NeuromuscularDisorders,Vol. I, No. 5, pp. 333-339~1991
0960-8966.91 $3.00 ~ 0.00 ic, 1992PergamonPress pic
Printed in Great Britain
DYSTROPHIN ABNORMALITIES IN POLYMYOSITIS AND DERMATOMYOSITIS C. A. SEWRY,*'I" A. CLERK,* J. Z. HECKMATT,++ T. VYSE,* V. DUBOWITZ* a n d P. N. STRONG* *Jerry Lewis Muscle Research Centre, Department of Paediatrics and Neonatal Medicine; and ~Department of Medicine, Rheumatology Unit, Royal Postgraduate Medical School, D u Cane Road, London W12 0NN, U.K.
(Received 29 July 1991; accepted 11 October 1991)
Abstract--The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten
cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and Cterminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to #-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.
Key words: Dystrophin, inflammatory myopathy, myositis, muscle.
[6, 10, 11] but relatively few cases ofpolymyositis or dermatomyositis have been documented. Recently, it has been shown that dystrophin is associated with a glycoprotein complex [14]. Our earlier studies with the lectin Ricinus communis I, showed that a 370 kDa glycoprotein is absent or greatly reduced in D M D [15] and electron microscopy demonstrated that a glycoprotein that binds the same lectin was abnormal not only in D M D and BMD but also in polymyositis [16, 17]. The possible association ofdystrophin with a glycoprotein, and the evidence suggesting a membrane glycoprotein defect in inflammatory myopathies led us to carry out a detailed examination of dystrophin expression in patients with polymyositis and dermatomyositis. Polymyositis and dermatomyositis are acquired myopathies usually characterised by sudden or acute onset, muscle weakness and fatigue [17]. Serum creatine kinase levels may be elevated and muscle biopsies may show varying degrees of muscle fibre degeneration and cellular infiltration [18]. In dermatomyositis a skin rash is also present. Onset may be in childhood or during adult life and both males and females can be affected. The'pathogenesis of these diseases probably has an immunological basis. Many patients, particularly those with childhood
INTRODUCTION
Dystrophin, the defective protein in Duchenne muscular dystrophy (DMD) is a high molecular weight protein (427 kDa) coded by a gene of at least 2.3 Mb at position Xp21 on the short arm of the X chromosome [1, 2]. Approximately 60% of D M D patients have detectable deletions in the gene. Becker muscular dystrophy (BMD), which is phenotypically less severe than DMD, is also caused by a defect in the same Xp21 gene [3, 4]. In normal skeletal muscle dystrophin has been localised immunocytochemically to the cytoplasmic face of the sarcolemmal plasma membrane [5-8]. On immunoblots, using antibodies raised against fusion proteins containing dystrophin peptide sequences, dystrophin appears as a doublet [9, 10]. In D M D , as well as the Xlinked muscular dystrophies in the mouse (mdx), the dog (xmd) and the cat, dystrophin is absent or greatly reduced [11-13]. In BMD, dystrophin is often either reduced in abundance or has an altered molecular weight [9, 11]. In a variety of other neuromuscular disorders the expression of dystrophin has been reported to be normal
t A u t h o r to whom correspondence should be addressed. 333
C. A. SEWRY et al.
334
Table 1. Clinical details and the results of dystrophin analysis in patients with polymyositis and dermatomyositis Case
Age (yr)
Sex
Diagnosis
Steroids"
I.MH 2.RA 3.NJ 4.JS
3 3 31 33
5.AJ
CKt
M M F F
Poly Poly Poly Poly
No Yes Yes No
133 14000 1090 74
38
F
Poly
No
156
6.FA
42
F
Poly
Yes
7755
7.GG 8.EW
58 76
M F
Poly Poly
Yes Yes
40000 1395
9.FC 10.FK I I.SW 12.GT
77 1 5 6
F M F M
Poly JDM JDM JDM
Yes No No Yes
1450 1783 50 266
13.DD
8
F
JDM
No
29
14.GI 15.CJ
8 9
M F
JDM JDM
No No
89 64
16.EH 17.TC
12 13
F F
JDM JDM
Yes No
166 88
18.SS 19.MG
14 16
F M
JDM JDM
No No
155 101
20.JW 21.DS
51 57
M F
Derm Derm
No Yes
1560 175
22.FB
62
M
Derm
Yes
37
lmmunoblot
$ Mol.wt ~ Mol.wt ~ Mol.wt Normal size reduced amount ~, Mol.wt reduced amount Normal size reduced amount Normal Normal size reduced amount Normal $ Mol.wt $ Mol.wt Normal size reduced amount ,L Mol.wt reduced amount ~ Mol.wt Normal size reduced amount Normal Normal size reduced amount Normal Normal size reduced amount Normal ~ Mol.wt reduced amount Normal
Immunostaining
Not done Variability Normal Normal Normal Normal Variability Not done Variability Variability Not done Variability Normal Normal Normal Normal Variability Variability Variability Variability Variability Variability
Poly = polymyositis; JDM = juvenile dermatomyositis; and Derm = adult dermatomyositis. * Steroid treatment at time of biopsy. t International units, upper limit of normal 200 IU 1 ~.
