n
n
P
Quadriceps Myopathy: Porme Pruste of: Becker Muscular Dystrophy Nobuhiko Sunohara, MD,* Kiichi Arahata, MD,? Eric P. Hoffman, PhD,q Hitoshi Yamada, MD,” Jin Nishimiya, MD,$ Eri Arikawa, MD,t Misako Kaido, MD,t Ikuya Nonaka, MD,S and Hideo Sugita, M D t
We examined dystrophin, the protein product of the Duchenne muscular dystrophy gene, in muscle biopsy specimens from 4 male patients with quadriceps myopathy, all of whom showed a mild and slowly progressive myopathy confined to the quadriceps muscles. All 4 patients had clear abnormalities of dystrophin, and were diagnosed as having Becker muscular dystrophy by both immunofluorescence and immunoblot examinations; that is, dystrophin of an abnormal molecular mass was visualized in muscle cryosections as “patchy” or discontinuous immunostaining at the surface membrane of the muscle fibers. One patient had a brother who showed widespread myopathic changes consistent with typical Becker muscular dystrophy. We conclude that the syndrome called quadriceps myopathy includes a group of forme fruste Becker muscular dystrophy. Sunohara N, Arahata K, Hoffman EP, Yamada H, Nishimiya J, Arikawa E, Kaido M, Nonaka I, Sugita H. Quadriceps rnyopathy: forme fruste of Becker muscular dystrophy. Ann Neurol 1990;28:634-639
Cases of patients with muscle wasting and weakness limited to the quadriceps femoris have been occasionally reported 11-61. These patients with so-called “quadriceps myopathy” have been regarded as suffering from a heterogeneous group of disorders, including a form of limb-girdle muscular dystrophy El], a unique form of muscular dystrophy 12-41, polymyositis [ S , 61, or a form of chronic spinal muscular atrophy [7, 81. Nine of the 1 7 patients previously described r1-83 were male, and 7 patients had a positive family history. We identified 4 patients with a clinical picture consistent with quadriceps myopathy in the 3,048 patients who underwent biopsy and who were studied in our clinic over the last 12 years. In this report, we studied dystrophin, the protein product of the DuchenneIBecker muscular dystrophy gene, in the muscle biopsy specimens of these 4 patients. All 4 patients had dystrophin patterns consistent with Becker muscular dystrophy, indicating that quadriceps myopathy is an unusual presentation of Becker muscular dystrophy.
he was admitted to our hospital. H e showed moderate wasting and weakness of the quadriceps femoris muscles (Medical Research Council [MRC] Scale: 3/5). There was hypertrophy of the calf muscles. Gowers’ sign was not recognized. Deep tendon reflexes were normally active, except for diminished Achilles’ tendon reflexes. Serum creatine kinase (CK) levels were 10 to 20 times the upper limit of normal values. Needle electromyography (EMG) showed positive sharp waves at rest, and short-duration, low-amplitude polyphasic motor units with long-duration, high-amplitude potentials on voluntary contraction. When he was reexamined at the age of 37 years, he exhibited similar neurological findings to those observed at the previous admission plus a mild Gowers’ sign. Computed tomographic (CT) scan revealed marked atrophy of the quadriceps femoris muscles, but other muscles were well preserved (Fig 2). Electrocardiography (ECG) disclosed no abnormality. The pedigree of this individual is shown in Figure 1. The mother of the patient (11-7) showed no abnormality on neurological examination, but her serum CK level was slightly elevated (153 unitdliter; normal, 12 to 75 unitdliter). The maternal uncle had suffered from similar symptoms, and died at age 40.
Patients and Methods Case History 1. A 37-year-old man (I114 in Fig 1A) became aware of hypertrophy of the calf muscles at the age of 10 years. At the age of 18 years he had sllght difficulty in climbing stairs and wasting of the thigh muscles. At 27 years old
PATIENT 2. A 41-year-old man noticed hypertrophy of the calf muscles at the age of 11 years. From the age of 15 years he could not run and from the age of 20 he had difficulty in climbing stairs. At the age of 32 he was admitted to the National Center Hospital (Musashi). He displayed marked muscular wasting of the quadriceps femoris muscles and
From the *Department of Neurology, National Center Hospital (Musashi),and the tDivisions of Neuromuscular Research and $Ul-
Received Feb 21, 1990, and in revised form Apr 26. Accepted for publication May 14. 1990.
PATIENT
Children’s Hospital, Boston, MA.
634 Copyright 0 1990 by the American Neurological Association
a 0
I
I
0 3
?
0 4
11
A
111
I
i
. j
I
. .I
I1 111
Oo0.D 2 I
B
3 7 4
0 5
N
I I1
C
Ill
Fig I. Pedigrees of families. (A) Family of Patient 1. (B) Family of Patient 2. (C) Family aPatient 3. Note that Patient 5 is 111-1 and Patient 6 is 11-12 in farnib C. W = Propositus, A = affected individuals, @ = death, = cam'er.
