Elsevier

Neuromuscular Disorders

Volume 9, Issue 3, 1 May 1999, Pages 150-158
Neuromuscular Disorders

Correlation of muscle fiber type measurements with clinical and molecular genetic data in Duchenne muscular dystrophy

https://doi.org/10.1016/S0960-8966(98)00114-XGet rights and content

Abstract

Clinical improvement following surgery in patients with Duchenne muscular dystrophy (DMD) may be influenced by the severity of muscle fiber damage. This study correlates morphometric alterations of muscle fiber types, severity of fat tissue proliferation and fibrosis with Western blots, multiplex polymerase chain reaction (PCR), and postoperative state in DMD. The main results of this study show that the mean diameter of type 2 fibers is usually markedly larger than that of type 1 fibers in DMD although the number of type 2 fibers is severely reduced. The mean percentage of the remaining type 1 fibers was in the range of 57–82%. The morphometric and histopathological results were in agreement with the clinically estimated postoperative state, especially in the patients who presented a severe state or suggestive clinical improvement. It is concluded that combination of both quantitative and qualitative evaluation of muscle biopsies is suitable for better evaluation of the postoperative state in patients with DMD, whereas severity of exon deletions correlated poorly with morphometry and postoperative clinical state.

Introduction

The fatal disease of boys known as Duchenne muscular dystrophy (DMD) was first described in 1852 [1]in a single case and later in 1872 [2]in a larger series of cases by Duchenne, for whom the disorder is named. DMD is the most common muscle disorder affecting boys and is now known to be associated with mutations in the dystrophin gene at Xp21 with absence of the cytoskeletal protein dystrophin 3, 4, 5, 6, 7.

With the standard myofibrillar ATPase reaction, usually type 1 and type 2 muscle fibers are distinguished. The spatial distribution of fiber types in normal human muscle is random [8], but DMD may show alterations of this proportion of fiber types. In addition, there is severe variation in the size of fibers. Moreover, there is increasing endomysial and perimysial proliferation of fat and fibrous connective tissues with disease progression. The connective tissue is composed predominantly of type 3 collagen [9]. Eventually the architecture of the fascicles becomes disorganized and fatty and fibrous connective tissue replaces the degenerated fibers. All of these changes are closely related to sitting and walking ability of DMD patients. Surgical treatment in DMD, such as lower limb and scoliosis surgery, is one of major approaches in order to improve quality of life 10, 11. The postoperative state induced by the surgical procedure may be influenced by the severity of muscle fiber change and progression of the disease or the time of the surgical treatment.

Due to molecular genetically identified mutations of the dystrophin gene and their effect on the dystrophin protein, molecular assays of dystrophin and its gene have provided an improved diagnostic approach for DMD patients and their families. Western blot can be used for examining the existence and alteration of dystrophin. Originally, detection of exon deletions was accomplished by analysis of the patients' DNA in Southern blots. Now, most deletions are detected using the multiplex polymerase chain reaction (multiplex PCR) to amplify selected exons of the dystrophin gene. Primers have been designed to amplify specific exons adjacent to hot spots of the dystrophin gene where the majority of deletions cluster. Deletions of the dystrophin gene are found in about 60% to 65% of DMD patients with the multiplex PCR [12].

The present study demonstrates that morphometric analysis and histopathological changes in the DMD cases correlate well to the clinically estimated postoperative state, especially in the patients who represent a severe state, whereas the position and size of deletions in the dystrophin gene and the number of revertant fibers do not.

Section snippets

Clinical material

Muscle samples of the quadriceps muscle in 30 patients with DMD were obtained by biopsy for diagnostic purposes. The average age of these boys was 8.5 years (range 3.75–14.2 years). Histochemical, immunohistochemical and morphometric studies were performed in all of the cases. Nineteen of them were also examined using Western blots. In these 30 patients with DMD, lower limb surgery was carried out 13, 14. The clinical state was assessed pre- and postoperatively as described comprehensively in

Morphometric analysis

In the 30 patients the mean diameter of type 2 fibers was markedly larger than that of type 1 fibers (mean: 65±21 μm and 48±22 μm); the variance of diameters for type 1 and type 2 fibers was on the average 295 and 486, respectively (Table 1 and Fig. 1). The number of type 2 fibers was more severely reduced than that of type 1 fibers (Fig. 2).

Histopathological observations

Various pathological changes of DMD were apparent in the 30 cases, such as various forms of degeneration, necrosis, and regeneration of muscle fibers.

Discussion

In order to improve quality of life in DMD patients, surgical treatment is considered as one of the major therapeutical approaches 10, 11, 29, 30, but the postoperative state following the surgical procedure may be influenced by the severity of muscle fiber damage, progression of the disease, or time of surgical intervention. In this study we morphometrically evaluated alterations of muscle fiber type, severity of fat tissue proliferation and fibrosis, and tried to assess their correlation with

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Acknowledgements

The technical assistance of Mrs. A. Knischewski in morphometry, Ms. M. Wiederholt in histochemistry, Miss H. Mader and Miss M. Henssen in photographical documentation, Mrs. S. Franke in Western blot and multiplex PCR is gratefully acknowledged. This study was supported by the `Start-Programm' of the University Hospital Aachen.

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