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Muscle histochemistry in myotubular (centronuclear) myopathy
Muscle Histochemistry in Myotuhular (Centronuclear) Myopathy Takashi Sasaki, MD, Keiko Shikura, MD, Kenji Sugai, MD, Ikuya Nonaka, MD and Komei Kumagai, MD
We report the clinical and histochemical findings in 7 patients with myotubular (centronuclear) myopathy aged from 2 months to 32 years. The clinical symptoms varied from patient to patient. Three patients developed severe muscle weakness and hypotonia with respiratory distress from infancy, and 4 had muscle weakness from 2-5 years of age with no apparent delay in developmental milestones. In addition to an increased number of fibers with centrally placed nuclei, there were 3 other histochemical characteristics of this disorder, i.e., type 1 fiber predominance, type 1 fiber hypotrophy and type 2B fiber deficiency. Other histological findings included a peripheral halo in the sarcoplasm on NADH- TR staining and an increased number of undifferentiated type 2C fibers, indicating a delay in muscle fiber growth and differentiation due to a probable defective neural supply in the developing muscles. Key words: Myotubular myopathy, centronuclear myopathy, muscle histochemistry, muscle fiber immaturity. Sasaki T, Shikura K, Sugai K, Nonaka I, Kumagai K Muscle histochemistry in myotubular (centronuclear) myopathy. Brain Dev 1989; 11: 26-32
The term, myotubular myopathy, was first coined by Spiro et al [1], who described a male patient with outstanding pathological fmdings, including a high proportion of muscle fibers with central nuclei and the presence of perinuclear halos mimicking myotubes in the fetal muscle. They thought that these abnormal fibers resulted from the arrest of muscle fiber growth. Since the muscle fibers in this disorder show good differentiation into either type 1 or 2 fibers, which are different from the myotubes with immature and undifferentiated histological characteristics, various synonyms have been proposed instead of "myotubular" myopathy, such as centronuclear myopathy [2], pericentronuclear myopathy [3], and type 1 fiber hypotrophy and central nuclei [4]. The disorder can be classified clinically into 3 subtypes based on the time of onset of the disease [5], Le., in-
From the Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo (TS, KS, KS, IN); Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo (TS); Kanagawa Rehabilitation Center, Nanasawa, Kanagawa (KK). Received for publication: February 3, 1988. Accepted for publication: November 18, 1988. Correspondence address: Dr. Takashi Sasaki, Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan.
fantile, childhood and adult forms. In the present study, 3 patients were classified as the infantile form, because they had muscle hypotonia and respiratory distress from birth, and the other 4 as the childhood form, who were noted to have muscle weakness at the age of 2- 5 years. To clarify as to whether or not the muscle pathology reflects a delay in muscle fiber growth, and to determine the fiber type differentiation and morphological differences between the infantile and childhood forms, morphometric analysis as to fiber type distribution was performed on patients of different ages. PATIENTS AND METHODS
The clinical symptoms are summarized in Table 1. Patients 1-3 exhibited respiratory distress, hypotonia and marked muscle weakness from birth. Patients 4-7 showed no delay in developmental milestones, but muscle weakness was noted when they learned to walk alone at 2 to 4 years of age. Facial muscle involvement was noted in 5 patients and ptosis in 1. The CK was normal and EMG showed a normal to mild myopathic pattern in all patients. A muscle biopsy was performed on the biceps brachii in all patients. The muscle specimens were frozen in isopentane cooled in liqUid nitrogen. Serial frozen sections were stained with hematoxylin and eosin (H & E), and various other histochemical methods, including modi-
Table I Clinical summaries 7
6
5
Patient
1
2
3
Sex
M
M
M
F
M
F
F
9 yrs
18 yrs
32 yrs
2 yrs 6 mos
3 yrs
4-5 yrs
I yr 2 mos
I yr
Not delayed
I yr 6 mos
Prox
Gen
Dist
Prox
4
2 yrs
Neo
5 mos Neo
8 yrs
Neo
5 yrs
+ +
+ +
+ +
Gen
Gen
Gen
+
+
+
±
Ptosis
+ +
CK
N
N
N
+ N
N
Age at biopsy Onset of disease Respiratory distress in infancy Delay in developmental milestones
2 mos
Walked alone Muscle weakness Facial involvement
N
N
M: male, F: female, Neo: neonatal, Gen: general, Dist: distal dominant, Prox: proximal dominant, N: normal
Table 2
Summary of the muscle histochemistry in 5 patients with myotubular myopathy
Patient
1
2
3
4
5
6
7
++
++
++
++
++ ++
+
+
Change in fiber size Fiber hypotrophy Type 1
++
++
Type 2A
++
++
Fiber hypertrophy Type 1 Type 2A
+
Small angular fibers Change in distribution Type 1 fiber predominance
++
++
++
++
++
++ +
++ +
++
++
++
+
+
+
Type 2A fiber deficiency Type 2B fiber deficiency Increase of type 2C fibers
+
++ ++ ++ +
++
+
++
++
Internal nuclei Single
+
+
Multiple Degeneration & regeneration Cellular response Inflammatory Fibrosis Architectural change Perin uclear clear zone Peripheral halo Radial cell structure "Targetoid"
+ + ++
++
++
++
++
++
+
+ + + +
-: none, +: mild to moderate, ++: marked.