pseudohypertrophy of the calf muscles. There was muscular weakness of the quadriceps femoris muscles (MRC: 3/5), iliopsoas muscles (3/5), and gluteal muscles (4+/5). Serum CK levels were 5 to 24 times the upper limit of normal. Needle EMG demonstrated fibrillation potentials and positive sharp waves at rest, and short-duration, small-amplitude polyphasic potentials on voluntary contraction. At age 40 he had an episode of cardiac failure due to cardiomyopathy with dilatation. Since then, he has been treated with 0.1 mg of metildigoxin and 40 mg of furosemide per day. At the age of 41 years we examined him again. H e had no muscular weakness or wasting of the neck and upper limb-girdle muscles. The lower girdle muscles revealed normal strength, except for the quadriceps femoris (MRC: 3/5), iliopsoas (2/5), and gluteal (4/5) muscles. The pedigree of his family is shown in Figure 1B. The family members examined (11-8, 11-11, 111-1, 111-2, 111-3, III-5,III-6, IV-1, and IV-2) had no neuromuscular disorders and their serum CK levels were within normal limits. PATIENT 3. A 31-year-old man noticed slight difficulty in climbing stairs at the age of 25 years, but he did not think it abnormal. H e was a ping-pong player while at the university, and he can play even now. Neurological examination disclosed marked atrophy of the quadriceps muscles bilaterally and mild pseudohypertrophy of the calf muscles. Mild weakness of the iliopsoas (MRC: 4/5) and quadriceps muscles (4/ 5 ) was detected. Deep tendon reflexes were normally active. Serum CK and aldolase levels were elevated (16 and 3 times
the upper limit of normal, respectively). Needle EMG examination revealed low-amplitude and short-duration polyphasic potentials in the muscles of both legs. CT scan showed marked atrophy of the quadriceps muscles (see Fig 2). ECG and echocardiogram disclosed no abnormality. The family tree is shown in Figure 1C. His brother (Patient 5, 111-1 in Fig lC), age 32, had predominantly proximal muscular weakness and wasting, and pseudohypertrophy of the calf muscles, features compatible with typical Becker muscular dystrophy. The serum CK level was 5 times the upper limit of normal. C T scan revealed marked atrophy of the quadriceps and biceps femoris, iliopsoas, and paravertebral muscles, and mild to moderate atrophy and patchy lowdensity areas in the biceps and triceps brachii muscles (see Fig 2). EMG showed short-duration, small-amplitude polyphasic potentials on voluntary contraction. On ECG and echocardiogram, there was no abnormality. The patient's mother (Patient 6, 11-12 in Fig lC), age 56, had slight weakness of the sternocleidomastoideus muscles. A needle EMG examination on voluntary contraction disclosed small amounts of short-duration, low-amplitude polyphasic potentials, but she had no elevation of serum CK levels or abnormality on muscle CT scan. ECG disclosed no abnormality. Family members of 11-1 and 111-3 had no abnormality on neurological examination and no elevation of serum CK levels. PATIENT 4. A 3 1-year-old man noticed difficulty in walking at the age of 19 years old. At age 21, his thighs became thin. At age 31 he had congestive heart failure due to dilated cardiomyopathy. Neurologically, he had marked muscular wasting and weakness (MRC grade: 3/5) of the quadriceps femoris muscles bilaterally. Serum CK level was elevated 7 times the upper limit of normal.
Mascle Biopsies Muscle biopsy specimens were obtained from the left quadriceps femoris (Patients 1, 2, 3, and 4) and biceps brachii (Patients 1, 2, 3, 5 , and 6) for diagnostic purposes, with the informed consent of all subjects. Serial frozen sections were stained by hematoxylin and eosin, modified Gomori trichrome, and various histochemical stainings. The same muscle biopsy samples were used for both immunofluorescence and immunoblot analyses.
Dystrophin Analyses IMMUNOREAGENTS AND CEDURES. Four different
IMMUNOHISTOCHEMICAL
PRO-
polyclonal anti-dystrophin antisera, which have been previously described [9}, were used in this study. The cryosections to be immunoreacted were picked up on gelatinized coverslips and were processed as described before C9- 11). IMMUNOBLOTTING. Immunoblot detection of dystrophin was done as published previously l12). The relative abundance values we have assigned to dystrophin are qualitative approximations relative to the muscle protein (myosin) in the control samples on the same gel. These calculations were based on the average values obtained in two or more experiments.
Sunohara et al: Quadriceps Myopathy
635
Fig 2. CT scans oftheforearm (A,D,and G), thigh (B, E, and H), and lumbar spinal (C, I;, and I ) muscles in Patient 1 (A,B, and C), Patient 2 ID, E , and P), and Patient 5 {G,H , and I). The quadriceps muscles of Patients 1 (B) and 3 /E) are markedly involved (arrows).Howeyer, the biceps and triceps brachii muscles of Patients I (A)and 3 (D), the biceps femoris muscles o f Patients 1 (B) and 3 (E), and the Paravertebral muscles of Patients 1 (C) and 3 (Pi are wellpreserved. The low-density awas in (A)are artifacts. The biceps and triceps brachri muscles (G),the biceps fernoris muscles (Hj, and the paravertebral muscles (I) are markedly damaged in Patient 5 (triangles).