dermatomyositis, respond to a carefully controlled regime of steroid treatment and recently the use of cyclosporin has proved to be effective in refractory cases [19]. We report here studies on the expression of dystrophin in both children and adults suffering from polymyositis or dermatomyositis, using immunocytochemical and immunoblotting techniques. METHODS
BergstrSm needle biopsies were taken under local anaesthesia from the quadriceps of two children and seven adults with polymyositis, ten children with the juvenile form of dermatomyositis and three adults with dermatomyositis [20]. Details of the patients are given in Table 1. Diagnosis was made according to established clinical and pathological criteria [18]. Samples for immunocytochemistry were orientated transversely on cork discs, rapidly frozen in isopentane cooled in liquid nitrogen, and stored at -80°C until required. Samples for immunoblotting were either frozen separately in liquid nitrogen and stored at -80°C or, in some
cases, were cut from the cork-mounted block (4 x 20/~m sections) and kept frozen at -80°C in vials. Similar results were obtained with either method, and 20/tm sections have been shown to provide sufficient material for the detection of dystrophin [9]. Antibodies. A sheep polyclonal antiserum raised against a fusion protein containing a 60 kDa dystrophin peptide sequence from the Nterminal region ofdystrophin (kindly donated by Drs L. Kunkel and E. Hoffman, Boston) was used to study all cases (this antibody is referred to subsequently as 60 KD). In addition, most cases were also studied with rabbit polyclonal antibodies (H12 and P6) raised against fusion proteins corresponding to amino acids 2604-3024 and 2814-3028, respectively, from the C-terminal end of the rod repeat region of dystrophin (Sherratt, in preparation). Monoclonal antibodies raised against the rod domain (Dy4) and against the last 17 C-terminal amino acids (Dy8) were kindly made available by Dr L. Nicholson (Newcastle upon Tyne), towards the end of the study. Monoclonal antibodies to human erythrocyte fl-spectrin (56A) were used to assess the morphological integrity of the sarcolemma (a gift from Dr M. Newton, Oxford).
Dystrophin in Myositis
335
Gel electrophoresis and Western blot analysis. These were performed as described previously [21]. Briefly, the frozen piece of muscle was crushed in foil by lead weights and the powder solubilised in 1% SDS containing protease inhibitors (1 mM iodoacetamide, 1 mM benzemethonium chloride, 0.5 mM phenyl methyl sulphonyl fluoride, 0.05 mM Pepstatin A). The 20 ~tm cryostat sections were solubilised directly in 1% SDS. Fifty micrograms of protein (Bradford protein assay) were boiled with an equal volume of Laemmli buffer for 2 min and resolved by SDS-PAGE on 4-20% linear gradient gels for 16 h at 20 mA gel -~. Gels were washed for 30 min in transfer buffer (20 mM Tris base, 150 mM glycine) and electroblotted onto nitrocellulose (20 h at 500 mA). Blots were incubated with dystrophin antibodies diluted in phosphate buffered saline (PBS) containing 0.05% Tween 20 (TP buffer) for 1 h at 20°C (60 KD 1 : 1000, H12 1 : 3000, P6 1 : 3000, Dy4 1 : 50, Dy8 1 : 50). This was followed by incubation for 1 h with an appropriate biotinylated, secondary antibody (Amersham) diluted 1 : 400 in TP/50% (v/v) human serum. Dystrophin was visualised with a preformed streptavidin-biotin-peroxidase complex (Dakopatts ABC kit) followed by freshly prepared diaminobenzidene (0.025%, w/v) containing 0.05% H202. Control muscle from normal volunteers and from patients with non-Xp21 neuromuscular disorders were probed on the same blot for comparison and control nitrocellulose strips probed without the primary antibody. Immunocytochemistry. Unfixed cryostat sections (6 pm) were incubated for 30 min at room temperature with antibodies to dystrophin diluted in PBS pH 7.4 (60 KD 1 : 1000, P6 1 : 1000, H12 1 : 1200; Dy4 and Dy8 were used undiluted). After washing in PBS, sections were incubated for a further 30 min with an appropriate biotinylated secondary antibody (Amersham, 1 : 200), washed again in PBS and incubated for 15 min with streptavidin conjugated to the fluorochrome Texas Red (Amersham, 1 : 200). Serial sections were immunostained in a similar manner with the monoclonal antibody to fl-spectrin (56A). Sections from biopsies from normal volunteers and from patients with non-Xp21 disorders were used for comparison. Control sections were incubated without the primary antibody and were negative except for autofluorescence.
