Muscular dystrophy, mental retardation and cardiomyopathy not associated with dystrophin deficiency

Neuromusc. Disord., Vol. 6, No. 3, pp. 167-172, 1996 Copyright :e. 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960--8966/96 S15.00 + .00

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PII: S0960-8966(96)00016-8

MUSCULAR DYSTROPHY, MENTAL RETARDATION A N D CARDIOMYOPATHY NOT ASSOCIATED WITH DYSTROPHIN DEFICIENCY M, VILLANOVA*t, A. MALANDRINI*, R. BIANCOTTI++, A. LOFGREN§, T. MONGINI¶, J. SIX[I, R. SALVESTRONI*, E. PARROTTA*, C. VAN BROECKHOVEN§, C. PAOLOZZI**, and G. GUAZZI* *Laboratory of Neuropathology, Institute of Neurological Sciences, University of Siena, Siena, Italy; ~Department of Neurosurgery, Siena Regional Hospital, Siena, Italy; §Laboratoryof Neurogenetics, Flemish Institute of Biotechnology, Born-Bunge Foundation, University of Antwerp (UIA), Antwerp, Belgium; INeuromuscular Center, 'Paolo Peirolo', University of Torino, Torino, Italy; IIDepartmentof Neurology, University of Kuopio, Kuopio, Finland; **Department of Neurophysiopathology,Universityof Naples, Naples, Italy (Received 27 November 1995; revised 15 January 1996," accepted 8 February 1996)

Abstract--We report on a male patient aged 38, affected by a syndrome whose characteristic features include onset in early childhood, slow progression, diffuse muscle weakness, mental retardation and cardiomyopathy. Muscle biopsy showed myopathic changes compatible with muscular dystrophy. However, immunostaining for dystrophin as well as 50 and 43 kDa dystrophin-associated glycoproteins (DAGs) was normal. Genetic analysis suggested that direct involvement of the dystrophin gene was highly unlikely. No other family members were affected. Although the clinical picture is reminiscent of Duchenne/Becker muscular dystrophy, the immunologically and genetically documented lack of dystrophin involvement suggests that this particular syndrome is as yet undescribed. Copyright © 1996 Elsevier Science Ltd. Key words: Muscular dystrophy, muscle biopsy, dystrophin, mental retardation, cardiomyopathy.

INTRODUCTION

Case history

Duchenne muscular dystrophy ( D M D ) is one of the major causes of muscular dystrophy. The genetic basis of D M D is a defect in the gene for dystrophin, a large membrane-associated protein, leading to a systemic deficiency of this protein. A number of very similar disorders have been described, which have been linked to defects in any of several dystrophin-associated glycoproteins (DAGs) rather than dystrophin itself. Here we describe a patient with a clinical picture reminiscent of Duchenne/Becker and related muscular dystrophies. By genetic and immunological criteria, however, dystrophin deficiency and similar known cases of muscular dystrophy have been ruled out, suggesting that this patient is suffering from a novel and previously undescribed disorder.

tAuthor to whom correspondence should be addressed at: Institute of Neurological Sciences, University of Siena, 53100 Siena, Italy.

The parents were healthy and unrelated. The maternal grandparents were first degree cousins. The patient was born after a normal delivery. His physical development was somewhat retarded. He had difficulties running and climbing a rope during childhood. He attended primary school with very poor results, having to repeat the first, the third and the fifth year twice. He then dropped out of school. He was exempted from military service because of mental retardation and muscular weakness. At age 20, these problems prompted him to consult a neurologist. When the patient was brought to our attention, neurological examination showed strength in the lower and upper limb was decreased to grade 3 [1]. Scapular winging was noted. A marked diffuse pseudohypertrophy of the muscle was observed associated with a marked hypertrophy of the calves (Fig. 1A, B). Muscle weakness was diffusely distributed, with only a slight tendency towards proximal in the arms. The facial muscles

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Fig. I(A,B). Note hypertrophy of the calves, lumbar lordosis and scapular winging.

were not clinically affected. No contractures were present. Muscle weakness reflexes were diminished. No myotonia was seen. Neuropsychological examination showed a lod score of 60 in the Wechsler adult intelligence test (WAIS). Verbal and non-verbal performances were impaired to the same degree. There were no sensory disturbances. An ECG and an echocardiogram showed diffuse enlargement of atrial and ventricular internal diameters, suggestive of dilated cardiomyopathy. The echocardiogram for both parents was normal. Serum creatine kinase (CK) was elevated to 1770 U 1-l (normal value <190), lactic dehydrogenases (LDH) 700 (normal value <40). CK levels were within the normal range in parents and sibling of the patient. Motor and nerve conduction velocities were normal in all nerves examined. Needle electromyography (EMG) examination revealed a myogenic pattern. No myotonic discharges were seen. A brain Magnetic Resonance Image (MRI) scan was normal. A muscle CT scan showed a diffuse fat infiltration, principally in the semimembranous, biceps femoris and gastrocnemius muscles.