ther, grouped atrophy of fibers indicative of denervation were only found in the quadriceps muscles in Patients l, 2, and 3 (Patients l and 2 are Cases 2 and l in [13], respectively). Fiber type grouping was also seen in the quadriceps femoris muscle specimen of Patient 2. Patient 5 , the elder brother of Patient 3 (111-1in Fig lC), was diagnosed as having mild Becker muscular dystrophy, and the biopsy specimen from his biceps brachii showed similar myopathic abnormalities. DystTophin Tests
Results General Mascle Pathological Featares
In all patients, biopsied muscle specimens showed marked variation in fiber size, fiber splitting, mild to moderate increase in the number of internal nuclei, occasional grouped fiber regeneration, aberrant myofibrillar network, and mild increase in endomysial and perimysial fibrous connective tissue, suggesting active and chronic myopathy consistent with muscular dystrophy. Although both the quadriceps and biceps muscles appeared to be involved histopathologically, the quadriceps samples were more severely affected. Fur-
IMMUNOFLUORESCENCE STUDIES. Dystrophin in the muscle specimens was analyzed with immunofluorescence. All 4 patients with quadriceps myopathy (Patients l, 2, 3, and 4 ) and Patient 5 showed a similar immunostaining pattern for dystrophin, which is consistent with Becker muscular dystrophy, that is, “patchy,” discontinuous, and faint immunoreaction at the surface membrane of the skeletal muscle fibers (Figs 3 and 4). In Patient 3, both biopsy specimens from the quadriceps femoris and biceps brachii had the similar “patchy” immunostaining pattern as shown in Figure 4. These muscle fibers that react abnormally for
636 Annals of Neurology Vol 28 No 5 November 1990
Fig 3. Indirect immunofluowscence staining of frozen muscle sectrons with anti-dystrophin antiserum. Note the continuous, cleur normal immunostaining ofdystrophin in normal muscle (A), and the discontinuous, ‘patchy” membrane staining of reduced intensity in patients with quadriceps myopathy (Patients 1, 2, and 4 correspond t o panels B, C, and D, respectzvelyj (Bar = 25 microns.) I
dystrophin arc considered to be nonnecrotic fibers by standard histochemistry or by immunocytochemistry for complement C9. In addition, “patchy” reacted fibers had continuous and clear immunostaining of the surface membrane for spectrin (data not shown), suggesting the primary role of “patchy” immunostaining for dystrophin in muscle of patients with Becker muscular dystrophy. In Patient 6, the mother of Patients 3 and 5 (see Fig l C ) , the biopsied specimen from the biceps brachii showed a normal pattern. IMMUNOBLOITING STUDIES. Immunoblotting tests identified abnormal molecular mass (370 to 380 kd; normal, 400 kd) and decreased amount of dystrophin (50 to 90%; normal, 100) in all 4 patients with quadriceps myopathy, a finding considered diagnostic of Becker muscular dystrophy {14]. Although the approximate quantities of dystrophin were reduced, all patients showed levels greater than 50% of normal
(Patient 1, 50%; Patient 2, 60%; Patient 3 , 50%; Patient 4, 90%). As all 4 patients showed dystrophin of abnormal molecular mass (Patient 1, 370 kd; Patient 2, 380 kd; Patient 3, 380 kd; Patient 4, 380 kd), it is expected that they should all have deletions of the dystrophin gene C14, 157. This was in fact the case; all 4 patients had deletions of the dystrophin gene (A. Beggs and L. Kunkel, personal communication). The molecular mass and amount of dystrophin in Patient 5 were 380 kd and loo%, respectively. Patient 6 expressed two different molecular masses of dystrophin, 380 kd and 400 kd, and the relative contents of these were 80 and 10096, respectively (Fig 5).
Discussion The present study provides the first evidence that patients with quadriceps myopathy have dystrophin abnormahties of Becker muscular dystrophy type. This was confirmed in two ways. First, immunofluorescent staining of dystrophin at the surface membrane of the muscle fiber showed a faint and discontinuous “patchy” pattern that is consistent with the immunohistological characteristics of muscle from patients with Becker muscular dystrophy [9, 101. Second, immunoblot tests revealed smaller sizes or Sunohara et al: Quadriceps Myopathy
637
Fig 4. Indirect immunoj’uorescent analysis of diffewnt muscles in Patient 3 with quadriceps myopathy. Two muscle specimens were obtainedseparatelyfrom the quadricepsfernoris (A and B) and the biceps brachii (C and 0).Frozen sections ofihese muscles stained with anti-dystrophin antiserum (B and D), together with sections stained by hematoxylin and eosin (A and C), are shown. Both the quadriceps and biceps muscles have equally shown chronic myopathic changes including muscle fiber hypertrophy, internal nuclei. and the “patchy”immunostaining of dystrophin. (Bar = 25 microns.)
reduced quantity, or both, of the dystrophin protein, a pattern that is diagnostic of Becker muscular dystrophy 1141. Three of 4 patients (Patients 1, 2, and 3) with quadriceps myopathy studied had intragenic deletions of dystrophin gene (Beggs AH, et al., unpublished data). From these results, we infer that these patients should in fact be considered to have Becker muscular dystrophy. Although the amount of dystrophin protein relative to the normal control samples was somewhat reduced, in our patients with quadriceps myopathy, it was still approximately 50 to 9095 of normal. Therefore, one can speculate that the benign clinical course of quadriceps myopathy results from the relatively high quantities of dystrophin 1141. Patient 5 had more generalized clinical manifesta-
tions of Becker muscular dystrophy in both arms and legs, but he showed dystrophic abnormalities similar to those of Patient 3, on muscle immunofluorescence. We found the more severely affected brother to have higher quantities of dystrophin (100%) relative to his brother with quadriceps myopathy (50%). Whereas previous reports have found a general correlation between dystrophin levels and clinical severity Cl2, 141, this correlation did not seem to hold for dystrophin levels above 20%, as in our patients. Indeed, the minimally affected biceps brachii muscle of Patient 3 still showed an abnormal dysuophin pattern indistinguishable from that of his severely affected quadriceps femoris muscles. Thus, we cannot provide a rational explanation for the differences in clinical expression between the two brothers. The probable role of extragenic factors or environmental factors, or both, in the expression of Becker dystrophy will be an important area of future research. It is interesting to note that we were able to detect a female carrier of Becker muscular dystrophy by dystrophin immunoblot testing of her biopsy specimen (see Fig 5). This is the first report of dystrophin testing as applied to carriers of Becker muscular dystrophy. Whether such a test for carriers could be of impor-
638 Annals of Neurology Vol 28 No 5 November 1990
touch on the cardiac abnormality. Severe cardiac involvement was observed in 2 of our 4 patients with quadriceps myopathy, a forme fruste of Becker muscular dystrophy. Although Emery and Skinner [l6] reported that none of their patients with Becker muscular dystrophy had early myocardial involvement, a more recent study found ECG abnormalities in over half of patients with true Becker muscular dystrophy [14]. Thus, special attention should be given to cardiac muscle involvement even when the clinical manifestations of skeletal muscle are very mild.
