Press, New York, 1976, 454-523.

15. Waggoner, A., Kingsett, T., Rottschaefer, S., Griffith, 0., Keith, A., Spin-labeled lipid for probing biological membranes. Chem. Phys. Lipids _3;245 (1969).

16. Folch, J. M., Lees, M. and Sloane-Stanley, G. H., A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497 (1957).

17. Morrison, W. R., and Smith, L. M . , Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J . Lipid Res. _5:600 (1964).

18. Bartlett, G. R., Phosphorous assay in column chromato­ graphy. J. Biol. Chem. 234:446 (1959). 88

19. Krolic, K. A., Wisnieski, B. J. and Sercarz, E. E., Differential lateral mobility of IgM and igG receptors in mouse B lymphocyte membranes. Proc. Natl. Acad. Sci. 24:4595 (1977).

20. Butterfield, D. A., Electron spin resonance investiga­ tions of membrane proteins in erythrocytes in muscle diseases-Duchenne and myotonic muscular dystrophy and congenital myotonia. Biochem. Biophys. Acta 470:1 (1977).

21. Roelofsen, B., and Van Deenan, L. M. , Lipid Requirement of Membrane-Bound ATPase. Studies on Human Erythrocyte Ghosts. Eur. J. Biochem. 4t0;245 (1973).

22. Matheson, D. W. and Howland, J. L., Erythrocyte Defor­ mation in Human Muscular Dystrophy. Sci. 184:165 (1973).

23. Cohen, L. and Morgan, J., Diethylstdlbestrol effects on serum enzymes and isozymes in muscular dystrophy. Arch. Neurol. 2 1 ' 480 (1976).

24. Drachman, D. B., Toyka, K. V. and Myer, E., Prednisone in Duchenne muscular dystrophy. Lancet 2:1409 (1974).

25. Howland, J. L. and Iyer, SI L., Erythrocyte Lipids in Heterozygous Carriers of Duchenne Muscular Dystrophy, Sci. 198:309 (1977).

26. Roses, A. D. and Appel, S. H., Erythrocyte Spectrin Peak II Phosphorylation in Duchenne Muscular Dystrophy, J. of the Neurol. Sci. 29:185 (1976).

27. Roses, A. D., Herbstreith, M. H., and Appel, S. H . , Membrane protein kinase alteration in Duchenne muscular dystrophy, Nature 254:350 (1975).

28. Mawatari, S.; Schonberg, M. and olarte, M., Biochemical Abnormalities of Erythrocyte Membranes in Duchenne Dystrophy, Arch. Neurol. 33:489 (1976).

29. Bosmann, H. B., Gersten, D. M., Griggs, R. C., Howland J. L., Hudecki, M. S., Katyare, S., and McLaughlin, J. Erythrocyte Surface Membrane Alterations, Arch. Neurol. 33:135 (1976). 89

30. Percy, A. K. and Miller, M. E., Reduced deformability of erythrocyte membranes from patients with Duchenne muscular dystrophy, Nature 258:147 (1975).