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Muscle histochemistry in the Prader-Willi syndrome
ELSEVIER
Brain & Development 1994; 16:183-8
Original Article
Muscle histochemistry in the Prader-Willi syndrome Sui Sone, MD
In a follow-up study of 259 floppy infants of undetermined cause in my laboratory, 11 patients were later diagnosed as having the Prader-Willi syndrome (PWS). To clarify the pathogenesis of muscle hypotonia in PWS, I examined muscle biopsies by histochemical and morphometric methods and the results were compared with those obtained from floppy infants with only mental retardation but with no other features. The histochemical abnormalities of PWS included (i) fiber size variation of both type 1 and 2 fibers, (ii) type 2 fiber atrophy, (iii) increased numbers of type 2C fibers, and (iv) decreased numbers of type 2B fiber. Although muscle hypotonia in PWS has been thought to be due to central nervous system abnormality, my findings suggest that primary muscle pathology, including muscle fiber immaturity and abnormal muscle fiber type distribution, at least in part, plays a role in muscle hypotonia and weakness. Key words: Prader-Willi syndrome; Muscle histochemistry; Fiber size variation in type 1 fiber; Increased type 2C fiber; Type 2B fiber deficiency; Floppy infant syndrome
1. I N T R O D U C T I O N The Prader-Willi syndrome (PWS) is characterized by muscle hypotonia, obesity, hypogonadism, short stature, small hands and feet, and mental deficiency [1]. The hypotonia is sometimes so severe that the patients exhibit the 'frog leg' posture, and their sucking reflex may be so weak as to require tube feeding [21. These patients are occasionally suspected of having some type of neuromuscular disease and may be evaluated with a muscle biopsy. In a follow-up study of 259 patients with unex-
Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan and Department of Pediatrics, Gifu University School of Medicine, Gifu, Gifu, Japan Received 25 March 1993; accepted 4 February 1994 Correspondence address: Dr. S. Sone, Department of Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan. Fax: (81) (423) 46-1749.
0387-7604/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0387-7604(94)00025-S
plained severe congenital hypotonia studied with a muscle biopsy, 11 patients were later found to have PWS on the basis of their clinical or chromosomal abnormalities. I have reviewed the muscle biopsies on these patients in an attempt to determine the reason for the muscle hypotonia in PWS.
2. PATIENTS AND M E T H O D S 259 muscle biopsies from infants under 3 years of age with muscle hypotonia, delayed developmental milestones and probable muscle weakness, from 1983 to 1987, were examined histochemically. Questionnaires were sent to their physicians to determine the present status of these patients. At least 5 years had elapsed since the biopsy. Among them, 11 patients were later diagnosed as having PWS clinically a n d / o r by chromosomal analysis. Muscle biopsies had been done between the ages of 8 - 3 4 months (average 13 months). Clinical features of PWS patients are summarised in Table 1. Patient 11 was reported by Miike et al. [3] as
184
S. Sone / Brain & l)evelopmettt 1994; 16. 183 ,~
"Fable
Summary (~f patients with the Prader-Willi syndrome
I
Patient
No. 1
2
3
4
5
6
7
S
9
10
11
Sex
M
F
F
F
M
M
F
M
M
M
M
Age at biopsy N e o n a t a l difficulties
8m
7m
25m
7m
6m
7m
Im
34m
5m
Im
2m
+
+
~-
+
+
Hypotonia
+
+
+
+
+
+
+
~
+
+
P o o r sucking Tubc feeding t Iigh-arched palate
+
+
+
+
+
+
+
~
+
+
-
+
-
+
+
+
+
-
Hypogonadism
+
NE
+
hands and feet Other malformations * * Small
Age at last examination
9y
NIE
+
NIE
+
-
+
+
--
+
+
Mental retardation Abnormal 15q
-
+
4 y :~
Obesit,,
+
+
-
+
+
+
~-
--
+
Iy
Im
4y
6y
43'
5y
S m :~
34m
+
+
+
+
NE
NE
+
+
+
+
+
+
-
NE
-
+
+
-
+
+
NE
NE
NE
NE
+
+
+
+
9).
known neuromuscular disease but who had had muscle biopsies were selected as controls. They were hypotonic during infancy but were able to walk unassisted and had no muscle weakness when last examined. They had had muscle biopsies between 8-27 months of age (average 13 months). The biceps brachii muscle was biopsied in all the patients. Muscle specimens were frozen in isopentane cooled in liquid nitrogen, and serial frozen sections of 10 /xm thickness were stained with hematoxylin and eosin ( H & E ) , modified Gomori trichrome (mGT) and a battery of histochemical methods, including N A D H tetrazolium reductase ( N A D H ) , succinic dehydrogenase, periodic acid Schiff, oil red O, routine ATPase, ATPase with preincubation at pH 4.6 and 4.3, nonspecific esterase, acid phosphatase, alkaline phos-
Summat3, ~)f histochemical jindings Controls
Patients
No.