References Fig 5 . Dystrophin immunoblot analysis of solubilized muscle biopsy specimensfrom the indicatedpatients. The lanes marked as “NormaP’ contain solubilized musclefrom a patient with a disorder unrelated to DuchennelBeckerdystrophy, and show the normal dystrophin pattern. Patient 3 has a clinical distribution of weakness consistent with quadriceps myopathy, yet shows dystrophin of a b n o m l molecular mass (black triangle) and reduced quantities that are characteristicof Becker muscular dystrophy. Patient 3’s mother (Patient 6) shows both normal dystrophin, and the dystrophin of abnormal molecular mass (black triangle), indicating that she is a cawierfar the mutant dystrophin gene. Patient 3’s brother (Patient 5 ) also has the same abnormal dystrophin, yet his clinicalphenotype is more consistent with typical Becker muscular dystrophy. Patient 4 is a second, unrelated patient with apparent quadricepJ-myopathy, but with a dystrophin pattern characteristicof Becker muscular dystrophy.
tance in family counseling will clearly require more comprehensive studies. Our results reinforce the importance of dystrophin tests for the proper clinical diagnosis and genetic counseling of patients with quadriceps myopathy. In general, it appears difficult to distinguish, clinically, quadriceps myopathy from Becker muscular dystrophy { 161, limb-girdle muscular dystrophy [11, chronic polymyositis [5,61, and chronic spinal muscular atrophy [7, 81, particularly when an X-linked inheritance pattern is not evident and when affected muscles are confined to the proximal regions of the legs. Indeed, a number of patients carrying diagnoses of either spinal muscular atrophy 1171 or limb-girdle muscular dystrophy [181 have been shown by molecular analyses to actually have Becker muscular dystrophy. Thus, although the “quadriceps syndrome” may result from a variety of heterogeneous diseases, at least a subset of these patients in fact have Becker muscular dystrophy. Of interest, careful neurological, EMG, and histological examinations of our patients with quadriceps myopathy revealed that muscle involvement was not confined to the quadriceps femoris but was more widespread. Therefore, we have to emphasize that the term “quadriceps myopathy” is not suitable for such patients. Finally, from the clinical point of view, we should
1. Walton JN. Two cases of myopathy limited to the quadriceps. J Neurol Neurosurg Psychiatry 1956;19:106-108 2. Van Wijngaarden GK, Hagen CJ, Bethlem J, Meijer AEFH. Myopathy of the quadriceps muscles. J Neurol Sci 1968;7:201206 3. Espir MLE, Matthews WB. Hereditary quadriceps myopathy. J Neurol Neurosurg Psychatry 1973;36:1041-1045 4. Swash M, Heathfield KWG. Quadriceps myopathy: a variant of the limb-girdle dystrophy syndrome. J Neurol Neurosurg Psychiatry 1983;46:355-3 5 7 5. Denny-Brown D. Mgopathic weakness of quadriceps. Proc R SOCMed (Neurol) 1939;32:49-50 6. Turner JWA, Heathfield KWG. Quadriceps myopathy occurring in middle age. J Neurol Neurosurg Psychiatry 1961;24:1821 7. Furukawa T, Akagami N, Maruyama S. Chronic neurogenic quadriceps amyotrophy. Ann Neurol 1977;2:528-530 8. Serratrice G, Pou-Serradel A, Pellissier JF, et al. Chronic neurogenic quadriceps amyotrophies. J Neurol 1985;232:150-153 9. Arahata K, Hoffman EP, Ishiura S, et al. Dystrophin diagnosis: comparison of dystrophin abnormalities by immunoblot and immunofluorescenr analyses. Proc Natl Acad Sci USA 1989;45: 498-506 10. Arahata K, Ishiura S, Ishiguro T, et al. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988;333:861863 11. Arahata K, Ishihara T, Kamakura K, et al. Mosaic expression of dystrophin in symptomatic carriers of Duchenne’s muscular dystrophy. N Engl J Med 1989;320:138-142 12. Hoffman EP, Fischbeck KH, Brown RH, et al. Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy patients. N Engl J Med 1988;318:13631368 13. Sunohara N, Takagi A, Nonaka I, et al. Two cases with quadriceps myopathy of juvenile onset. Clin Neurol 1981;21:321329 14. Hoffman EP, Kunkel LM, Angelini C, et al. Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology 1989;39:1011-1017 15. Monaco AP, Bertelson CJ, Liechti-Gallati S, et al. An explanation for the phenotypic differences between patients beating partial deletions of the DMD locus. Genomics 1988;2:90-95 16. Emery AEH, Skinner R. Clinical studies in benign (Becker type) X-linked muscular dystrophy. Clin Genet 1970;10:189201 17. Lunt PW, Cumming WJK, Kingston H , et al. DNA probes in differential diagnosis of Becker muscular dystrophy and spinal muscular atrophy. Lancet 1989;1:46-47 18. Norman A, Thomas N , Coakley J, Harper P. Distinction of Becker from limb-girdle muscular dystrophy by means of dystrophin cDNA probes. Lancet 1989;1:466-468 Sunohara e t al: Quadriceps Myopathy
639
n
P
