Twenty patients with Becker muscular dystrophy (BMD), confirmed by dystrophin tests, were studied histologically. There were several morphological differences between younger (515-year-old) and older (>15-year-old) patients. In the younger patients, active muscle fiber necrosis followed by a regenerating process was conspicuous. In the older patients, the active degenerative changes appeared less prominent and, instead, more chronic myopathic changes such as moth-eaten fibers, fiber splitting, and hypertrophic fibers were evident. These age-dependent differences in the pathology of BMD were irrespective of the duration of clinical symptoms, i.e., BMD patients of a similar age showed a similar morphological feature regardless of age at onset. Although the presence of mild fiber type grouping and some small angulated atrophic fibers suggested a certain degree of neurogenic involvement, none of biopsies showed significant grouped atrophy as seen in neuropathic disorders. There was no correlation between the histological changes and the specific dystrophin abnormality. Key words: Becker muscular dystrophy dystrophin muscle histochemistry myonecrosis fiber type analysis MUSCLE & NERVE 14:1067-1073 1991

MUSCLE HISTOLOGY IN BECKER MUSCULAR DYSTROPHY MISAKO KAIDO, MD, KllCHl ARAHATA, MD, ERIC P. HOFFMAN, PhD, IKUYA NONAKA, MD, and HIDE0 SUGITA, MD

Becker muscular dystrophy (BMD) is a benign form of X-linked muscular dystrophy, which was first described as a distinct clinical entity by Becker and Kiener in 1955." They distinguished BMD from Duchenne muscular dystrophy (DMD) on the basis of the milder clinical course. In 1979, Goebel et al.g examined muscle biopsies of 2 patients originally reported by Becker et al., and found that the myopathic changes were milder than those of age-matched DMD patients. In addition, there were some neurogenic changes, including scattered small angulated fibers and small groups of atrophic fibers. Other investigators have also reported morphological abnormalities similar

From the National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Japan (Drs. Kaido, Arahata, Nonaka, and Sugita), and the Division of Genetics, Harvard Medical School, Howard Hughes Medical Institute at Children's Hospital, Boston, Massachusetts (Dr. Hoffman). Dr. Hoffman is currently at the University Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Acknowledgments: The authors thank Drs. S. Ishiura, T. lshihara (NCNP), and Prof. Louis M. Kunkel (University of Pittsburgh School of Medicine) for their advice We also thank Prof. Seiichiro Tarui, Second Department of Internal Medicine, Osaka University School of Medicine, for his encouragement Address reprint requests to Dr. Misako Kaido. National Institute of Neuroscience (NCNP). 4-1 -1 Ogawa-higashi, Kodaira, Tokyo 187, Japan. Accepted for publication September 3, 1990 CCC 0148-639X/91/01101067-07 $04.00 0 1991 John Wiley & Sons, Inc.

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to those observed by Goebel et al. in a larger number of patients with BMD.5,'g Again, these authors found that while myopathic changes predominated, there were also some neuropathic abnormalities such as grouped atrophy, fiber type grouping, small angulated fibers, and pyknotic nuclear c l ~ r n p s . ~ * ' ~ More recently, BMD has been found to be caused by abnormalities of dystrophin, the protein product of the Duchenne muscular dystrophy gene. Dystrophin abnormalities in BMD patients have been visualized by both immunofluorescent and immunoblot techniques. 1*2,4,12,13On immunohistochemical staining of dy~trophin,'~'.~ BMD muscles show a discontinuous "patchy" pattern at the plasma membrane, while dystrophin is generally absent or undetectable in muscle from patients with DMD. In muscles from patients other than DMD and BMD, dystrophin immunostaining shows a continuous ring of fluorescence at the surface membrane of each fiber. On immunoblotting, BMD patients show abnormal molecular weight (larger or smaller size), and/or decreased amounts (quantities) of the dystrophin protein.2,'2- l 4 The purpose of this report is to re-examine the general histological abnormalities of BMD muscles whose dystrophin tests were consistent with BMD, and to investigate the correlation between clinical course and histological changes of the muscle.