I
Fiber size variation both in type I and 2 fibers Type 2 fiber atrophy T y p e 1 fiber atroph}. T y p e 2B fiber deficiency Increased type 2 C fiber Decreased CCO
+
~-
1 Iy
an atypical PWS. All patients were hypotonic and hypogonadism was noted in all male patients. Poor sucking and mental retardation were found in most patients. The number of patients documented to have small hands and feet a n d / o r high arched palate was small, since some of the clinical reports available at the time of biopsy made no reference to these features. About half of the patients were obese when last examined. Two patients had died, one from pneumonia (Patient 2) and the other from disseminated intravascular coagulopathy and acute renal failure (Patient 7). Six of 9 patients examined cytogenetically had an abnormality in chromosome 15. There was no difference in the clinical features between patients with and without this chromosomal abnormality. Eight patients who had mental retardation with no
2
-
N E
M . m a l e ; F . f e m a l e ; N E , not examined. :~ Age of death. * * Congenital hydronephrosis, micrognathia (Patient 4), hypospadia (Patient 8), cleft palate (Patient
Table
-
2
+
+
+
+
+
-
-
+
7
8
9
l0
11
+
+
+
+
+
-
ql +
,--
+
+
+
--
. +
+
+
+
+
-
activity
Increased lipid droplets
+
6
+ .
-
-
5
+ .
-
4
+
.
+
3
+
-
+
-
+
1
2
3
4
5
6
+
--
+
7
8
S. Sone / Brain & Development 1994; 16:183-8 Table 3 Patient No.
185
Morphometric analysis of muscle biopsies Age
Number of analyzed fibers
Muscle fiber type Type 1
Type 2A
Type 2B
Type 2C
23.7% 16.1±3.7" 28.8% 23.4±4.5 32.1% 22.2±5.6 42.6% 21.1±3.8 22.5% 22.0±4.1 30.9% 20.9±4.0 22.7% 20.2±3.5 47.1% 13.0±3.3 32.0% 18.0±3.4 25.1% 13.8±3.3 18.8% 11.8±2.5
45.9% 12.6±1.9 39.3% 19.2±2.8 63.2% 13.8±2.7 36.5% 16.1±2.7 53.3% 13.6±2.7 62.9% 14.7±1.9 38.2% 15.1±2.1 48.6% 9,0±1.4 50.8% 14.5±2.6 41.2% 12.2±2.4 71.1% 10.5±1.8
24.5% 12.1±2.3 31.0% 19.3±2.7 4.7% 12.8±2.2 3.9% 15.8±3.4 15.7% 11.6±2.2 2.4% 13.1±2.1 20.7% 15.0±2.1 2.3% 9.0±1.2 14.6% 16.6±2.7 31.1% 13.6±2.6 6.0% 10.2±0.9
5.9% 12.9±2.4 0.9% 18.3±3.9 0%
1
8m
1,210
2
7m
633
3
25m
700
4
7m
854
5
6m
612
6
7m
614
7
lm
767
8
34m
784
9
3m
650
10
lm
847
11
2m
996
17.0% 18.2±2.7 8.5% 14.7±2.5 3.7% 15.9±5.3 12.4% 16.9±2.2 2.0% 8.7±2.1 2.6% 13.8+2.4 2.6% 11.8±3.6 4.1% 9.5±1.9
* Mean diameter, ~ m + S.D.
Fig. 1. In addition to variation in type 1 (1) fiber size, type 2A (2A) fibers are atrophic. Type 2B (2B) fibers are decreased in number and type 2C (2C) fibers are increased in number. Patient 1. A: H&E. B: routine ATPase. C: ATPase (pH 4.6). D: ATPase (pH 4.3). A - D (serial sections) ×588.