Quadriceps Myopathy: Porme Pruste of: Becker Muscular Dystrophy Nobuhiko Sunohara, MD,* Kiichi Arahata, MD,? Eric P. Hoffman, PhD,q Hitoshi Yamada, MD,” Jin Nishimiya, MD,$ Eri Arikawa, MD,t Misako Kaido, MD,t Ikuya Nonaka, MD,S and Hideo Sugita, M D t
We examined dystrophin, the protein product of the Duchenne muscular dystrophy gene, in muscle biopsy specimens from 4 male patients with quadriceps myopathy, all of whom showed a mild and slowly progressive myopathy confined to the quadriceps muscles. All 4 patients had clear abnormalities of dystrophin, and were diagnosed as having Becker muscular dystrophy by both immunofluorescence and immunoblot examinations; that is, dystrophin of an abnormal molecular mass was visualized in muscle cryosections as “patchy” or discontinuous immunostaining at the surface membrane of the muscle fibers. One patient had a brother who showed widespread myopathic changes consistent with typical Becker muscular dystrophy. We conclude that the syndrome called quadriceps myopathy includes a group of forme fruste Becker muscular dystrophy. Sunohara N, Arahata K, Hoffman EP, Yamada H, Nishimiya J, Arikawa E, Kaido M, Nonaka I, Sugita H. Quadriceps rnyopathy: forme fruste of Becker muscular dystrophy. Ann Neurol 1990;28:634-639
Cases of patients with muscle wasting and weakness limited to the quadriceps femoris have been occasionally reported 11-61. These patients with so-called “quadriceps myopathy” have been regarded as suffering from a heterogeneous group of disorders, including a form of limb-girdle muscular dystrophy El], a unique form of muscular dystrophy 12-41, polymyositis [ S , 61, or a form of chronic spinal muscular atrophy [7, 81. Nine of the 1 7 patients previously described r1-83 were male, and 7 patients had a positive family history. We identified 4 patients with a clinical picture consistent with quadriceps myopathy in the 3,048 patients who underwent biopsy and who were studied in our clinic over the last 12 years. In this report, we studied dystrophin, the protein product of the DuchenneIBecker muscular dystrophy gene, in the muscle biopsy specimens of these 4 patients. All 4 patients had dystrophin patterns consistent with Becker muscular dystrophy, indicating that quadriceps myopathy is an unusual presentation of Becker muscular dystrophy.
he was admitted to our hospital. H e showed moderate wasting and weakness of the quadriceps femoris muscles (Medical Research Council [MRC] Scale: 3/5). There was hypertrophy of the calf muscles. Gowers’ sign was not recognized. Deep tendon reflexes were normally active, except for diminished Achilles’ tendon reflexes. Serum creatine kinase (CK) levels were 10 to 20 times the upper limit of normal values. Needle electromyography (EMG) showed positive sharp waves at rest, and short-duration, low-amplitude polyphasic motor units with long-duration, high-amplitude potentials on voluntary contraction. When he was reexamined at the age of 37 years, he exhibited similar neurological findings to those observed at the previous admission plus a mild Gowers’ sign. Computed tomographic (CT) scan revealed marked atrophy of the quadriceps femoris muscles, but other muscles were well preserved (Fig 2). Electrocardiography (ECG) disclosed no abnormality. The pedigree of this individual is shown in Figure 1. The mother of the patient (11-7) showed no abnormality on neurological examination, but her serum CK level was slightly elevated (153 unitdliter; normal, 12 to 75 unitdliter). The maternal uncle had suffered from similar symptoms, and died at age 40.
Patients and Methods Case History 1. A 37-year-old man (I114 in Fig 1A) became aware of hypertrophy of the calf muscles at the age of 10 years. At the age of 18 years he had sllght difficulty in climbing stairs and wasting of the thigh muscles. At 27 years old
PATIENT 2. A 41-year-old man noticed hypertrophy of the calf muscles at the age of 11 years. From the age of 15 years he could not run and from the age of 20 he had difficulty in climbing stairs. At the age of 32 he was admitted to the National Center Hospital (Musashi). He displayed marked muscular wasting of the quadriceps femoris muscles and
From the *Department of Neurology, National Center Hospital (Musashi),and the tDivisions of Neuromuscular Research and $Ul-
Received Feb 21, 1990, and in revised form Apr 26. Accepted for publication May 14. 1990.
PATIENT
Children’s Hospital, Boston, MA.
634 Copyright 0 1990 by the American Neurological Association
a 0
I
I
0 3
?
0 4
11
A
111
I
i
. j
I
. .I
I1 111
Oo0.D 2 I
B
3 7 4
0 5
N
I I1
C
Ill
Fig I. Pedigrees of families. (A) Family of Patient 1. (B) Family of Patient 2. (C) Family aPatient 3. Note that Patient 5 is 111-1 and Patient 6 is 11-12 in farnib C. W = Propositus, A = affected individuals, @ = death, = cam'er.