C
FK
200J,,-
P6 Fig. 1. Skeletal muscle from a 1-yr-old boy with juvenile dermatomyositis (case 10, FK) immunoblotted with antibodies to a C-terminal region of dystrophin (P6) showing a lower molecular weight protein (C = control). (Blemishes in some areas of this and subsequent blots are the result of fire damage to the photographic emulsion.)
C
CJ
C
CJ
....L
200
Dy4
"Dy8
Fig. 2. Muscle from a 9-yr-old girl with juvenile dermatomyositis (case 15, C J) immunoblotted with the two monoclonal antibodies to N- and C-terminal regions of dystrophin (Dy4, Dy8). Dystrophin is of normal molecular weight but reduced in amount (C = control). RESULTS
Control muscle from normal volunteers and from a variety of non-Xp21 neuromuscular disorders on Western blot analysis showed the characteristic doublet when probed with the 60 KD, H12, P6 and Dy4 dystrophin antibodies (Figs 1, 2 and 3). In contrast, Dy8 identified a single band (Fig. 2). Four out of nine cases of polymyositis and five out of thirteen cases of dermatomyositis, however, showed an immuno-
C. A. SEWRY et al.
336
Table 2 Diagnosis
Patients with abnormal immunoblot
Polymyositis Dermatomyositis Total
C SS
7/9 (78%) 9/13 (69%) 16/22 (73%)
C SS iiili ¸¸ i i ¸i
iiii
200 ?
Oy4 Fig. 3. Muscle from a 14-yr-old female with juvenile dermatomyositis (case 18, SS) immunoblotted with antibodies to an N-terminal region (Dy4), and a C-terminal region (H 12) of dystrophin. The size and amount of dystrophin are both normal (C = control).
reactive band of slightly smaller molecular weight (Fig. 1). In addition, four cases of polymyositis and six cases of dermatomyositis had a reduced amount of dystrophin, compared with the control muscle run on the same gel and with an equal loading of protein (Fig. 2). These abnormalities were consistent with more than one antibody (Fig. 2). A total of 16 out of 22 patients (73%) showed an abnormality on Western blot analysis (Table 2). The remaining patients had dystrophin of normal size and normal amount (Fig. 3). In the cases showing an abnormality in dystrophin there was no correlation with age, sex, serum CK levels, steroid treatment, disease type or degree of pathology (Table 1). There was no evidence of general protein breakdown nor of any characteristic dystrophin immunoreactive breakdown products [10]. Immunostaining of muscle from normal individuals and from controls showed labelling of the sarcolemma of all fibres with all antibodies (Fig. 4). Abnormalities in immunostaining were seen in three out of seven cases of polymyositis and eight out of twelve cases ofdermatomyositis. The abnormalities varied from patchy or dis-
Patients with abnormal immunostaining 3/7 (43%) 8/12 (67%) 11/19 (58%)
continuous staining on some fibres (Fig. 5), to extensive areas with reduced labelling (Fig. 6). These defects were more pronounced with the 60 KD antibody but were also observed with the other antibodies (Fig. 5). However, not all cases were assessed with the monoclonal antibodies Dy4 and Dy8 as these were only available towards the end of the investigation. Immunostaining with /~-spectrin antibodies showed an even distribution at the periphery of fibres (except necrotic fibres which were negative) implying that alterations in dystrophin were not due to any major loss of integrity of the sarcolemma (Fig. 5). However, the possibility that the abnormalities of the sarcolemma affected dystrophin more that ]~-spectrin cannot be excluded. A total of 11 out of 19 patients (58%) showed defects with immunostaining (Table 2) but these did not always correspond with those with abnormalities on immunoblotting (cf. Figs 3 and 5). Some samples that were abnormal on immunoblots showed no significant defects with immunostaining (Table 1). There was no apparent correlation with age, serum CK levels, drug therapy or pathological features in the biopsies. In one case of polymyositis (case 6, FA) and one case of dermatomyositis (case 16, EH) vacuoles were present. These were immunolabelled with antibodies to dystrophin and to ]~spectrin (Fig. 7). Splits in fibres were also labelled. Vacuoles were also observed in one case of dermatomyositis (case 12, GT) but in this biopsy the vacuoles did not show dystrophin, only /~-spectrin. Dystrophin, however, was greatly reduced in this biopsy (Fig. 6). The vacuoles in these cases were observed in transverse sections and no connections with the sarcolemma were seen. The latter possibility, however, cannot be excluded. DISCUSSION
An abnormal expression of dystrophin was demonstrated by Western blot analysis in 16 out of 22 of our patients with polymyositis or dermatomyositis (73%), and in 11 out of 19
Dystrophin in Myositis
337
Fig. 4. Biopsy from a normal adult immunostained with antibodies to an N-terminal region (60 KD), and a C-terminal region (Dy8) of dystrophin. The peripheries of all fibres are labelled with dystrophin and with antibodies to p-spectrin (56A). × 180.