186
S. Sone /Brain & Decelopment 1994; 16:183 8
Fig. 2. Myopathic changes consisting of marked variation m the size of type 1 (1) fibers with deficiency of type 2B fibers. Type 2A (2A) fibers are atrophic and 2C (2C) fibers increased in number. Patient 8. A: ATPase (pH 4.6). B: ATPase (pH 4.3). A,B (serial sections) ×400.
phatase, cytochrome c oxidase (CCO), phosphofructokinase, A M P deaminase and acetylcholinesterase. Photographs were taken of identical fields in serial sections stained with ATPase after preincubation at the three pH settings at a final magnification of × 1,000, and then type 1, 2A, 2B and 2C fibers were classified.
3. RESULTS The major histological findings are summarized in Table 2, and morphometric analysis of PWS patients in Table 3. Except for mild fiber size variation in all biopsies from PWS patients, no apparent morphologic change was seen on H & E and m G T (Fig. 1A). Muscle fibers appeared to be mature with no increase in the number
Table 4 Mean size and t'ariation of type 1 fibers in P W S and control groups (mean diameter, Ixm +_S.D.) Patient no.
PWS group
Control group
1 2 3 4 5 6 7 8 9 10
16.1 _+3.7 23.4 + 4.5 22.2_+5.6 21.1 _+3.8 22.0+4.1 211.9 + 4.11 20.2 _+3.5 13.0!3.3 18.11+3.4 13.8+3.3
19.5+2.6 20.1 + 2.9 23.6+ 3.1 13.6+2.2 17.4+_ 1.8 22.9 + 3.3 20.5 +_3.2 23.6+4.1
of fibers with centrally placed nuclei. All patients exhibited variation in the size of type 1 fibers (Fig. 1) and 3 patients had small type 1 fibers. In contrast to PWS patients, the mean size of type 1 fibers in the control group was normal, with less variation in their size (Table 4). In the coefficient of variance of the standard deviation of type 1 fibers (Wilcoxon' s U-test) there was a statistically significant difference between the two groups ( P < 0.01). There was type 2B fiber deficiency in 4 (Fig. 2, Table 3) of the PWS group, whereas there was none in the control group. Increased numbers of undifferentiated type 2C fibers and type 2 fiber atrophy were present in both groups (Fig. 1, Table 2), but they were more predominant in the PWS group. With N A D H , intermyofibrillar networks were normally developed. None of fibers had increased acid or alkaline phosphatase activity. With the other stains, there was no abnormality except for one patient who had decreased CCO activity (Fig. 3). There was no morphologic difference between PWS patients with chromosomal abnormality (Patients 4, 5, 8, 9, 10 and 11) and those without (Patients 1, 3 and 6).
4. DISCUSSION The clinical course of PWS has been divided into the hypotonic stage of early infancy and the mentally retarded stage after 1 to 2 years of age [1]. The degree
S. Sone ~Brain & Deuelopment 1994; 16:183-8
187
Fig. 3. Decreased cytochrome c oxidase activity seen in Patient 4 (A). Control muscle (B). A,B (cytochrome c oxidase) x 490.
of muscle hypotonia associated with poor suck, the cardinal symptom of the first stage, varied significantly from patient to patient. When the muscle hypotonia was so marked and appeared to be associated with muscle weakness, a muscle biopsy was sometimes done to rule out a neuromuscular disorder. Muscle symptoms gradually improve, usually between 8 and 11 months of age [4,5], and then mental retardation, obesity and hypogonadism become manifest [1]. Some patients have prolonged hypotonia with severely retarded motor development [3]. Since Ledbetter et al. [6] reported chromosome 15 deletion in PWS and Butler et al. [7] reported that PWS was manifested when chromosome 15 with the deletion is derived from the father, genetic studies of PWS have progressed rapidly. The deletion on the long arm of chromosome 15 is present in approximately half of the PWS patients and is assumed to be related to the hypopigmentation [8]. However, a portion of PWS patients have no abnormality in chromosome 15. These patients may be shown to have a maternal disomy if a genetic survey of these patients were possible. Several studies have been done to clarify the patho-