fied Comori trichrome, NADH-tetrazolium reductase (NADH-TR), oil red 0, periodic acid Schiff, acid phosphatase, alkaline phosphatase, nonspeCific esterase, cytochrome c oxidase, and myosin adenosine triphosphatase (AJPase) with alkaline and acid preincubation. Photographs were taken of identical fields in serial sec-
tions stained with ATPase at a final magnification of x 1,000, and then type 1, 2A, 2B and 2C fibers were classified. The diameter of approximately 500 muscle fibers in each muscle specimen was measured with a MOPAMO 3 semiautomatic image analyzer.
Sasaki et al: Myotubular myopathy 27
Fig 1 Increased number of muscle fibers with internally placed single to multiple nuclei. Patient 7. H & E, x 50. Fig 2 Fibers with a peripheral halo devoid of enzyme activity in the subsarcolemmal region and a central pale zone mimicking a "targetoid" structure. Patient 2. NADH-TR, x 100.
28 Brain & Development, Vol 11, No 1,1989
Fig 3 Fibers with a radial cell strncture (arrow). Patient 6. NADH-TR, x 100. Fig 4 Sl1Ulll type 1 (1) fibers and type 1 fiber predominance. Type 2C (C) fibers are scattered throughout. Patient 5. ATPase (pH4.2). x 50.
Sasaki et al: Myotubular myopathy 29
Table 3 Morphometric analysis of biopsied muscles Patient
Sex
Age *
No of fibers measured
No of centrally nucleated fibers
1
M
2 mos
1,082
10.3%
2
M
5 mos
801
3
M
2 yrs
4
F
5
Type 2A
Type 2B
Type 2C
60.0% (5.2 ± 5.3) **
33.3% (6.7 ± 7.0)
0
6.7% (5.7 ± 6.2)
10.4%
75.0% (11.5±3.7)
20.1% (5.3 ± 1.2)
0.7% (6.6 ± 3.2)
4.1% (6.7 ± 1.7)
1,439
10.0%
86.3% (14.6 ± 4.6)
12.8% (20.9 ± 12.5)
0.2% (20.4 ± 8.8)
0.7% 02.0 ± 3.5)
8 yrs
451
14.6%
71.2% 07.5 ± 2.9)
28.8% (49.5 ± 5.7)
0
0
M
9 yrs
457
29.9%
69.8% (27.3 ± 5.6)
12.3% (38.2 ± 11.0)
1.3% (42.6 ± 12.3)
16.2% (28.5 ± 9.4)
6
F
18 yrs
513
33.7%
92.2% (17.4 ± 7.4)
1.8% 07.9 ± 9.3)
0.8% (30.7 ± 3.5)
5.3% 00.5 ± 3.3)
7
F
32 yrs
853
50.6%
71.4% (37.2 ± 7.4)
28.3% (42.2 ± 7.4)
0.2% (44.2 ± 8.9)
* age at the time of biopsy, **mean fiber diameter
(~m) ±
Type 1
0.1% (13.3)
SD.