Fig. 5. Serial sections from the same biopsy as Fig. 3 with juvenile dermatomyositis (case 18, SS) immunostained with antibodies to an N-terminal region (60 KD), and a C-terminal region (H12). Labelling for dystrophin is patchy and discontinuous on some fibres (arrows) but the same fibres have normal expression of fl-spectrin (56A). x 240.
patients (58%) by immunostaining. There was not always a direct correlation between the immunoblotting and immunocytochemical results. This lack of correlation emphasises that both techniques should be used but that they should not be equated. Immunocytochemistry detects epitopes on native proteins and gives the cellullar localisation of a protein. Subtle differences in the antibody binding can be seen with immunocytochemistry but it gives no indication of the molecular weight of the detected protein or
of its quantity. Immunoblots, in contrast, detect epitopes on denatured proteins and differences in both the size and total quantity of a protein can be examined. The abnormalities in dystrophin expression cannot be explained on the basis of any consistent correlation with age, sex, steroid treatment, disease type or pathological severity. Hitherto, abnormalities in dystrophin have only been reported in D M D and BMD [6, 9-11], although a preliminary report has suggested abnormalities in some cases of the Fukuyama
338
C.A. SEWRY et al.
60KD
[ Fig. 6. Biopsy from a 6-yr-old boy with juvenile dermatomyositis (case 12, GT) immunostained with antibodies to an N-terminal region of dystrophin (60 KD) showing an extensive area of reduced labelling. Vacuoles in this sample are not labelled with dystrophin antibodies, x 260.
Fig. 7. Biopsy from an adult patient with polymyositis (case 6, FA) immunolabelled with antibodies to an N-terminal region of dystrophin (60 KD) showing staining of a vacuole (arrow) as well as the periphery of the fibres, x 330.
type of congenital muscular dystrophy [22]. It is therefore of considerable interest that alterations in the expression of dystrophin should be found in two inflammatory diseases unrelated to defects in the Xp21 locus. The abnormalities in dystrophin expression in polymyositis and derma-
tomyositis were found in both male and female patients further suggesting that the abnormalities are secondary effects of the disease, rather than genetically determined. The reduced amount of dystrophin in many patients could be the result of general muscle degradation, but there was no evidence of this. The smaller molecular weight form ofdystrophin might be due to a specific proteolytic cleavage resulting from increased protease activity originating from infiltrating cells, but there was no correlation with the degree of inflammation. We also considered the possibility that the presence of the smaller molecular weight protein might relate to regeneration and could be similar to the smaller form ofdystrophin that we have detected in human foetal muscle [23]. There was no correlation with the presence of foetal myosin or the degree of regeneration to support this hypothesis, but until more is known about the isoforms of dystrophin this possibility cannot be excluded. There are no known genetic similarities between the Xp21 dystrophies and the inflammatory myopathies but both are associated with defects in lectin binding glycoproteins [15, 16]. Recently it has been shown that dystrophin is associated with a sarcolemmal glycoprotein complex [14] but no relationship between the glycoproteins in these different studies has yet been established. The localisation ofdystrophin to vacuoles is of interest. Vacuoles are believed to originate from the T-tubule system or the sarcoplasmic reticulum but dystrophin and fl-spectrin are both associated with the sarcolemmal plasma membrane. Although evidence from biochemical fractionation procedures has suggested that dystrophin was localised to the T-tubule system [24, 25], all immunocytochemical data show that it is localised to the cytoplasmic face of the sarcolemma [7, 8]. No connections between the plasma membrane and the vacuoles were observed in the series of sections examined, thus the presence of dystrophin on vacuoles in two of our cases suggests that they may originate from the plasma membrane. Although there is no defect in the dystrophin gene in polymyositis and dermatomyositis, the reasons for the alterations that we have observed are not clear yet. Our studies show that secondary defects in the expression of dystrophin can occur and that abnormalities in the gene product are not confined to the X-linked dystrophies. Our results emphasise the essential need for detailed
Dystrophin in Myositis
clinical assessments for the interpretation of the results from dystrophin analysis and that the latter cannot be used in isolation as a diagnostic marker. work was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland. We thank Miss P. Badiani, Miss A. Mequita and Miss L. Wilson for technical assistance, Mrs K. Davidson for photography and Mr T. Sherratt for the P6 and HI2 dystrophin antibodies. The gifts of antibodies from Drs L. Kunkel, M. Newton and L. Nicholson are also gratefully acknowledged.
l l.