physiology of the hypotonia in the PWS patients. Afifi and Zellweger [9] examined PWS muscles by light as well as electron microscopy. Although they found some ultrastructural changes including subsarcolemmal mitochondrial aggregates, abnormal Z-line and myofilamentous disarray and loss, they concluded that these abnormalities were nonspecific and too mild to explain the muscle symptoms in PWS. Since histochemical examination did not show any architectural change such as mitochondrial aggregation or disorganized intermyofibrillar networks, ultrastructural examination may not provide any information to explain pathogenetic mechanism. Hirayama et al. [10] also found nonspecific minor pathologic changes and proposed that there were some defects in trophic influence on the developing muscle from the central nervous system (CNS). Since other histochemical and electron microscopic studies also showed no pathognomonic fndings to explain the muscle hypotonia, the muscle symptom in PWS has been thought to result from some undetermined CNS abnormality [9,11,12], or from the small muscle bulk [13]. In this study, there were two striking findings, in-
188
S. Sone / B r a m & Det,elopment 1994; 16:183 8
cluding m a r k e d variation in the size of type 1 fibers and a deficiency in type 2B fibers which were not seen in the control group of m e n t a l l y r e t a r d e d floppy infants. A l t h o u g h there was no disease-specific pathologic abormality in PWS patients, the striking c o m m o n findings of variation in the size of type 1 and 2 fibers, a b n o r m a l fiber type distribution, including occasional type 2B fiber deficiency, a n d increased n u m b e r s of u n d i f f e r e n t i a t e d type 2C fibers suggest a primary muscle involvement. Type 2 fiber atrophy itself is a n o n specific finding c o m m o n l y seen in a variety of conditions, including disuse, cerebral palsy, m e n t a l retardation, and d e g e n e r a t i v e diseases (these may all be examples of disuse). Type 2B fiber deficiency is a c o m m o n finding in muscles from patients with c o n g e n i t a l non-progressive myopathies (CNM), including n e m a l i n e myopathy a n d central core disease, and the end-stage of progressive m u s c u l a r dystrophies [14,15]. Type 1 fiber atrophy a n d p r e d o m i n a n c y , the major p a t h o g n o m o n i c findings in CNM, were not f o u n d in the PWS patients. T h e r e are increased n u m b e r s of u n d i f f e r e n t i a t e d type 2C fibers in muscles u n d e r g o i n g active r e g e n e r a t i o n after myonecrosis, d u r i n g r e - i n n e r v a t i o n following d e n e r v a t i o n and in i m m a t u r i t y [14-16]. Since there was no necrotizing or d e n e r v a t i n g process in PWS muscles, increased n u m b e r s of type 2C fiber are t h o u g h t to reflect muscle fiber immaturity, as suggested by H i r a y a m a et al. [10]. In conclusion, variations in the size of type 1 fibers (probable myopathic change) and type 2B fiber deficiency suggest that the pathogenesis of the hypotonia of PWS is not entirely due to CNS a b n o r m a l i t y but is, at least in part, due to an a b n o r m a l i t y in the muscle itself. ACKNOWLEDGEMENTS The author is grateful to Dr. Ikuya Nonaka (National Institute of Neuroscience, NCNP) and Professor Tadao Orii (Department of Pediatrics, Gifu University School of Medicine) for their advice and suggestions on this work. The author wishes to express cordial thanks to Professor S.M. Sumi (Department of Pathology, University of Washington) for his kind suggestions and criticisms on this work.
REFERENCES 1. Butler MG. Prader-Willi syndrome: current understanding of cause and diagnosis. A m J Med Genet 1990; 35: 319-32. 2. Cassidy SB. Prader-Willi syndrome. In: Gluck, L. Coen RW, eds. Current problems in pediatrics. New York: Year Book Medical Publishers. 1984; 5-55. 3. Miike T, Ogata T, Ohtani Y, Yamaguchi H, Yokoyama Y. Atypical Prader-Willi syndrome with severe developmental delay and emaciation. Brain Det~ (Tokyo) 1988; 10: 186-8. 4. Butler MG, Meaney FJ, Palmer CG. Clinical and cytogenetic survey of 39 individuals with Prader-Labhart-Willi syndrome. A m J Med Genet 1986; 23: 793-809. 