RESULTS
DISCUSSION
The major histochemical findings are summarized in Table 2. Central nuclei were present in 10 to 50% of the fibers. They tended to increase in number with age. In patient 7, aged 32 years, fibers with centrally placed nuclei amounted to 50.6%, 11)ost of which were multiple (Fig 1). The nuclei were occasionally surrounded by a clear zone (perinuclear halo) in 2 patients. No evidence of muscle degeneration or regeneration was seen. On NADH-TR staining, the aggregation of enzyme activity in the center of fibers surrounded by a pale halo (peripheral halo) or a central unstained area mimicking a targetoid structure was observed in 5 patients (Fig 2). These architectural changes were predominantly seen in younger patients 1- 3. In some fibers, a peculiar distribution of intermyofibri1lar networks forming a radial cell structure was seen in patient 6 (Fig 3). Type 1 fiber predominance, ranging from 60.0 to 92.2%, was observed in all patients (Table 3). In addition, in these patients, type 2B fibers were absent. Four patients had a moderately increased number of type 2C fibers, from 4.1 to 16.2% (Fig 4). Although both type 1 and 2 fibers in 3 patients were small, the former were much more hypoplastic (type 1 fiber hypotrophy) in 6 patients. In patient 2, type 2A fibers measured 5.3 11m in diameter, which was smaller than type 1 fibers, which had a mean diameter of 11.5 11m. In diameter histograms for individual muscle specimens, type 1 fibers showed a unimodal distribution in 6 patients and a bimodal one in 1 who had both type 2A and 2B fiber deficiency (Fig 5).
Although the clinical symptoms varied from patient to patient, all exhibited the clinical and histopathological characteristics of myotubular myopathy, including the early onset of non- to slowly progressive generalized muscle weakness involving facial muscle, no serum CK elevation and normal to myopathic EMG findings. Muscle fibers with centrally placed nuclei were present at a high frequency, with an abnormal histochemistry in the central portion of the fibers; perinuclear halos, high oxidative enzyme activities representing dark-centered fibers (peripheral halos) and radial cell structures. In addition, we found the following common histochemical abnormalities; i) type 1 fiber hypotrophy, ii) type 1 fiber predominance and iii) type 2B fiber deficiency. Type 2C fibers were slightly increased in number in 4 patients. The term "myotubular" myopathy has been applied to this disorder because the muscle fibers with an abnormal morphology in the central region are similar to those in immature muscle, and are thought to result from the arrest of muscle fiber maturation [1]. However, the present study again confirmed that most muscle fibers had differentiated well into type 1 or 2 fibers, unlike undifferentiated myotubes. Heckmatt et al [6] also reported the good differentiation of fiber types in all their patients. Since Engel et al [4] found that type 1 fibers with central nuclei were smaller than type 2 fibers in a patient, they proposed the term, type 1 fiber hypotrophy and central nuclei. As Bethlem et al [7] and Karpati et al [8] reported, type 1 fiber hypotrophy and predominance are commonly present in myotubular myopathy, the
30 Brain & Development, Volll, No 1, 1989
B
A 500
Patient 3,
2Y
200
Patient 7,
32Y
400
en
"II)
!II "-
....
-
"-
.0 100
.0
II)
300 :e ....
o
o
"II)
II)
.0
E
;:, 200
Z
:::J
E
Z
100
--
10
.. ..........
___ 2a __ _
--
40
50
60
I'm
Diameter
10
20
30
40
50
60
70
80
90
pm
Diameter
Fig 5 Representative firber type histograms. Patient 3 at the age of 2 years (A) and patient 7 at 32 years (E).