12.
Acknowledgements--This
13. 14.
15. REFERENCES
1. Koenig M, Hoffman E P, Bertelson C J, Monaco A P, Feener C, Kunkel L M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organisation of the DMD gene in normal and affected individuals, Cell 1987; 50:509-517. 2. Hoffman E P, Brown R H, Kunkel L M. Dystrophin: the protein product of the Duchenne dystrophy locus. Cell 1987; 51: 919-928. 3. Kingston H M, Sarfarazi M, Thomas N S T, Harper P S. Localisation of the Becker muscular dystrophy gene on the short arm of the X chromosome by linkage to cloned DNA sequences. Hum Genet 1984; 67: 6-17. 4. Koenig M, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989; 45: 498-506. 5. Zubrzycka-Gaarn E E, Bulman D E, Karpati G, et al. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988; 333: 466-469. 6. 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: 861-863. 7. Watkins S C, Hoffman E D, Slayter H S, Kunkel L M. Immunoelectron microscopic localization of dystrophin in myofibres. Nature 1988; 333: 863-866. 8. Cullen M J, Walsh J, Nicholson L V B, Harris J B. Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling. Proc Roy Soc L o n d B 1990; 240: 197-210. 9. Patel K, Voit T, Dunn M J, Strong P N, Dubowitz V. Dystrophin and nebulin in the muscular dystrophies. J Neurol Sci 1988; 87: 315-326. 10. Nicholson L V B, Davison K, Falkous G, et al. Dystrophin in skeletal muscle. I. Western blot analysis using a monoclonal antibody. J Neurol Sci 1989: 94: 125 136.
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Hoffman E P, Fischbeck K H, Brown R H, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J M e d 1988; 318: 1363-1368. Cooper B J, Winand N J, Stedman H, et al. The homologue of the Duchenne locus is defective in Xlinked muscular dystrophy of dogs. Nature 1988; 334: 154 156. Carpenter J L, Hoffman E P, Romanul F C A, et al. Feline muscular dystrophy with dystrophin deficiency. Am J Patho11989; 135: 909-919. Ohlendieck K, Ervasti J M, Snook J B, Campbell K P. Dystrophin glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J Cell Biol 1991: 112: 135-148. Capaldi M J, Dunn M J, Sewry C A, Dubowitz V. Lectin blotting of human muscle: identification of a high molecular weight glycoprotein which is absent or altered in Duchenne muscular dystrophy. J Neurol Sci 1985; 68:225 231. Capaldi M J, Dunn M J, Sewry C A, Dubowitz V. Binding of Ricinus communis I lectin to the muscle cell plasma membrane in diseased muscle. J Neurol Sci 1984; 64:315 324. Currie S. The inflammatory myopathies. In: Walton J N ed. Disorders of Voluntary Muscle, 5th Edn. Edinburgh: Churchill Livingstone, 1988: 588-610. Dubowitz V. Muscle Biopsy. A Practical Approach 2nd Edn. London: Bailli~re Tindall, 1985. Heckmatt J Z~ Hasson N, Saunders C, et al. Cyclosporin in juvenile dermatomyositis. Lancet 1989: 1063-1066. Heckmatt J Z, Moosa A, Hutson C, Maunder-Sewry C A, Dubowitz V. Diagnostic needle muscle biopsy: a practical and reliable alternative to open biopsy. Arch Dis Child 1984; 59:528 532. Clerk A, Rodillo E, Heckmatt J Z, Dubowitz V, Strong P N, Sewry C A. Characterisation of dystrophin in carriers of Duchenne muscular dystrophy. J Neurol Sci 1991; 102:197 205. Arikawa E, Arahata K, Nonaka I, Sugita H. Immunohistochemical analysis of dystrophin in congenital muscular dystrophy (CMD). J Neurol Sci 1990: 98 (Suppl): 438. Clerk A, Strong P N, Sewry C A. Characterisation of dystrophin during development of human skeletal muscle. Development 1992 (in press). Knudsen C M, Hoffman E P, Kahl S D, Kunkel L M, Campbell K P. Evidence for the association of dystrophin with the transverse tubular system in skeletal muscle. J Biol Chem 1988; 263:8480-8484 Salviati G, Betto R, Ceoldo S, et al. Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle. Biochem J 1989; 258: 837 841.