5. Hall BD, Smith DW. Prader-Willi syndrome. JPediatr 1972; 81: 286-93. 6. Ledbetter DH, Riccardi VM, Airhart SD, et al. Deletions of chromosome 15 as a cause of the Prader-Willi syndrome. N Engl J Med 1981; 304: 325-9. 7. Butler MG, Palmer CG. Parental origin of chromosome 15 deletion in Prader-Willi syndrome. Lancet 1983; i: 1285-6. 8. Wiesner GL, Bende CM, Olds DP, et al. Hypopigmentation in the Prader-Willi syndrome. A m J Hum Genet 1987; 40:431-4 9. Afifi AK, Zellweger H. Pathology of muscular hypotonia in the Prader-Willi syndrome. J Neurol Sci 1969; 9: 49-61. 10. Hirayama Y, Suzuki H, Ohsawa M. Histopathological background of muscle hypotonia in children with the Prader-Willi syndrome. Acta Paediatr Jpn (Tokyo) 1981; 23: 294-300. 11. Dubowitz V. Prader-Willi syndrome (hypotonia-obesity syndrome). In: Dubowitz V. Muscle disorders in childhood. London: Saunders, 1978: 227-31. 12. Zellweger H, Schneider HJ. Syndrome of hypotonia-hypomentia-hypogonadism-obesity (HHHO) or Prader-Willi syndrome. A m J Dis Child 1968; 115: 588-98. 13. Klish W, Brown B, Lin T, Lee P, Greenberg F. Studies of body composition in patients with Prader-Willi syndrome: implications for management. A m J Med Genet 1992; 42: 245-6. 14. Shimomura C, Nonaka I. Nemalinc myopathy: comparative muscle histochemistry in the severe neonatal, moderate congenital and adult-onset forms. Pediatr Neurol 1989; 5: 25-31. 15. Nonaka I, Sugita H, Takada K, Kumagai K. Muscle histochemistry in congenital muscular dystrophy with central nervous system involvement. Musele Nerve 1982; 5: 102-6. 16. Nonaka I, Takagi A, Sugita H. The significance of type 2C muscle fibers in Duchenne muscular dystrophy. Muscle Nert,e 1981; 4: 326-33.
Brain & Development 1994; 16:183-8
Original Article
Muscle histochemistry in the Prader-Willi syndrome Sui Sone, MD
In a follow-up study of 259 floppy infants of undetermined cause in my laboratory, 11 patients were later diagnosed as having the Prader-Willi syndrome (PWS). To clarify the pathogenesis of muscle hypotonia in PWS, I examined muscle biopsies by histochemical and morphometric methods and the results were compared with those obtained from floppy infants with only mental retardation but with no other features. The histochemical abnormalities of PWS included (i) fiber size variation of both type 1 and 2 fibers, (ii) type 2 fiber atrophy, (iii) increased numbers of type 2C fibers, and (iv) decreased numbers of type 2B fiber. Although muscle hypotonia in PWS has been thought to be due to central nervous system abnormality, my findings suggest that primary muscle pathology, including muscle fiber immaturity and abnormal muscle fiber type distribution, at least in part, plays a role in muscle hypotonia and weakness. Key words: Prader-Willi syndrome; Muscle histochemistry; Fiber size variation in type 1 fiber; Increased type 2C fiber; Type 2B fiber deficiency; Floppy infant syndrome
1. I N T R O D U C T I O N The Prader-Willi syndrome (PWS) is characterized by muscle hypotonia, obesity, hypogonadism, short stature, small hands and feet, and mental deficiency [1]. The hypotonia is sometimes so severe that the patients exhibit the 'frog leg' posture, and their sucking reflex may be so weak as to require tube feeding [21. These patients are occasionally suspected of having some type of neuromuscular disease and may be evaluated with a muscle biopsy. In a follow-up study of 259 patients with unex-
Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan and Department of Pediatrics, Gifu University School of Medicine, Gifu, Gifu, Japan Received 25 March 1993; accepted 4 February 1994 Correspondence address: Dr. S. Sone, Department of Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan. Fax: (81) (423) 46-1749.
0387-7604/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0387-7604(94)00025-S
plained severe congenital hypotonia studied with a muscle biopsy, 11 patients were later found to have PWS on the basis of their clinical or chromosomal abnormalities. I have reviewed the muscle biopsies on these patients in an attempt to determine the reason for the muscle hypotonia in PWS.