EXPERIMENTAL PROCEDURES

Skeletal muscles biopsies were obtained from the deltoid muscle of the patient. Muscle biopsies were snap frozen in liquid nitrogen-cooled isopentane. Transverse cryostat sections (10/~m thick) were stained with routine histochemical methods. Fibre-type classification was made in serial sections stained with myofibrillar ATPase.

Immunofluorescence For immunofluorescence, serial cryostat sections (7 /~m) were cut and incubated with monoclonal antibodies against: dystrophin (COOH terminus, ROD domain and NH2terminus; Novocastra, Newcastle), 50 kDa or adhalin (Novocastra), 43 kDa or fl-dystroglycan (Novocastra) DAGs, merosin (laminin t~ 2) (Chemicon) and desmin (Dakopatts). These antibodies were used at the dilutions shown in Table 1. The cryostat sections were processed for immunocytochemistry with an indirect immunofluorescence technique. Blocking was performed by a 30-min incubation with 5% bovine serum albumin in PBS (50 nM sodium phosphate, pH 7.4; 0.9% NaC1). Incubation

Muscular Dystrophy W i t h o u t Lack of Dystrophin

with primary antibodies was performed for 1 h, followed by incubation with 1/100 diluted fluorescein-labelled goat anti-mouse IgG (Boehringer-Mannheim) for 1 h. Incubation was performed at room temperature. Each incubation step was followed by extensive washing with PBS. Specimens were examined under a Zeiss Axioplan fluorescence microscope. Negative controls consisted of preincubation with PBSand 0.1% bovine serum albumine, incubation with non-immune mouse IgG, or omission of the primary antibody.

Western blot A small muscle specimen, crushed in liquid nitrogen, was solubilized in boiling Laemmli buffer. A volume corresponding to 250 mg protein per lane was electrophoresed in a 5% SDS-polyacrylamide gel and transferred to nitrocellulose. The blot was incubated with the primary monoclonal antibody against the rod domain and the COOH-terminal domain. Immune complexes were visualized by colorimetric development.

DNA analysis Genomic DNA of the patient was extracted from total blood, digested with restriction enzymes BgllI and HindlII, separated by agarose gel electrophoresis, blotted on a nylon membrane and hybridized with radiolabelled plasmid probes 9-7, 30-2, 30-1, 47-7, 44-1 and 63-1/1. The DNA fragments were visualized by autoradiography. Appropriate control samples from normal human male and female muscle were used to calibrate the density measurements. RESULTS

Light microscopy Light microscopy showed an overtly dystrophic muscle with increased variation in fibre size, some atrophic and hypertrophic Table 1. Antibodies used in this study Antibody DYS 1 DYS 2 DYS 3 50 kDa 43 kDa Merosin Desmin

Dilution 1/5 1/5 1/100 1/100 1/100 1/1000 1/200

Source Novocastra Novocastra Novocastra Novocastra Novocastra Chemicon Dakopatts

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muscle fibres and slightly increased interstitial connective tissue (Fig. 2A). A significant increase of internalized nuclei was observed. Frequent fibre splitting and basophilic muscle fibres were also seen. Necrotic fibres undergoing phagocytosis were observed only rarely (Fig. 2B). PAS reaction and Sudan red reactions were normal. ATPase reaction showed type 1 predominance.

Immunofluorescence Normal immunostaining was observed with all anti-dystrophin antibodies as well as the adhalin and 43 kDa (DAGs) antibodies (Fig. 3A-E). With the antibodies against merosin and desmin, a normal immunostaining pattern was also observed.

DNA analysis The restriction pattern observed in the patient's DNAwith the dystrophin cDNA probes completely matched that of a control DNA sample, indicating that no structural alterations such as deletion or duplication were present in the dystrophin gene.