pseudohypertrophy of the calf muscles. There was muscular weakness of the quadriceps femoris muscles (MRC: 3/5), iliopsoas muscles (3/5), and gluteal muscles (4+/5). Serum CK levels were 5 to 24 times the upper limit of normal. Needle EMG demonstrated fibrillation potentials and positive sharp waves at rest, and short-duration, small-amplitude polyphasic potentials on voluntary contraction. At age 40 he had an episode of cardiac failure due to cardiomyopathy with dilatation. Since then, he has been treated with 0.1 mg of metildigoxin and 40 mg of furosemide per day. At the age of 41 years we examined him again. H e had no muscular weakness or wasting of the neck and upper limb-girdle muscles. The lower girdle muscles revealed normal strength, except for the quadriceps femoris (MRC: 3/5), iliopsoas (2/5), and gluteal (4/5) muscles. The pedigree of his family is shown in Figure 1B. The family members examined (11-8, 11-11, 111-1, 111-2, 111-3, III-5,III-6, IV-1, and IV-2) had no neuromuscular disorders and their serum CK levels were within normal limits. PATIENT 3. A 31-year-old man noticed slight difficulty in climbing stairs at the age of 25 years, but he did not think it abnormal. H e was a ping-pong player while at the university, and he can play even now. Neurological examination disclosed marked atrophy of the quadriceps muscles bilaterally and mild pseudohypertrophy of the calf muscles. Mild weakness of the iliopsoas (MRC: 4/5) and quadriceps muscles (4/ 5 ) was detected. Deep tendon reflexes were normally active. Serum CK and aldolase levels were elevated (16 and 3 times
the upper limit of normal, respectively). Needle EMG examination revealed low-amplitude and short-duration polyphasic potentials in the muscles of both legs. CT scan showed marked atrophy of the quadriceps muscles (see Fig 2). ECG and echocardiogram disclosed no abnormality. The family tree is shown in Figure 1C. His brother (Patient 5, 111-1 in Fig lC), age 32, had predominantly proximal muscular weakness and wasting, and pseudohypertrophy of the calf muscles, features compatible with typical Becker muscular dystrophy. The serum CK level was 5 times the upper limit of normal. C T scan revealed marked atrophy of the quadriceps and biceps femoris, iliopsoas, and paravertebral muscles, and mild to moderate atrophy and patchy lowdensity areas in the biceps and triceps brachii muscles (see Fig 2). EMG showed short-duration, small-amplitude polyphasic potentials on voluntary contraction. On ECG and echocardiogram, there was no abnormality. The patient's mother (Patient 6, 11-12 in Fig lC), age 56, had slight weakness of the sternocleidomastoideus muscles. A needle EMG examination on voluntary contraction disclosed small amounts of short-duration, low-amplitude polyphasic potentials, but she had no elevation of serum CK levels or abnormality on muscle CT scan. ECG disclosed no abnormality. Family members of 11-1 and 111-3 had no abnormality on neurological examination and no elevation of serum CK levels. PATIENT 4. A 3 1-year-old man noticed difficulty in walking at the age of 19 years old. At age 21, his thighs became thin. At age 31 he had congestive heart failure due to dilated cardiomyopathy. Neurologically, he had marked muscular wasting and weakness (MRC grade: 3/5) of the quadriceps femoris muscles bilaterally. Serum CK level was elevated 7 times the upper limit of normal.
Mascle Biopsies Muscle biopsy specimens were obtained from the left quadriceps femoris (Patients 1, 2, 3, and 4) and biceps brachii (Patients 1, 2, 3, 5 , and 6) for diagnostic purposes, with the informed consent of all subjects. Serial frozen sections were stained by hematoxylin and eosin, modified Gomori trichrome, and various histochemical stainings. The same muscle biopsy samples were used for both immunofluorescence and immunoblot analyses.
Dystrophin Analyses IMMUNOREAGENTS AND CEDURES. Four different
IMMUNOHISTOCHEMICAL
PRO-
polyclonal anti-dystrophin antisera, which have been previously described [9}, were used in this study. The cryosections to be immunoreacted were picked up on gelatinized coverslips and were processed as described before C9- 11). IMMUNOBLOTTING. Immunoblot detection of dystrophin was done as published previously l12). The relative abundance values we have assigned to dystrophin are qualitative approximations relative to the muscle protein (myosin) in the control samples on the same gel. These calculations were based on the average values obtained in two or more experiments.
Sunohara et al: Quadriceps Myopathy
635
Fig 2. CT scans oftheforearm (A,D,and G), thigh (B, E, and H), and lumbar spinal (C, I;, and I ) muscles in Patient 1 (A,B, and C), Patient 2 ID, E , and P), and Patient 5 {G,H , and I). The quadriceps muscles of Patients 1 (B) and 3 /E) are markedly involved (arrows).Howeyer, the biceps and triceps brachii muscles of Patients I (A)and 3 (D), the biceps femoris muscles o f Patients 1 (B) and 3 (E), and the Paravertebral muscles of Patients 1 (C) and 3 (Pi are wellpreserved. The low-density awas in (A)are artifacts. The biceps and triceps brachri muscles (G),the biceps fernoris muscles (Hj, and the paravertebral muscles (I) are markedly damaged in Patient 5 (triangles).