2. PATIENTS AND M E T H O D S 259 muscle biopsies from infants under 3 years of age with muscle hypotonia, delayed developmental milestones and probable muscle weakness, from 1983 to 1987, were examined histochemically. Questionnaires were sent to their physicians to determine the present status of these patients. At least 5 years had elapsed since the biopsy. Among them, 11 patients were later diagnosed as having PWS clinically a n d / o r by chromosomal analysis. Muscle biopsies had been done between the ages of 8 - 3 4 months (average 13 months). Clinical features of PWS patients are summarised in Table 1. Patient 11 was reported by Miike et al. [3] as
184
S. Sone / Brain & l)evelopmettt 1994; 16. 183 ,~
"Fable
Summary (~f patients with the Prader-Willi syndrome
I
Patient
No. 1
2
3
4
5
6
7
S
9
10
11
Sex
M
F
F
F
M
M
F
M
M
M
M
Age at biopsy N e o n a t a l difficulties
8m
7m
25m
7m
6m
7m
Im
34m
5m
Im
2m
+
+
~-
+
+
Hypotonia
+
+
+
+
+
+
+
~
+
+
P o o r sucking Tubc feeding t Iigh-arched palate
+
+
+
+
+
+
+
~
+
+
-
+
-
+
+
+
+
-
Hypogonadism
+
NE
+
hands and feet Other malformations * * Small
Age at last examination
9y
NIE
+
NIE
+
-
+
+
--
+
+
Mental retardation Abnormal 15q
-
+
4 y :~
Obesit,,
+
+
-
+
+
+
~-
--
+
Iy
Im
4y
6y
43'
5y
S m :~
34m
+
+
+
+
NE
NE
+
+
+
+
+
+
-
NE
-
+
+
-
+
+
NE
NE
NE
NE
+
+
+
+
9).
known neuromuscular disease but who had had muscle biopsies were selected as controls. They were hypotonic during infancy but were able to walk unassisted and had no muscle weakness when last examined. They had had muscle biopsies between 8-27 months of age (average 13 months). The biceps brachii muscle was biopsied in all the patients. Muscle specimens were frozen in isopentane cooled in liquid nitrogen, and serial frozen sections of 10 /xm thickness were stained with hematoxylin and eosin ( H & E ) , modified Gomori trichrome (mGT) and a battery of histochemical methods, including N A D H tetrazolium reductase ( N A D H ) , succinic dehydrogenase, periodic acid Schiff, oil red O, routine ATPase, ATPase with preincubation at pH 4.6 and 4.3, nonspecific esterase, acid phosphatase, alkaline phos-
Summat3, ~)f histochemical jindings Controls
Patients
No.
I
Fiber size variation both in type I and 2 fibers Type 2 fiber atrophy T y p e 1 fiber atroph}. T y p e 2B fiber deficiency Increased type 2 C fiber Decreased CCO
+
~-
1 Iy
an atypical PWS. All patients were hypotonic and hypogonadism was noted in all male patients. Poor sucking and mental retardation were found in most patients. The number of patients documented to have small hands and feet a n d / o r high arched palate was small, since some of the clinical reports available at the time of biopsy made no reference to these features. About half of the patients were obese when last examined. Two patients had died, one from pneumonia (Patient 2) and the other from disseminated intravascular coagulopathy and acute renal failure (Patient 7). Six of 9 patients examined cytogenetically had an abnormality in chromosome 15. There was no difference in the clinical features between patients with and without this chromosomal abnormality. Eight patients who had mental retardation with no
2
-
N E
M . m a l e ; F . f e m a l e ; N E , not examined. :~ Age of death. * * Congenital hydronephrosis, micrognathia (Patient 4), hypospadia (Patient 8), cleft palate (Patient
Table
-
2
+
+
+
+
+
-
-
+
7
8
9
l0
11
+
+
+
+
+
-
ql +
,--
+
+
+
--
. +
+
+
+
+
-
activity
Increased lipid droplets
+
6
+ .
-
-
5
+ .
-
4
+
.