Western blot Immunoblot analysis of muscle proteins showed normal quantities of dystrophin with a normal molecular weight (427 kDa). DISCUSSION

Duchenne and Becker muscle dystrophy are caused by mutations in the D M D gene [2-4]. The D M D gene is by far the largest known human gene, composed of approximately 79 exons [5, 6]. It is expressed in cardiac muscle, skeletal muscle and in brain tissue [7, 8]. The D M D gene encodes a protein named dystrophin, which is generally absent from the tissues of D M D patients. Salient features of our patient's illness were early childhood onset, slow progression, diffuse muscle weakness, mental retardation and cardiomyopathy, with myopathic changes on muscle biopsy typical for muscular dystrophy. Among these features, cardiomyopathy has been reported in BMD patients [9-11]. Mental retardation associated with Duchenne/Becker muscular dystrophy has likewise been described in some patients [12, 13]. Thus, in the patient we describe here, the

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Fig. 2. Cryostat sections stained with hematoxylin-eosin showing increased variation in fibre size, increase of internalized nuclei, a necrotic fibre undergoing phagocytosis (long arrow) and a regenerative muscle fibre (short arrow). × 170.

phenotype, the clinical history and the muscle biopsy findings made Becket muscular dystrophy the most likely diagnosis. However, BMD invariably correlates with a histologically demonstrable lack of dystrophin and neither immunohistochemistry nor Western blot using various anti-dystrophin antibodies could demonstrate any dystrophin deficiency in our patient. Furthermore, genetic analysis enabled us to exclude any deletion/duplication in the dystrophin gene. While the possibility of a point mutation could not be absolutely ruled out on our genetic evidence, our immunological findings showed normal dystrophin expression in muscle biopsy, making it extremely unlikely that our patient's pathology was caused

directly by dystrophin deficiency. We therefore proceeded to investigate other possible causes of our patient's disorder. Another syndrome that features mental retardation, cardiomyopathy and myopathic changes on muscle biopsy is Steinert's myotonic dystrophy. Our observations that there was no family history for this syndrome, no myotonic signs were clinically present and no myotonic discharges were demonstrable by EMG enabled us to exclude this syndrome as a possible cause for our patient's pathology. There have been occasional reports of cardiomyopathy associated with mental retardation which shows vacuolar myopathy and desmin accumulation. However, none of these patients showed hypertrophy of

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Fig. 3. Serial cryostat sections stained with (A) DYS 1, (B) DYS 2, (C) DYS 3, (D) 43 kDa (E) 50 kDa, all showing normal dystrophinimmunostaining.× 270. the calves and their cardiomyopathy tended to be hypertrophic, while our patient appeared to have dilated cardiomyopathy. Also, our patient appeared to have an earlier age of onset. Furthermore, both PAS and desmin immunostaining were normal in our patient. Finally, no vacuoles were observed in the damaged muscle tissue of our patient. We therefore excluded the diagnosis of cardiomyopathy and mental retardation associated with vacuolar myopathy. A recently described Duchenne/Becker-like

disease called severe childhood autosomal recessive muscular dystrophy (SCARMD), which affects males and females equally, is associated with total (17q-linked SCARMD) [14, 15] or partial (13q-linked SCARMD) deficiency of adhalin (50 kDa DAG) [16]. The patients affected by this muscle disorder are clinically characterized by a phenotype reminiscent of Duchenne/Becker muscular dystrophy. They have hypertrophy of the calves and high serum CK. The loss of ambulatory abilities

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may be variable. Muscle biopsy shows a pattern of necrosis regeneration compatible with muscular dystrophy. To exclude these muscle disorders as a possible cause for our patient's complaints, we performed immuno-histochemical studies with several antibodies against DAGs. However, adhalin (50 kDa DAG) immunostaining was completely normal in our patient, ruling out the diagnosis of SCARMD. Furthermore, in previously described SCARMD patients, ECG was invariably normal and no association with mental retardation has ever been reported. Finally, the 43 kDa DAG, associated with a particular form of limb-girdle muscular dystrophy [17, 18] was also normal in our patient. We conclude that the disorder affecting the patient we describe here presents highly suggestive features reminiscent of BMD/DMD /SCARMD, but is not associated with any of the genetic/biochemical defects as yet known to cause these disorders. For the moment, the lack of data from other similar cases makes our patient's disorder difficult to evaluate, and the lack of family history for the disorder at present precludes more extensive genetic studies. It may be significant that the patient's maternal grandparents were first cousins. The similarity of clinical features with those of DMD/BMD/SCARMD strongly suggests, however, that the hypothetical defect which constitutes the primary cause of our patient's disorder may somehow be involved with the dystrophin-DAG complex of membrane-associated proteins, albeit without directly causing a deficiency of dystrophin or the associated DAGs previously implicated in these disorders. Moreover, since at least three different organ systems appear to be involved, to wit (1) brain, as evidenced by mental retardation; (2) cardiac muscle, as evidenced by cardiomyopathy; and (3) skeletal muscle, as evidenced by muscular dystrophy; we propose that the hypothetical protein whose dysfunction would be the primary cause involved in this syndrome has to be expressed at least in these three organs, and quite possibly systemically. Acknowledgements--We are grateful to Telethon-Italy for

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its financial support to M. V.

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