ther, grouped atrophy of fibers indicative of denervation were only found in the quadriceps muscles in Patients l, 2, and 3 (Patients l and 2 are Cases 2 and l in [13], respectively). Fiber type grouping was also seen in the quadriceps femoris muscle specimen of Patient 2. Patient 5 , the elder brother of Patient 3 (111-1in Fig lC), was diagnosed as having mild Becker muscular dystrophy, and the biopsy specimen from his biceps brachii showed similar myopathic abnormalities. DystTophin Tests
Results General Mascle Pathological Featares
In all patients, biopsied muscle specimens showed marked variation in fiber size, fiber splitting, mild to moderate increase in the number of internal nuclei, occasional grouped fiber regeneration, aberrant myofibrillar network, and mild increase in endomysial and perimysial fibrous connective tissue, suggesting active and chronic myopathy consistent with muscular dystrophy. Although both the quadriceps and biceps muscles appeared to be involved histopathologically, the quadriceps samples were more severely affected. Fur-
IMMUNOFLUORESCENCE STUDIES. Dystrophin in the muscle specimens was analyzed with immunofluorescence. All 4 patients with quadriceps myopathy (Patients l, 2, 3, and 4 ) and Patient 5 showed a similar immunostaining pattern for dystrophin, which is consistent with Becker muscular dystrophy, that is, “patchy,” discontinuous, and faint immunoreaction at the surface membrane of the skeletal muscle fibers (Figs 3 and 4). In Patient 3, both biopsy specimens from the quadriceps femoris and biceps brachii had the similar “patchy” immunostaining pattern as shown in Figure 4. These muscle fibers that react abnormally for
636 Annals of Neurology Vol 28 No 5 November 1990
Fig 3. Indirect immunofluowscence staining of frozen muscle sectrons with anti-dystrophin antiserum. Note the continuous, cleur normal immunostaining ofdystrophin in normal muscle (A), and the discontinuous, ‘patchy” membrane staining of reduced intensity in patients with quadriceps myopathy (Patients 1, 2, and 4 correspond t o panels B, C, and D, respectzvelyj (Bar = 25 microns.) I
dystrophin arc considered to be nonnecrotic fibers by standard histochemistry or by immunocytochemistry for complement C9. In addition, “patchy” reacted fibers had continuous and clear immunostaining of the surface membrane for spectrin (data not shown), suggesting the primary role of “patchy” immunostaining for dystrophin in muscle of patients with Becker muscular dystrophy. In Patient 6, the mother of Patients 3 and 5 (see Fig l C ) , the biopsied specimen from the biceps brachii showed a normal pattern. IMMUNOBLOITING STUDIES. Immunoblotting tests identified abnormal molecular mass (370 to 380 kd; normal, 400 kd) and decreased amount of dystrophin (50 to 90%; normal, 100) in all 4 patients with quadriceps myopathy, a finding considered diagnostic of Becker muscular dystrophy {14]. Although the approximate quantities of dystrophin were reduced, all patients showed levels greater than 50% of normal
(Patient 1, 50%; Patient 2, 60%; Patient 3 , 50%; Patient 4, 90%). As all 4 patients showed dystrophin of abnormal molecular mass (Patient 1, 370 kd; Patient 2, 380 kd; Patient 3, 380 kd; Patient 4, 380 kd), it is expected that they should all have deletions of the dystrophin gene C14, 157. This was in fact the case; all 4 patients had deletions of the dystrophin gene (A. Beggs and L. Kunkel, personal communication). The molecular mass and amount of dystrophin in Patient 5 were 380 kd and loo%, respectively. Patient 6 expressed two different molecular masses of dystrophin, 380 kd and 400 kd, and the relative contents of these were 80 and 10096, respectively (Fig 5).
Discussion The present study provides the first evidence that patients with quadriceps myopathy have dystrophin abnormahties of Becker muscular dystrophy type. This was confirmed in two ways. First, immunofluorescent staining of dystrophin at the surface membrane of the muscle fiber showed a faint and discontinuous “patchy” pattern that is consistent with the immunohistological characteristics of muscle from patients with Becker muscular dystrophy [9, 101. Second, immunoblot tests revealed smaller sizes or Sunohara et al: Quadriceps Myopathy
637
Fig 4. Indirect immunoj’uorescent analysis of diffewnt muscles in Patient 3 with quadriceps myopathy. Two muscle specimens were obtainedseparatelyfrom the quadricepsfernoris (A and B) and the biceps brachii (C and 0).Frozen sections ofihese muscles stained with anti-dystrophin antiserum (B and D), together with sections stained by hematoxylin and eosin (A and C), are shown. Both the quadriceps and biceps muscles have equally shown chronic myopathic changes including muscle fiber hypertrophy, internal nuclei. and the “patchy”immunostaining of dystrophin. (Bar = 25 microns.)
reduced quantity, or both, of the dystrophin protein, a pattern that is diagnostic of Becker muscular dystrophy 1141. Three of 4 patients (Patients 1, 2, and 3) with quadriceps myopathy studied had intragenic deletions of dystrophin gene (Beggs AH, et al., unpublished data). From these results, we infer that these patients should in fact be considered to have Becker muscular dystrophy. Although the amount of dystrophin protein relative to the normal control samples was somewhat reduced, in our patients with quadriceps myopathy, it was still approximately 50 to 9095 of normal. Therefore, one can speculate that the benign clinical course of quadriceps myopathy results from the relatively high quantities of dystrophin 1141. Patient 5 had more generalized clinical manifesta-
tions of Becker muscular dystrophy in both arms and legs, but he showed dystrophic abnormalities similar to those of Patient 3, on muscle immunofluorescence. We found the more severely affected brother to have higher quantities of dystrophin (100%) relative to his brother with quadriceps myopathy (50%). Whereas previous reports have found a general correlation between dystrophin levels and clinical severity Cl2, 141, this correlation did not seem to hold for dystrophin levels above 20%, as in our patients. Indeed, the minimally affected biceps brachii muscle of Patient 3 still showed an abnormal dysuophin pattern indistinguishable from that of his severely affected quadriceps femoris muscles. Thus, we cannot provide a rational explanation for the differences in clinical expression between the two brothers. The probable role of extragenic factors or environmental factors, or both, in the expression of Becker dystrophy will be an important area of future research. It is interesting to note that we were able to detect a female carrier of Becker muscular dystrophy by dystrophin immunoblot testing of her biopsy specimen (see Fig 5). This is the first report of dystrophin testing as applied to carriers of Becker muscular dystrophy. Whether such a test for carriers could be of impor-
638 Annals of Neurology Vol 28 No 5 November 1990
touch on the cardiac abnormality. Severe cardiac involvement was observed in 2 of our 4 patients with quadriceps myopathy, a forme fruste of Becker muscular dystrophy. Although Emery and Skinner [l6] reported that none of their patients with Becker muscular dystrophy had early myocardial involvement, a more recent study found ECG abnormalities in over half of patients with true Becker muscular dystrophy [14]. Thus, special attention should be given to cardiac muscle involvement even when the clinical manifestations of skeletal muscle are very mild.