+
3
+
-
+
-
+
1
2
3
4
5
6
+
--
+
7
8
S. Sone / Brain & Development 1994; 16:183-8 Table 3 Patient No.
185
Morphometric analysis of muscle biopsies Age
Number of analyzed fibers
Muscle fiber type Type 1
Type 2A
Type 2B
Type 2C
23.7% 16.1±3.7" 28.8% 23.4±4.5 32.1% 22.2±5.6 42.6% 21.1±3.8 22.5% 22.0±4.1 30.9% 20.9±4.0 22.7% 20.2±3.5 47.1% 13.0±3.3 32.0% 18.0±3.4 25.1% 13.8±3.3 18.8% 11.8±2.5
45.9% 12.6±1.9 39.3% 19.2±2.8 63.2% 13.8±2.7 36.5% 16.1±2.7 53.3% 13.6±2.7 62.9% 14.7±1.9 38.2% 15.1±2.1 48.6% 9,0±1.4 50.8% 14.5±2.6 41.2% 12.2±2.4 71.1% 10.5±1.8
24.5% 12.1±2.3 31.0% 19.3±2.7 4.7% 12.8±2.2 3.9% 15.8±3.4 15.7% 11.6±2.2 2.4% 13.1±2.1 20.7% 15.0±2.1 2.3% 9.0±1.2 14.6% 16.6±2.7 31.1% 13.6±2.6 6.0% 10.2±0.9
5.9% 12.9±2.4 0.9% 18.3±3.9 0%
1
8m
1,210
2
7m
633
3
25m
700
4
7m
854
5
6m
612
6
7m
614
7
lm
767
8
34m
784
9
3m
650
10
lm
847
11
2m
996
17.0% 18.2±2.7 8.5% 14.7±2.5 3.7% 15.9±5.3 12.4% 16.9±2.2 2.0% 8.7±2.1 2.6% 13.8+2.4 2.6% 11.8±3.6 4.1% 9.5±1.9
* Mean diameter, ~ m + S.D.
Fig. 1. In addition to variation in type 1 (1) fiber size, type 2A (2A) fibers are atrophic. Type 2B (2B) fibers are decreased in number and type 2C (2C) fibers are increased in number. Patient 1. A: H&E. B: routine ATPase. C: ATPase (pH 4.6). D: ATPase (pH 4.3). A - D (serial sections) ×588.
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Fig. 2. Myopathic changes consisting of marked variation m the size of type 1 (1) fibers with deficiency of type 2B fibers. Type 2A (2A) fibers are atrophic and 2C (2C) fibers increased in number. Patient 8. A: ATPase (pH 4.6). B: ATPase (pH 4.3). A,B (serial sections) ×400.
phatase, cytochrome c oxidase (CCO), phosphofructokinase, A M P deaminase and acetylcholinesterase. Photographs were taken of identical fields in serial sections stained with ATPase after preincubation at the three pH settings at a final magnification of × 1,000, and then type 1, 2A, 2B and 2C fibers were classified.
3. RESULTS The major histological findings are summarized in Table 2, and morphometric analysis of PWS patients in Table 3. Except for mild fiber size variation in all biopsies from PWS patients, no apparent morphologic change was seen on H & E and m G T (Fig. 1A). Muscle fibers appeared to be mature with no increase in the number
Table 4 Mean size and t'ariation of type 1 fibers in P W S and control groups (mean diameter, Ixm +_S.D.) Patient no.
PWS group
Control group
1 2 3 4 5 6 7 8 9 10
16.1 _+3.7 23.4 + 4.5 22.2_+5.6 21.1 _+3.8 22.0+4.1 211.9 + 4.11 20.2 _+3.5 13.0!3.3 18.11+3.4 13.8+3.3
19.5+2.6 20.1 + 2.9 23.6+ 3.1 13.6+2.2 17.4+_ 1.8 22.9 + 3.3 20.5 +_3.2 23.6+4.1
of fibers with centrally placed nuclei. All patients exhibited variation in the size of type 1 fibers (Fig. 1) and 3 patients had small type 1 fibers. In contrast to PWS patients, the mean size of type 1 fibers in the control group was normal, with less variation in their size (Table 4). In the coefficient of variance of the standard deviation of type 1 fibers (Wilcoxon' s U-test) there was a statistically significant difference between the two groups ( P < 0.01). There was type 2B fiber deficiency in 4 (Fig. 2, Table 3) of the PWS group, whereas there was none in the control group. Increased numbers of undifferentiated type 2C fibers and type 2 fiber atrophy were present in both groups (Fig. 1, Table 2), but they were more predominant in the PWS group. With N A D H , intermyofibrillar networks were normally developed. None of fibers had increased acid or alkaline phosphatase activity. With the other stains, there was no abnormality except for one patient who had decreased CCO activity (Fig. 3). There was no morphologic difference between PWS patients with chromosomal abnormality (Patients 4, 5, 8, 9, 10 and 11) and those without (Patients 1, 3 and 6).