0960-8966.91 $3.00 ~ 0.00 ic, 1992PergamonPress pic
Printed in Great Britain
DYSTROPHIN ABNORMALITIES IN POLYMYOSITIS AND DERMATOMYOSITIS C. A. SEWRY,*'I" A. CLERK,* J. Z. HECKMATT,++ T. VYSE,* V. DUBOWITZ* a n d P. N. STRONG* *Jerry Lewis Muscle Research Centre, Department of Paediatrics and Neonatal Medicine; and ~Department of Medicine, Rheumatology Unit, Royal Postgraduate Medical School, D u Cane Road, London W12 0NN, U.K.
(Received 29 July 1991; accepted 11 October 1991)
Abstract--The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten
cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and Cterminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to #-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.
Key words: Dystrophin, inflammatory myopathy, myositis, muscle.
[6, 10, 11] but relatively few cases ofpolymyositis or dermatomyositis have been documented. Recently, it has been shown that dystrophin is associated with a glycoprotein complex [14]. Our earlier studies with the lectin Ricinus communis I, showed that a 370 kDa glycoprotein is absent or greatly reduced in D M D [15] and electron microscopy demonstrated that a glycoprotein that binds the same lectin was abnormal not only in D M D and BMD but also in polymyositis [16, 17]. The possible association ofdystrophin with a glycoprotein, and the evidence suggesting a membrane glycoprotein defect in inflammatory myopathies led us to carry out a detailed examination of dystrophin expression in patients with polymyositis and dermatomyositis. Polymyositis and dermatomyositis are acquired myopathies usually characterised by sudden or acute onset, muscle weakness and fatigue [17]. Serum creatine kinase levels may be elevated and muscle biopsies may show varying degrees of muscle fibre degeneration and cellular infiltration [18]. In dermatomyositis a skin rash is also present. Onset may be in childhood or during adult life and both males and females can be affected. The'pathogenesis of these diseases probably has an immunological basis. Many patients, particularly those with childhood
INTRODUCTION
Dystrophin, the defective protein in Duchenne muscular dystrophy (DMD) is a high molecular weight protein (427 kDa) coded by a gene of at least 2.3 Mb at position Xp21 on the short arm of the X chromosome [1, 2]. Approximately 60% of D M D patients have detectable deletions in the gene. Becker muscular dystrophy (BMD), which is phenotypically less severe than DMD, is also caused by a defect in the same Xp21 gene [3, 4]. In normal skeletal muscle dystrophin has been localised immunocytochemically to the cytoplasmic face of the sarcolemmal plasma membrane [5-8]. On immunoblots, using antibodies raised against fusion proteins containing dystrophin peptide sequences, dystrophin appears as a doublet [9, 10]. In D M D , as well as the Xlinked muscular dystrophies in the mouse (mdx), the dog (xmd) and the cat, dystrophin is absent or greatly reduced [11-13]. In BMD, dystrophin is often either reduced in abundance or has an altered molecular weight [9, 11]. In a variety of other neuromuscular disorders the expression of dystrophin has been reported to be normal
t A u t h o r to whom correspondence should be addressed. 333
C. A. SEWRY et al.
334
Table 1. Clinical details and the results of dystrophin analysis in patients with polymyositis and dermatomyositis Case
Age (yr)
Sex
Diagnosis
Steroids"
I.MH 2.RA 3.NJ 4.JS
3 3 31 33
5.AJ
CKt
M M F F
Poly Poly Poly Poly
No Yes Yes No
133 14000 1090 74
38
F
Poly
No
156
6.FA
42
F
Poly
Yes
7755
7.GG 8.EW
58 76
M F
Poly Poly
Yes Yes
40000 1395
9.FC 10.FK I I.SW 12.GT
77 1 5 6
F M F M
Poly JDM JDM JDM
Yes No No Yes
1450 1783 50 266
13.DD
8
F
JDM
No
29
14.GI 15.CJ
8 9
M F
JDM JDM
No No
89 64
16.EH 17.TC
12 13
F F
JDM JDM
Yes No
166 88
18.SS 19.MG
14 16
F M
JDM JDM
No No
155 101
20.JW 21.DS
51 57
M F
Derm Derm
No Yes
1560 175
22.FB
62
M
Derm
Yes
37
lmmunoblot
$ Mol.wt ~ Mol.wt ~ Mol.wt Normal size reduced amount ~, Mol.wt reduced amount Normal size reduced amount Normal Normal size reduced amount Normal $ Mol.wt $ Mol.wt Normal size reduced amount ,L Mol.wt reduced amount ~ Mol.wt Normal size reduced amount Normal Normal size reduced amount Normal Normal size reduced amount Normal ~ Mol.wt reduced amount Normal
Immunostaining
Not done Variability Normal Normal Normal Normal Variability Not done Variability Variability Not done Variability Normal Normal Normal Normal Variability Variability Variability Variability Variability Variability
Poly = polymyositis; JDM = juvenile dermatomyositis; and Derm = adult dermatomyositis. * Steroid treatment at time of biopsy. t International units, upper limit of normal 200 IU 1 ~.
