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International 300 N INFORMATION TO USERS This was produced from a copy of a document sent to us for microfilming. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the material su b m itte d . The following explanation of techniques is provided to help you understand markings or notations which may appear on this reproduction. 1. The sign or “target” for pages apparently lacking from the document photographed is “Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure you of complete continuity. 2. When an image on the film is obliterated with a round black mark it is an indication that the film inspector noticed either blurred copy because of movement during exposure, or duplicate copy. Unless we meant to delete copyrighted materials that should not have been filmed, you will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., is part of the material being photo­ graphed the photographer has followed a definite method in “sectioning” the material. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete. 4. For any illustrations that cannot be reproduced satisfactorily by xerography, photographic prints can be purchased at additional cost and tipped into your xerographic copy. Requests can be made to our Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases we have filmed the best available copy. 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
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