4. DISCUSSION The clinical course of PWS has been divided into the hypotonic stage of early infancy and the mentally retarded stage after 1 to 2 years of age [1]. The degree
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Fig. 3. Decreased cytochrome c oxidase activity seen in Patient 4 (A). Control muscle (B). A,B (cytochrome c oxidase) x 490.
of muscle hypotonia associated with poor suck, the cardinal symptom of the first stage, varied significantly from patient to patient. When the muscle hypotonia was so marked and appeared to be associated with muscle weakness, a muscle biopsy was sometimes done to rule out a neuromuscular disorder. Muscle symptoms gradually improve, usually between 8 and 11 months of age [4,5], and then mental retardation, obesity and hypogonadism become manifest [1]. Some patients have prolonged hypotonia with severely retarded motor development [3]. Since Ledbetter et al. [6] reported chromosome 15 deletion in PWS and Butler et al. [7] reported that PWS was manifested when chromosome 15 with the deletion is derived from the father, genetic studies of PWS have progressed rapidly. The deletion on the long arm of chromosome 15 is present in approximately half of the PWS patients and is assumed to be related to the hypopigmentation [8]. However, a portion of PWS patients have no abnormality in chromosome 15. These patients may be shown to have a maternal disomy if a genetic survey of these patients were possible. Several studies have been done to clarify the patho-
physiology of the hypotonia in the PWS patients. Afifi and Zellweger [9] examined PWS muscles by light as well as electron microscopy. Although they found some ultrastructural changes including subsarcolemmal mitochondrial aggregates, abnormal Z-line and myofilamentous disarray and loss, they concluded that these abnormalities were nonspecific and too mild to explain the muscle symptoms in PWS. Since histochemical examination did not show any architectural change such as mitochondrial aggregation or disorganized intermyofibrillar networks, ultrastructural examination may not provide any information to explain pathogenetic mechanism. Hirayama et al. [10] also found nonspecific minor pathologic changes and proposed that there were some defects in trophic influence on the developing muscle from the central nervous system (CNS). Since other histochemical and electron microscopic studies also showed no pathognomonic fndings to explain the muscle hypotonia, the muscle symptom in PWS has been thought to result from some undetermined CNS abnormality [9,11,12], or from the small muscle bulk [13]. In this study, there were two striking findings, in-
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cluding m a r k e d variation in the size of type 1 fibers and a deficiency in type 2B fibers which were not seen in the control group of m e n t a l l y r e t a r d e d floppy infants. A l t h o u g h there was no disease-specific pathologic abormality in PWS patients, the striking c o m m o n findings of variation in the size of type 1 and 2 fibers, a b n o r m a l fiber type distribution, including occasional type 2B fiber deficiency, a n d increased n u m b e r s of u n d i f f e r e n t i a t e d type 2C fibers suggest a primary muscle involvement. Type 2 fiber atrophy itself is a n o n specific finding c o m m o n l y seen in a variety of conditions, including disuse, cerebral palsy, m e n t a l retardation, and d e g e n e r a t i v e diseases (these may all be examples of disuse). Type 2B fiber deficiency is a c o m m o n finding in muscles from patients with c o n g e n i t a l non-progressive myopathies (CNM), including n e m a l i n e myopathy a n d central core disease, and the end-stage of progressive m u s c u l a r dystrophies [14,15]. Type 1 fiber atrophy a n d p r e d o m i n a n c y , the major p a t h o g n o m o n i c findings in CNM, were not f o u n d in the PWS patients. T h e r e are increased n u m b e r s of u n d i f f e r e n t i a t e d type 2C fibers in muscles u n d e r g o i n g active r e g e n e r a t i o n after myonecrosis, d u r i n g r e - i n n e r v a t i o n following d e n e r v a t i o n and in i m m a t u r i t y [14-16]. Since there was no necrotizing or d e n e r v a t i n g process in PWS muscles, increased n u m b e r s of type 2C fiber are t h o u g h t to reflect muscle fiber immaturity, as suggested by H i r a y a m a et al. [10]. In conclusion, variations in the size of type 1 fibers (probable myopathic change) and type 2B fiber deficiency suggest that the pathogenesis of the hypotonia of PWS is not entirely due to CNS a b n o r m a l i t y but is, at least in part, due to an a b n o r m a l i t y in the muscle itself. ACKNOWLEDGEMENTS The author is grateful to Dr. Ikuya Nonaka (National Institute of Neuroscience, NCNP) and Professor Tadao Orii (Department of Pediatrics, Gifu University School of Medicine) for their advice and suggestions on this work. The author wishes to express cordial thanks to Professor S.M. Sumi (Department of Pathology, University of Washington) for his kind suggestions and criticisms on this work.
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