dermatomyositis, respond to a carefully controlled regime of steroid treatment and recently the use of cyclosporin has proved to be effective in refractory cases [19]. We report here studies on the expression of dystrophin in both children and adults suffering from polymyositis or dermatomyositis, using immunocytochemical and immunoblotting techniques. METHODS
BergstrSm needle biopsies were taken under local anaesthesia from the quadriceps of two children and seven adults with polymyositis, ten children with the juvenile form of dermatomyositis and three adults with dermatomyositis [20]. Details of the patients are given in Table 1. Diagnosis was made according to established clinical and pathological criteria [18]. Samples for immunocytochemistry were orientated transversely on cork discs, rapidly frozen in isopentane cooled in liquid nitrogen, and stored at -80°C until required. Samples for immunoblotting were either frozen separately in liquid nitrogen and stored at -80°C or, in some
cases, were cut from the cork-mounted block (4 x 20/~m sections) and kept frozen at -80°C in vials. Similar results were obtained with either method, and 20/tm sections have been shown to provide sufficient material for the detection of dystrophin [9]. Antibodies. A sheep polyclonal antiserum raised against a fusion protein containing a 60 kDa dystrophin peptide sequence from the Nterminal region ofdystrophin (kindly donated by Drs L. Kunkel and E. Hoffman, Boston) was used to study all cases (this antibody is referred to subsequently as 60 KD). In addition, most cases were also studied with rabbit polyclonal antibodies (H12 and P6) raised against fusion proteins corresponding to amino acids 2604-3024 and 2814-3028, respectively, from the C-terminal end of the rod repeat region of dystrophin (Sherratt, in preparation). Monoclonal antibodies raised against the rod domain (Dy4) and against the last 17 C-terminal amino acids (Dy8) were kindly made available by Dr L. Nicholson (Newcastle upon Tyne), towards the end of the study. Monoclonal antibodies to human erythrocyte fl-spectrin (56A) were used to assess the morphological integrity of the sarcolemma (a gift from Dr M. Newton, Oxford).
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335
Gel electrophoresis and Western blot analysis. These were performed as described previously [21]. Briefly, the frozen piece of muscle was crushed in foil by lead weights and the powder solubilised in 1% SDS containing protease inhibitors (1 mM iodoacetamide, 1 mM benzemethonium chloride, 0.5 mM phenyl methyl sulphonyl fluoride, 0.05 mM Pepstatin A). The 20 ~tm cryostat sections were solubilised directly in 1% SDS. Fifty micrograms of protein (Bradford protein assay) were boiled with an equal volume of Laemmli buffer for 2 min and resolved by SDS-PAGE on 4-20% linear gradient gels for 16 h at 20 mA gel -~. Gels were washed for 30 min in transfer buffer (20 mM Tris base, 150 mM glycine) and electroblotted onto nitrocellulose (20 h at 500 mA). Blots were incubated with dystrophin antibodies diluted in phosphate buffered saline (PBS) containing 0.05% Tween 20 (TP buffer) for 1 h at 20°C (60 KD 1 : 1000, H12 1 : 3000, P6 1 : 3000, Dy4 1 : 50, Dy8 1 : 50). This was followed by incubation for 1 h with an appropriate biotinylated, secondary antibody (Amersham) diluted 1 : 400 in TP/50% (v/v) human serum. Dystrophin was visualised with a preformed streptavidin-biotin-peroxidase complex (Dakopatts ABC kit) followed by freshly prepared diaminobenzidene (0.025%, w/v) containing 0.05% H202. Control muscle from normal volunteers and from patients with non-Xp21 neuromuscular disorders were probed on the same blot for comparison and control nitrocellulose strips probed without the primary antibody. Immunocytochemistry. Unfixed cryostat sections (6 pm) were incubated for 30 min at room temperature with antibodies to dystrophin diluted in PBS pH 7.4 (60 KD 1 : 1000, P6 1 : 1000, H12 1 : 1200; Dy4 and Dy8 were used undiluted). After washing in PBS, sections were incubated for a further 30 min with an appropriate biotinylated secondary antibody (Amersham, 1 : 200), washed again in PBS and incubated for 15 min with streptavidin conjugated to the fluorochrome Texas Red (Amersham, 1 : 200). Serial sections were immunostained in a similar manner with the monoclonal antibody to fl-spectrin (56A). Sections from biopsies from normal volunteers and from patients with non-Xp21 disorders were used for comparison. Control sections were incubated without the primary antibody and were negative except for autofluorescence.