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MATERIALS AND METHODS

Twenty male patients with BMD were examined. Fourteen of these 20 patients were included in a previous report.2 Subjects ranged in age from 2 to 32 years. Twelve had a positive family history of probable X-linked inheritance. All patients showed proximal muscle weakness and pseudohypertrophic calf muscles, except patient 2, who was suspected to have a muscle disease by chance from the result of a blood test. With or without subjective complaints, most patients presented with difficulties in keeping up with peers, or difficulty in physical exercise in their early childhood. Five patients complained of myalgia on exercise which began during the later half of the first decade. Muscle weakness and atrophy progressed slowly, but all patients could stand and walk by themselves at the time of muscle biopsy (Table 1). Serum creatine kinase (CK) levels were elevated in all patients ranging from 549 to 18,365 U/L (normal < 110 UIL). The clinical features of each patient are shown in Table l . All patients underwent muscle biopsy on the biceps brachii muscle for diagnostic purposes with informed consent. Histochemical Staining. T h e biopsied muscles were frozen in isopentane chilled

Conventional

with liquid nitrogen. Ten-micron thick sections were stained with hematoxylin and eosin (H&E), modified Gomori trichrome (mGt), and various histochemical methods including NADH-tetrazolium reductase (NADH-TR), alkaline phosphatase, acid phosphatase, and nonspecific esterase (NSE). On ATPase with alkaline and acid preincubation at pH 4.2 and 4.6, muscle fibers were classified into type 1, 2A, 2B, and 2C fibers, to determine distributions of the fiber types. The histogram' of each patient was made by measuring and manually counting at least 500 muscle fibers from each patient's biopsy on photographs. Atrophic and hypertrophic factors' were calculated in only those patients over age 15 years. Necrotic, phagocytic, and moth-eaten fibers were counted per 1000 fibers. Six-micron cryostat sections on gelatinized cover slips were fixed in 100% ice-cold acetone for 5 minutes, and preincubated with PBS containing 2% BSA and 5% heat-inactivated goat serum. The sections were sequentially incubated with antidystrophin for 2 hours at 37"C, then affinitypurified in FITC-labeled goat (Fab')' antirabbit IgG (Tago Inc., 10 pg/mL, in PBYBSA) for 45 lmmunofluorescent Staining of Dystrophin.

~~

Table 1. Clinical and biochemical features of BMD patients

Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Age at biopsy

Family history

2 3 4 6 7 8 8 10 12 12 13 14 17 23 27 27 28 31 32 32

-

+ -

+ -

+ + + -

+ + + + + + -

+

Dystrophin CK (UIL)

EMG

MW (kd)

Amount (%)

11200 3372 3200 18365 600 549 41 50 31 03 8004 3349 9999 3044 ND 3699 748 4367 788 4420 1790 1373

NP M ND ND ND ND M NP M ND M M M M+N M+N M ND M M M

390 360 380 350 370 380 400 410 380 420 380 370 420 390 370 380 380 380 380 380

20 50 10 (5 10 30 10

10 40 20 40 30 10 20 50 20 10 50 60 20

Patients 10, 13, and patients 18, 20 are sfblfngs, respectively CK: creabne kmase, normal i 110 UIL; MW molecular weight: ND. not done; NP: not particular; M: myogenfc, N. neurogenic

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minutes at room temperature. The sections were then mounted in a glycerol-based medium and observed under a Zeiss Axiophot microscope with epifluorescence.

letions for patients with smaller molecular weight dystrophin proteins, duplications for larger dystrophin patients).‘

For each muscle biopsy specimen (stored at -8O”C), Western blot analysis was performed. Dystrophin quality (molecular weight) was determined by comparing the dystrophin contained in each patient’s biopsy to that of a normal biopsy sample (normal molecular weight = 400 kd). Myosin heavy chain (205 kd) was used as a quantity reference for each sample. The quantity (relative amount) of dystrophin contained in each biopsy was expressed as a percentage of the amount of dystrophin in normal control samples. The amount of dystrophin in each biopsy was estimated in a semiquantitative way, comparing both the adjacent lane of normal control samples and the amount of muscle tissue loaded in each lane, determined by the post-transfer myosin stain.13 Actually, the data varied by 10% to 20% of the determined value in separate determinations on the same samples. Dystrophin gene analysis was done for 14 of the 20 patients. In each case, the DNA results were consistent with the protein results (gene de-

RESULTS

Dystrophin lmmunoblotting and DNA Analysis.