name proposed by Engel et al is no longer used. In addition, type 1 fiber hypotrophy and predominance, and type 2B fiber deficiency are not specific changes in this disorder, but are commonly seen in various congenital non progressive myopathies (CNM), including nemaline myopathy, central core disease and congenital fiber type disproportion [9-14]. A certain abnormal neural influence upon the developing muscles causing the abnormal fiber type distribution may playa role in the pathogenetic mechanism. Since central nuclei, which are regarded as one of the diagnostic findings in this disorder, are seen at a higher frequency in older patients, they seem to increase and become multiple with age [5]. Therefore the presence of central nuclei may not be closely related to the centrally placed nuclei in myotubes. The fibers with a peripheral halo characterized by the central aggregation of the stain on the NADH-TR reaction, the increase in immature type 2C fibers seen in patients 1, 2, 5 and 6, and the small muscle fibers are highly suggestive of a delay in muscle fiber growth. In spite of the small number of centrally nucleated fibers (10.0-14.6%), the muscles in the infantile form have outstanding histochemical characteristics of myotube-like immaturity, Le., a peripheral halo in the sarcoplasm on NADH-TR staining. The histochemical
difference in muscle immaturity between the infantile and childhood forms may be closely related to the muscle weakness and the onset of the disease. A defective neural influence on immature fibers may induce a delay in muscle fiber growth and differentiation resulting in the arrest of fetal muscle fiber maturation after birth. Because of the histochemical evidence of muscle fiber immaturity in the infantile form, the term "myotubular" myopathy should be reserved. ACKNOWLEDGMENTS The authors wish to express their thanks to Professor Haruo Ichihashi (Kyorin University School of Medicine) for the suggestions and advice on this work.
REFERENCES 1. Spiro AJ, Shy GM, Gonatas NK. Myotubuiar myopathy.
Arch Neural 1966;14:1-14. 2. Bethlem J, Meijer AEFH, Schellens JPM. Centronuc1ear myopathy. Eur Neural 1968; 1: 325-33. 3. Campbell MJ, Rebeiz JJ, Walton IN. Myotubular, centronuclear or pericentronuclear myopathy? J Neural Sci 1969; 8:425-43. 4. Engel WK, Gold GN, Karpati G. Type 1 fiber hypotrophy and central nuclei. Arch Neural 1968; 18:435-44.
Sasaki et al: Myotubular myopathy
31
5. Goebel HH, Meinck HM, Reinecke M, Schimrigk K, Mielke U. Centronuc1ear myopathy with special consideration of the adult form. Eur Neurol 1984;23:425-34. 6. Heckmatt IZ, Sewry CA, Hodes D, Dubowitz V. Congenital centronuc1ear (myotubular) myopathy. Brain 1985; 108: 941-64. 7. Bethlem I, van Wijngaarden GK, Mumenthaler M, Meijer AEFH. Centronuclear myopathy with type 1 fiber atrophy and myotubes. Arch Neurol 1970;23: 70-3. 8. Karpati G, Carpenter S, Nelson RF. Type 1 muscle fiber atrophy and central nuclei. J Neurol Sci 1970; 10:489-500. 9. Fardeau M. Relevance of morphologic studies in the classification and pathophysiology of congenital myopathies. In: Serratrice G, Cros D, Desnuelle C, et ai, eds. Neuromuscular diseases. New York: Raven Press, 1984:201-6.
32 Brain & Development, Vol 11, No 1,1989
10. Brooke MH. Congenital fiber type disproportion. In: Kakulas BA, ed. Ginical studies in myology. Vol 2. Amsterdam: Excepta Medica, 1973:147-59. 11. Dubowitz V. Muscle biopsy: a practical approach. London: Bailliere Tindall, 1985. 12. Carpenter S, Karpati G. Pathology of skeletal muscle. New York· Edinburgh. London· Melbourne: Churchill Livingstone, 1984. 13. Wroblewski R, Edstrom L, Mair WGP. Five different types of centrally nucleated muscle fibers in man: Elemental composition and morphological criteria. J Submicrosc Cytol 1982; 14:377-87. 14. Sarnat HB, Roth SI, Jimenez IF. Neonatal myotubular myopathy: neuropathy and failure of postnatal maturation of fetal muscle. Can J Neural Sci 1981;8 :313-20.