References Fig 5 . Dystrophin immunoblot analysis of solubilized muscle biopsy specimensfrom the indicatedpatients. The lanes marked as “NormaP’ contain solubilized musclefrom a patient with a disorder unrelated to DuchennelBeckerdystrophy, and show the normal dystrophin pattern. Patient 3 has a clinical distribution of weakness consistent with quadriceps myopathy, yet shows dystrophin of a b n o m l molecular mass (black triangle) and reduced quantities that are characteristicof Becker muscular dystrophy. Patient 3’s mother (Patient 6) shows both normal dystrophin, and the dystrophin of abnormal molecular mass (black triangle), indicating that she is a cawierfar the mutant dystrophin gene. Patient 3’s brother (Patient 5 ) also has the same abnormal dystrophin, yet his clinicalphenotype is more consistent with typical Becker muscular dystrophy. Patient 4 is a second, unrelated patient with apparent quadricepJ-myopathy, but with a dystrophin pattern characteristicof Becker muscular dystrophy.
tance in family counseling will clearly require more comprehensive studies. Our results reinforce the importance of dystrophin tests for the proper clinical diagnosis and genetic counseling of patients with quadriceps myopathy. In general, it appears difficult to distinguish, clinically, quadriceps myopathy from Becker muscular dystrophy { 161, limb-girdle muscular dystrophy [11, chronic polymyositis [5,61, and chronic spinal muscular atrophy [7, 81, particularly when an X-linked inheritance pattern is not evident and when affected muscles are confined to the proximal regions of the legs. Indeed, a number of patients carrying diagnoses of either spinal muscular atrophy 1171 or limb-girdle muscular dystrophy [181 have been shown by molecular analyses to actually have Becker muscular dystrophy. Thus, although the “quadriceps syndrome” may result from a variety of heterogeneous diseases, at least a subset of these patients in fact have Becker muscular dystrophy. Of interest, careful neurological, EMG, and histological examinations of our patients with quadriceps myopathy revealed that muscle involvement was not confined to the quadriceps femoris but was more widespread. Therefore, we have to emphasize that the term “quadriceps myopathy” is not suitable for such patients. Finally, from the clinical point of view, we should
1. Walton JN. Two cases of myopathy limited to the quadriceps. J Neurol Neurosurg Psychiatry 1956;19:106-108 2. Van Wijngaarden GK, Hagen CJ, Bethlem J, Meijer AEFH. Myopathy of the quadriceps muscles. J Neurol Sci 1968;7:201206 3. Espir MLE, Matthews WB. Hereditary quadriceps myopathy. J Neurol Neurosurg Psychatry 1973;36:1041-1045 4. Swash M, Heathfield KWG. Quadriceps myopathy: a variant of the limb-girdle dystrophy syndrome. J Neurol Neurosurg Psychiatry 1983;46:355-3 5 7 5. Denny-Brown D. Mgopathic weakness of quadriceps. Proc R SOCMed (Neurol) 1939;32:49-50 6. Turner JWA, Heathfield KWG. Quadriceps myopathy occurring in middle age. J Neurol Neurosurg Psychiatry 1961;24:1821 7. Furukawa T, Akagami N, Maruyama S. Chronic neurogenic quadriceps amyotrophy. Ann Neurol 1977;2:528-530 8. Serratrice G, Pou-Serradel A, Pellissier JF, et al. Chronic neurogenic quadriceps amyotrophies. J Neurol 1985;232:150-153 9. Arahata K, Hoffman EP, Ishiura S, et al. Dystrophin diagnosis: comparison of dystrophin abnormalities by immunoblot and immunofluorescenr analyses. Proc Natl Acad Sci USA 1989;45: 498-506 10. Arahata K, Ishiura S, Ishiguro T, et al. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988;333:861863 11. Arahata K, Ishihara T, Kamakura K, et al. Mosaic expression of dystrophin in symptomatic carriers of Duchenne’s muscular dystrophy. N Engl J Med 1989;320:138-142 12. Hoffman EP, Fischbeck KH, Brown RH, et al. Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy patients. N Engl J Med 1988;318:13631368 13. Sunohara N, Takagi A, Nonaka I, et al. Two cases with quadriceps myopathy of juvenile onset. Clin Neurol 1981;21:321329 14. Hoffman EP, Kunkel LM, Angelini C, et al. Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology 1989;39:1011-1017 15. Monaco AP, Bertelson CJ, Liechti-Gallati S, et al. An explanation for the phenotypic differences between patients beating partial deletions of the DMD locus. Genomics 1988;2:90-95 16. Emery AEH, Skinner R. Clinical studies in benign (Becker type) X-linked muscular dystrophy. Clin Genet 1970;10:189201 17. Lunt PW, Cumming WJK, Kingston H , et al. DNA probes in differential diagnosis of Becker muscular dystrophy and spinal muscular atrophy. Lancet 1989;1:46-47 18. Norman A, Thomas N , Coakley J, Harper P. Distinction of Becker from limb-girdle muscular dystrophy by means of dystrophin cDNA probes. Lancet 1989;1:466-468 Sunohara e t al: Quadriceps Myopathy
639