In all biopsies, the surface membrane showed faint and discontinuous “patchy” immunostaining of dystrophin at the surface membrane, which is characteristic of BMD’*2z4(Fig. 1). Immunoblot analysis also confirmed the diagnosis of each patient (Table l). Abnormality in molecular weight of dystrophin was found in all patients except patient 7, whose muscle showed decreased relative amounts of dystrophin of about 10% of normal levels on Western blotting, and a faint and “patchy” pattern on dystrophin immunostaining. A decrease in the relative amount of dystrophin was observed in all patients. There was no obvious age-dependent differences in staining intensity (Table 1). Ten of the 14 BMD patients had in-frame deletions in the deletion-prone “hot spot” (exons 45-49) in the distal portion of the rod domain. Three patients had no detectable deletion, and 1 patient showed an out-of-frame deletion between exons 3 and 7.3 On conventional histochemical stainings, there

FIGURE 1. lmmunofluorescence localization of dystrophin in muscles from a myotonic patient (a: 18-year-old male), a younger Becker patient (b: 12-year-old male), and an older Becker patient (c: 31-year-old male) are shown. Six-micron frozen sections were immunostained for dystrophin. All surface membrane is clearly stained in muscle from the diseased control (a), but faint, patchy staining was seen in the muscle sections from patients with both younger (b), and older (c) Becker muscular dystrophy. Bar = 25 pm.

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FIGURE 2. “Neurogenic”changes in BMD. Scattered small angular fibers with high nonspecific esterase activity (arrow) (a); occasional, enclosed fibers (e) showing mild fiber type grouping (b); and small atrophic fibers, with nuclear clumps (c) are suggestive of neuropathic involvement. However, typical group atrophy, as seen in neurogenic muscular atrophy, was not observed in any of the biopsies studies. (a) Patient 13, nonspecific esterase; (b) patient 20, ATPase at pH 4.6; (c) patient 18, H&E. Bar = 50 +m.

was moderate or marked variation in fiber size, and both necrotic and basophilic regenerating fibers were recognized in most biopsies. Fibers with centrally placed nuclei were increased in number in all muscle biopsies. Infiltration of mononuclear cells was rarely observed. Mild to moderate interstitial fibrosis was present without striking infiltration of fatty tissue. Muscle fiber splitting and a mild moth-eaten appearance on NADH-TR staining were seen in 70% and 30% of muscle biopsies, respectively. Cytochrome c-oxidase activity was well preserved, except in 1 patient. Some fiber type grouping, which is considered a characteristic finding of the neuropathic process, was seen in 35% of biopsies. Small angulated fibers appeared in 25%, and pyknotic nuclear clumps in 15% of biopsies, but both were not as prominent as usually seen in denervated muscles (Fig. 2). While the observations suggest that there may be a neuropathic component to BMD, it seems variable and relatively mild. Ring fibers, target and targetoid fibers, and groups of atrophic fibers were not observed. As to fiber diameter, 80% of the patients had hypertrophic fibers measuring from 100 to 200 km in caliber, especially in type 2 fibers. On the other hand, type 1 fiber atrophy was seen in 35% of the muscle biopsies, and type 1 fiber predominance was noted in 15% of them (patients 1, 11, and 20). Type 2B fibers were well preserved in all patients, except patients 6 and 7, who had less than 10%. Interestingly, there were striking morphologi-

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CHANGE IN FIBER SIZE small angulated fibers selective fiber atrophy type 1 type 2A type 28 hypertrophic fibers CHANGE IN DISTRIBUTION group atrophy fiber type grouping fiber type Predominance fiber type deficiency (28)

PERCENTAGE OF PATIENTS 0 50 100

h

(%)

Bm I 5 I 1:16.7%

1:12.5%, 28:12.5%

NUCLEI internal nuclei pyknotic nuclear clumps DEGENERATION & REGENERATION necrosis YYYYYYCC(d phagocytosis splitting ____________n basophilic fibers increased type 2C fibers CELLULAR RESPONSE inflammatory fibrosis ARCHITECTURAL CHANGE targetold fibers moth-eaten fibers CCO DEFICIENT FIBERS

=young (5 15y.O.)

r I

k

old (> 15y.o.)

FIGURE 3. Bar graph of histological analysis of 20 patients with BMD.