We report the clinical and histochemical findings in 7 patients with myotubular (centronuclear) myopathy aged from 2 months to 32 years. The clinical symptoms varied from patient to patient. Three patients developed severe muscle weakness and hypotonia with respiratory distress from infancy, and 4 had muscle weakness from 2-5 years of age with no apparent delay in developmental milestones. In addition to an increased number of fibers with centrally placed nuclei, there were 3 other histochemical characteristics of this disorder, i.e., type 1 fiber predominance, type 1 fiber hypotrophy and type 2B fiber deficiency. Other histological findings included a peripheral halo in the sarcoplasm on NADH- TR staining and an increased number of undifferentiated type 2C fibers, indicating a delay in muscle fiber growth and differentiation due to a probable defective neural supply in the developing muscles. Key words: Myotubular myopathy, centronuclear myopathy, muscle histochemistry, muscle fiber immaturity. Sasaki T, Shikura K, Sugai K, Nonaka I, Kumagai K Muscle histochemistry in myotubular (centronuclear) myopathy. Brain Dev 1989; 11: 26-32
The term, myotubular myopathy, was first coined by Spiro et al [1], who described a male patient with outstanding pathological fmdings, including a high proportion of muscle fibers with central nuclei and the presence of perinuclear halos mimicking myotubes in the fetal muscle. They thought that these abnormal fibers resulted from the arrest of muscle fiber growth. Since the muscle fibers in this disorder show good differentiation into either type 1 or 2 fibers, which are different from the myotubes with immature and undifferentiated histological characteristics, various synonyms have been proposed instead of "myotubular" myopathy, such as centronuclear myopathy [2], pericentronuclear myopathy [3], and type 1 fiber hypotrophy and central nuclei [4]. The disorder can be classified clinically into 3 subtypes based on the time of onset of the disease [5], Le., in-
From the Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo (TS, KS, KS, IN); Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo (TS); Kanagawa Rehabilitation Center, Nanasawa, Kanagawa (KK). Received for publication: February 3, 1988. Accepted for publication: November 18, 1988. Correspondence address: Dr. Takashi Sasaki, Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan.
fantile, childhood and adult forms. In the present study, 3 patients were classified as the infantile form, because they had muscle hypotonia and respiratory distress from birth, and the other 4 as the childhood form, who were noted to have muscle weakness at the age of 2- 5 years. To clarify as to whether or not the muscle pathology reflects a delay in muscle fiber growth, and to determine the fiber type differentiation and morphological differences between the infantile and childhood forms, morphometric analysis as to fiber type distribution was performed on patients of different ages. PATIENTS AND METHODS
The clinical symptoms are summarized in Table 1. Patients 1-3 exhibited respiratory distress, hypotonia and marked muscle weakness from birth. Patients 4-7 showed no delay in developmental milestones, but muscle weakness was noted when they learned to walk alone at 2 to 4 years of age. Facial muscle involvement was noted in 5 patients and ptosis in 1. The CK was normal and EMG showed a normal to mild myopathic pattern in all patients. A muscle biopsy was performed on the biceps brachii in all patients. The muscle specimens were frozen in isopentane cooled in liqUid nitrogen. Serial frozen sections were stained with hematoxylin and eosin (H & E), and various other histochemical methods, including modi-
Table I Clinical summaries 7
6
5
Patient
1
2
3
Sex
M
M
M
F
M
F
F
9 yrs
18 yrs
32 yrs
2 yrs 6 mos
3 yrs
4-5 yrs
I yr 2 mos
I yr
Not delayed
I yr 6 mos
Prox
Gen
Dist
Prox
4
2 yrs
Neo
5 mos Neo
8 yrs
Neo
5 yrs
+ +
+ +
+ +
Gen
Gen
Gen
+
+
+
±
Ptosis
+ +
CK
N
N
N
+ N
N
Age at biopsy Onset of disease Respiratory distress in infancy Delay in developmental milestones
2 mos
Walked alone Muscle weakness Facial involvement
N
N
M: male, F: female, Neo: neonatal, Gen: general, Dist: distal dominant, Prox: proximal dominant, N: normal
Table 2
Summary of the muscle histochemistry in 5 patients with myotubular myopathy
Patient
1
2
3
4
5
6
7
++
++
++
++
++ ++
+
+
Change in fiber size Fiber hypotrophy Type 1
++
++
Type 2A
++
++
Fiber hypertrophy Type 1 Type 2A
+
Small angular fibers Change in distribution Type 1 fiber predominance
++
++
++
++
++
++ +
++ +
++
++
++
+
+
+
Type 2A fiber deficiency Type 2B fiber deficiency Increase of type 2C fibers
+
++ ++ ++ +
++
+
++
++
Internal nuclei Single
+
+
Multiple Degeneration & regeneration Cellular response Inflammatory Fibrosis Architectural change Perin uclear clear zone Peripheral halo Radial cell structure "Targetoid"
+ + ++
++
++
++
++
++
+
+ + + +
-: none, +: mild to moderate, ++: marked.