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FIGURE 4. In the younger group (a), in addition to a variation in fiber size, active degenerative and regenerative processes are seen. Necrotic fibers are often in groups. While in the older group (b), necrotic fibers are decreased in number and chronic rnyopathic changes of fiber splitting, central nuclei, and hypertrophic fibers become prominent. The well-preserved interrnyofibrillarnetworks observed in the younger patient muscle (c) are markedly disorganized showing whorled, moth-eaten structures in the older patient (d). (a) Patient 5 (7-years-old), (b,d) patient 20 (32-years old), (c) patient 7 (8-years-old). (a,b) H&E; (c,d) NADH-TR stain. Bar = 100 prn.

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cal differences between the younger (515 years) and the older patients (>15 years) (Fig. 3). First of all, active degenerating and regenerating processes such as muscle fiber necrosis, phagocytosis, and basophilia were seen at higher frequencies in the younger patients (16.6 to 87.7 per 1000 muscle fibers) than in the older ones (0 to 22.0 per 1000 fibers). In the younger BMD patients, necrotic and regenerating fibers often aggregated in small clusters (Fig. 4). Undifferentiated type 2C fibers were more frequently observed in the younger patients than in the older ones (10.9% and 2.7%, respectively). By contrast, chronic myopathic changes including fiber-splitting, hypertrophy, and moth-eaten appearances were more commonly observed in the older patients (Fig. 4). Muscle fibers having central nuclei were more frequently observed in the older group (24.5%) than in the younger group (7.2%). DISCUSSION

Our results indicate that the histopathological features of BMD are dependent upon the age of the patient. In the younger patients (115 years), active necrotic and regenerating processes of muscle fibers were quite prominent. On the other hand, the elder BMD patients (>15 years) had more chronic changes with numerous internal nuclei, fiber hypertrophy, fiber-splitting, and disorganization of intermyofibrillar networks. Given these differences, the younger BMD patients had myopathic histology more closely resembling that of severe DMD, while the elder BMD patients’ histology resembled that of limb-girdle dystrophy (LGD).’ ATPase analysis of the younger patients showed that type 2C fibers were increased to 10% to 30% of all fibers. But, in the older patients, the percentage was, instead, dramatically decreased to less than 6%. Although type 2B fibers were relatively well preserved in both younger and older BMD patients when compared with DMD and LGD,’ in the 2 younger patients, type 2B fibers were decreased to less than lo%, which fulfills the criteria’ of type 2B fiber deficiency. T h e

change in proportion of type 1 fibers was not significant. It is of interest to look for relationships between the age of presentation, rate of progression, and muscle pathology of the 20 patients studied. Interestingly, histological differences between the younger and older BMD patients simply reflected their age at the time of biopsy-irrespective of their onset of the clinical symptoms. For example, patients 15, 19, and 20 presented in early infancy, yet experienced a very slow clinical progression of the illness over 20 years. On the other hand, patients 16, 17, and 18 noticed their muscular weakness in the third decade. Patients 18 and 20 were male siblings with different onset-of-disease ages and clinical courses. Although unexpected, there were few morphological differences between the former and the latter groups, rather, their muscle biopsies all showed the characteristics of the other older BMD patients. These results suggest that epigenic or polygenic factors may play an important role in the histological changes as well as the clinical differences of BMD patients, as recently suggested by Medori et al? Finally, neurogenic changes in BMD have been emphasized in past Although there was fiber type grouping in 35% of BMD muscles examined, the grouping was mild and most was present as “enclosed fibers” which were surrounded on all sides by fibers of its own histochemical type“ (Fig. 2). While it is conceivable that the observed fiber type grouping could be a direct result of dystrophin deficiency in neurons,’ small-scale grouping of “enclosed fibers” can also occur secondarily to a primary myopathy. Specifically, it is thought that fiber type grouping can result from grouped necrosis followed by grouped regeneration, or from extensive fiber ~plitting.’~ A third possible cause of the neurogenic changes in BMD may be secondary to reinnervation of regenerating fibers, where intramuscular peripheral nerves are involved, to some extent, during chronic degenerating and regenerating processes of muscle fibers.6 The relationship of these neurogenic changes to specific dystrophin abnormalities in BMD muscle remains to be elucidated.

REFERENCES 1. Arahata K, Ishiura S, Ishiguro T, Tsukahara T, Suhara Y, Eguchi C, Ishihara T, Nonaka I, Ozawa E, Sugita H: Immunostaining of skeletal and cardiac muscle surface mernbrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988;333:861-863.

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2. Arahata K, Hoffman EP, Kunkel LM, Ishiura S, Tsukahara T, Ishihara T, Sunohara N, Nonaka I, Ozawa F., Sugita H: Dystrophin diagnosis: Comparison of dystrophin abnormalities by imrnunofluorescence and immunohlot analyses (Duchenne muscular dystrophy/Becker muscular

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NERVE

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dystrophy). Proc Natl Acad Sci USA 1989;86:7154-7158. 3. Beggs AH, Hoffman EP, Snyder JR, Arahata K, Specht L, Shapiro F, Angelini C, Sugita H, Kunkel LM: Exploring the molecular basis for variability among patients with Becker muscular dystrophy: Dystrophin gene and protein studies. A m / H u m Genet (in press). 4. Bonilla E, Samitt CE, Miranda AF, Hays AP, Salviati G, DiMauro S, Kunkel LM, Hoffman EP, Rowland PR: Duchenne muscular dystrophy: Deficiency of dystrophin at the muscle cell surface. Cell 1988;54:447-452. 5. Bradley WG, Jones MZ, Mussini JM, Fawcett PRW: Becker-type muscular dystrophy. Muscle Neroe 1978; 1 : l l l - 132. 6. Desmedt JE, Borenstein S: Regeneration in Duchenne muscular dystrophy. Arch Neurol 1976;33:642-650. 7. Dubowitz V: Definition of pathological changes seen in muscle biopsies, in Dubowitz V (ed): Muscle Biopsy: A Practical Approach London, Bailliere Tindall, 1985, pp 82- 128. 8. Dubowitz V: The muscular dystrophies, in Dubowitz V (ed): Mwcle Biopsy: A Practical Approach London, Bailliere Tindall, 1985, pp 289-404. 9. Goebel HH, Prange H, Gullotta F, Kiefer H, Jones MZ: Becker’s X-linked muscular dystrophy. Histological, enzyme-histochemical, and ultrastructural studies of two cases, originally reported by Becker. Acta Neuropathol (Berl) 1979;46:69-77. 10. Grimm T: Becker dystrophy, in Engel AG, Banker BQ (eds): Myology New York, McGraw-Hill, 1986, pp 12411250. 1 1 . Hoffman EP, Hudecki MS, Rosenberg PA, Pollina CM, Kunkel LM: Cell and fiber-type distribution of dystrophin. Neuron 1988;1:411-420.

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12. Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterston R, Brooke M, Specht L, Kupsky W, Chamberlain J, Caskey CT, Shapiro F, Kunkel LM: Characterization of dystrophin in musclebiopsy specimens from patients with Duchenne or Becker’s muscular dystrophy. N Engl J Med 1988;318: 1363- 1368. 13. Hoffman EP, Kunkel LM, Angelini C, Clarke A, Johnson M, Harris JB: Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology 1989;39: 101 1 1017. 14. Hoffman EP, Kunkel LM: Dystrophin abnormalities in Duchenne/Becker muscular dystrophy. Neuron 1989; 2~1019-1029. 15. Isaacs ER, Bradley WG, Henderson G: Longitudinal fiber splitting in muscular dystrophy: A serial cinematographic study. J Neurol Neurosurg Psychiatry 1973;36:813-819. 16. Jennekens FGI, Tomlinson BE, Walton JN: Data on the distribution of fiber type in five human limb muscles. An autopsy study. J Neurol Sci 1971;14:245-257. 17. Medori R, Brooke MH, Waterston RH: Two dissimilar brothers with Becker’s dystrophy have an identical genetic defect. Neurology 1989;39: 1493- 1496. 18. Sugita H, Arahata K, Ishiguro T, Suhara Y, Tsukahara T, Ishiura S, Eguchi C, Nonaka I, Ozawa E: Negative immunostaining of Duchenne muscular dystrophy (DMD) and mdx muscle surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA. Proc Jpn Acad 1988;64-B :37 - 39. 19. Ten Houton, De Visser M: Histopathological findings in Becker-type muscular dystrophy. Arch Neurol 1984; 41:729-733.

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Muscle histology in Becker muscular dystrophy.

Twenty patients with Becker muscular dystrophy (BMD), confirmed by dystrophin tests, were studied histologically. There were several morphological dif...
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