fied Comori trichrome, NADH-tetrazolium reductase (NADH-TR), oil red 0, periodic acid Schiff, acid phosphatase, alkaline phosphatase, nonspeCific esterase, cytochrome c oxidase, and myosin adenosine triphosphatase (AJPase) with alkaline and acid preincubation. Photographs were taken of identical fields in serial sec-
tions stained with ATPase at a final magnification of x 1,000, and then type 1, 2A, 2B and 2C fibers were classified. The diameter of approximately 500 muscle fibers in each muscle specimen was measured with a MOPAMO 3 semiautomatic image analyzer.
Sasaki et al: Myotubular myopathy 27
Fig 1 Increased number of muscle fibers with internally placed single to multiple nuclei. Patient 7. H & E, x 50. Fig 2 Fibers with a peripheral halo devoid of enzyme activity in the subsarcolemmal region and a central pale zone mimicking a "targetoid" structure. Patient 2. NADH-TR, x 100.
28 Brain & Development, Vol 11, No 1,1989
Fig 3 Fibers with a radial cell strncture (arrow). Patient 6. NADH-TR, x 100. Fig 4 Sl1Ulll type 1 (1) fibers and type 1 fiber predominance. Type 2C (C) fibers are scattered throughout. Patient 5. ATPase (pH4.2). x 50.
Sasaki et al: Myotubular myopathy 29
Table 3 Morphometric analysis of biopsied muscles Patient
Sex
Age *
No of fibers measured
No of centrally nucleated fibers
1
M
2 mos
1,082
10.3%
2
M
5 mos
801
3
M
2 yrs
4
F
5
Type 2A
Type 2B
Type 2C
60.0% (5.2 ± 5.3) **
33.3% (6.7 ± 7.0)
0
6.7% (5.7 ± 6.2)
10.4%
75.0% (11.5±3.7)
20.1% (5.3 ± 1.2)
0.7% (6.6 ± 3.2)
4.1% (6.7 ± 1.7)
1,439
10.0%
86.3% (14.6 ± 4.6)
12.8% (20.9 ± 12.5)
0.2% (20.4 ± 8.8)
0.7% 02.0 ± 3.5)
8 yrs
451
14.6%
71.2% 07.5 ± 2.9)
28.8% (49.5 ± 5.7)
0
0
M
9 yrs
457
29.9%
69.8% (27.3 ± 5.6)
12.3% (38.2 ± 11.0)
1.3% (42.6 ± 12.3)
16.2% (28.5 ± 9.4)
6
F
18 yrs
513
33.7%
92.2% (17.4 ± 7.4)
1.8% 07.9 ± 9.3)
0.8% (30.7 ± 3.5)
5.3% 00.5 ± 3.3)
7
F
32 yrs
853
50.6%
71.4% (37.2 ± 7.4)
28.3% (42.2 ± 7.4)
0.2% (44.2 ± 8.9)
* age at the time of biopsy, **mean fiber diameter
(~m) ±
Type 1
0.1% (13.3)
SD.
RESULTS
DISCUSSION
The major histochemical findings are summarized in Table 2. Central nuclei were present in 10 to 50% of the fibers. They tended to increase in number with age. In patient 7, aged 32 years, fibers with centrally placed nuclei amounted to 50.6%, 11)ost of which were multiple (Fig 1). The nuclei were occasionally surrounded by a clear zone (perinuclear halo) in 2 patients. No evidence of muscle degeneration or regeneration was seen. On NADH-TR staining, the aggregation of enzyme activity in the center of fibers surrounded by a pale halo (peripheral halo) or a central unstained area mimicking a targetoid structure was observed in 5 patients (Fig 2). These architectural changes were predominantly seen in younger patients 1- 3. In some fibers, a peculiar distribution of intermyofibri1lar networks forming a radial cell structure was seen in patient 6 (Fig 3). Type 1 fiber predominance, ranging from 60.0 to 92.2%, was observed in all patients (Table 3). In addition, in these patients, type 2B fibers were absent. Four patients had a moderately increased number of type 2C fibers, from 4.1 to 16.2% (Fig 4). Although both type 1 and 2 fibers in 3 patients were small, the former were much more hypoplastic (type 1 fiber hypotrophy) in 6 patients. In patient 2, type 2A fibers measured 5.3 11m in diameter, which was smaller than type 1 fibers, which had a mean diameter of 11.5 11m. In diameter histograms for individual muscle specimens, type 1 fibers showed a unimodal distribution in 6 patients and a bimodal one in 1 who had both type 2A and 2B fiber deficiency (Fig 5).
Although the clinical symptoms varied from patient to patient, all exhibited the clinical and histopathological characteristics of myotubular myopathy, including the early onset of non- to slowly progressive generalized muscle weakness involving facial muscle, no serum CK elevation and normal to myopathic EMG findings. Muscle fibers with centrally placed nuclei were present at a high frequency, with an abnormal histochemistry in the central portion of the fibers; perinuclear halos, high oxidative enzyme activities representing dark-centered fibers (peripheral halos) and radial cell structures. In addition, we found the following common histochemical abnormalities; i) type 1 fiber hypotrophy, ii) type 1 fiber predominance and iii) type 2B fiber deficiency. Type 2C fibers were slightly increased in number in 4 patients. The term "myotubular" myopathy has been applied to this disorder because the muscle fibers with an abnormal morphology in the central region are similar to those in immature muscle, and are thought to result from the arrest of muscle fiber maturation [1]. However, the present study again confirmed that most muscle fibers had differentiated well into type 1 or 2 fibers, unlike undifferentiated myotubes. Heckmatt et al [6] also reported the good differentiation of fiber types in all their patients. Since Engel et al [4] found that type 1 fibers with central nuclei were smaller than type 2 fibers in a patient, they proposed the term, type 1 fiber hypotrophy and central nuclei. As Bethlem et al [7] and Karpati et al [8] reported, type 1 fiber hypotrophy and predominance are commonly present in myotubular myopathy, the
30 Brain & Development, Volll, No 1, 1989
B
A 500
Patient 3,
2Y
200
Patient 7,
32Y
400
en
"II)
!II "-
....
-
"-
.0 100
.0
II)
300 :e ....
o
o
"II)
II)
.0
E
;:, 200
Z
:::J
E
Z
100
--
10
.. ..........
___ 2a __ _
--
40
50
60
I'm
Diameter
10
20
30
40
50
60
70
80
90
pm
Diameter
Fig 5 Representative firber type histograms. Patient 3 at the age of 2 years (A) and patient 7 at 32 years (E).
name proposed by Engel et al is no longer used. In addition, type 1 fiber hypotrophy and predominance, and type 2B fiber deficiency are not specific changes in this disorder, but are commonly seen in various congenital non progressive myopathies (CNM), including nemaline myopathy, central core disease and congenital fiber type disproportion [9-14]. A certain abnormal neural influence upon the developing muscles causing the abnormal fiber type distribution may playa role in the pathogenetic mechanism. Since central nuclei, which are regarded as one of the diagnostic findings in this disorder, are seen at a higher frequency in older patients, they seem to increase and become multiple with age [5]. Therefore the presence of central nuclei may not be closely related to the centrally placed nuclei in myotubes. The fibers with a peripheral halo characterized by the central aggregation of the stain on the NADH-TR reaction, the increase in immature type 2C fibers seen in patients 1, 2, 5 and 6, and the small muscle fibers are highly suggestive of a delay in muscle fiber growth. In spite of the small number of centrally nucleated fibers (10.0-14.6%), the muscles in the infantile form have outstanding histochemical characteristics of myotube-like immaturity, Le., a peripheral halo in the sarcoplasm on NADH-TR staining. The histochemical
difference in muscle immaturity between the infantile and childhood forms may be closely related to the muscle weakness and the onset of the disease. A defective neural influence on immature fibers may induce a delay in muscle fiber growth and differentiation resulting in the arrest of fetal muscle fiber maturation after birth. Because of the histochemical evidence of muscle fiber immaturity in the infantile form, the term "myotubular" myopathy should be reserved. ACKNOWLEDGMENTS The authors wish to express their thanks to Professor Haruo Ichihashi (Kyorin University School of Medicine) for the suggestions and advice on this work.
REFERENCES 1. Spiro AJ, Shy GM, Gonatas NK. Myotubuiar myopathy.
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