C
FK
200J,,-
P6 Fig. 1. Skeletal muscle from a 1-yr-old boy with juvenile dermatomyositis (case 10, FK) immunoblotted with antibodies to a C-terminal region of dystrophin (P6) showing a lower molecular weight protein (C = control). (Blemishes in some areas of this and subsequent blots are the result of fire damage to the photographic emulsion.)
C
CJ
C
CJ
....L
200
Dy4
"Dy8
Fig. 2. Muscle from a 9-yr-old girl with juvenile dermatomyositis (case 15, C J) immunoblotted with the two monoclonal antibodies to N- and C-terminal regions of dystrophin (Dy4, Dy8). Dystrophin is of normal molecular weight but reduced in amount (C = control). RESULTS
Control muscle from normal volunteers and from a variety of non-Xp21 neuromuscular disorders on Western blot analysis showed the characteristic doublet when probed with the 60 KD, H12, P6 and Dy4 dystrophin antibodies (Figs 1, 2 and 3). In contrast, Dy8 identified a single band (Fig. 2). Four out of nine cases of polymyositis and five out of thirteen cases of dermatomyositis, however, showed an immuno-
C. A. SEWRY et al.
336
Table 2 Diagnosis
Patients with abnormal immunoblot
Polymyositis Dermatomyositis Total
C SS
7/9 (78%) 9/13 (69%) 16/22 (73%)
C SS iiili ¸¸ i i ¸i
iiii
200 ?
Oy4 Fig. 3. Muscle from a 14-yr-old female with juvenile dermatomyositis (case 18, SS) immunoblotted with antibodies to an N-terminal region (Dy4), and a C-terminal region (H 12) of dystrophin. The size and amount of dystrophin are both normal (C = control).
reactive band of slightly smaller molecular weight (Fig. 1). In addition, four cases of polymyositis and six cases of dermatomyositis had a reduced amount of dystrophin, compared with the control muscle run on the same gel and with an equal loading of protein (Fig. 2). These abnormalities were consistent with more than one antibody (Fig. 2). A total of 16 out of 22 patients (73%) showed an abnormality on Western blot analysis (Table 2). The remaining patients had dystrophin of normal size and normal amount (Fig. 3). In the cases showing an abnormality in dystrophin there was no correlation with age, sex, serum CK levels, steroid treatment, disease type or degree of pathology (Table 1). There was no evidence of general protein breakdown nor of any characteristic dystrophin immunoreactive breakdown products [10]. Immunostaining of muscle from normal individuals and from controls showed labelling of the sarcolemma of all fibres with all antibodies (Fig. 4). Abnormalities in immunostaining were seen in three out of seven cases of polymyositis and eight out of twelve cases ofdermatomyositis. The abnormalities varied from patchy or dis-
Patients with abnormal immunostaining 3/7 (43%) 8/12 (67%) 11/19 (58%)
continuous staining on some fibres (Fig. 5), to extensive areas with reduced labelling (Fig. 6). These defects were more pronounced with the 60 KD antibody but were also observed with the other antibodies (Fig. 5). However, not all cases were assessed with the monoclonal antibodies Dy4 and Dy8 as these were only available towards the end of the investigation. Immunostaining with /~-spectrin antibodies showed an even distribution at the periphery of fibres (except necrotic fibres which were negative) implying that alterations in dystrophin were not due to any major loss of integrity of the sarcolemma (Fig. 5). However, the possibility that the abnormalities of the sarcolemma affected dystrophin more that ]~-spectrin cannot be excluded. A total of 11 out of 19 patients (58%) showed defects with immunostaining (Table 2) but these did not always correspond with those with abnormalities on immunoblotting (cf. Figs 3 and 5). Some samples that were abnormal on immunoblots showed no significant defects with immunostaining (Table 1). There was no apparent correlation with age, serum CK levels, drug therapy or pathological features in the biopsies. In one case of polymyositis (case 6, FA) and one case of dermatomyositis (case 16, EH) vacuoles were present. These were immunolabelled with antibodies to dystrophin and to ]~spectrin (Fig. 7). Splits in fibres were also labelled. Vacuoles were also observed in one case of dermatomyositis (case 12, GT) but in this biopsy the vacuoles did not show dystrophin, only /~-spectrin. Dystrophin, however, was greatly reduced in this biopsy (Fig. 6). The vacuoles in these cases were observed in transverse sections and no connections with the sarcolemma were seen. The latter possibility, however, cannot be excluded. DISCUSSION
An abnormal expression of dystrophin was demonstrated by Western blot analysis in 16 out of 22 of our patients with polymyositis or dermatomyositis (73%), and in 11 out of 19
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337
Fig. 4. Biopsy from a normal adult immunostained with antibodies to an N-terminal region (60 KD), and a C-terminal region (Dy8) of dystrophin. The peripheries of all fibres are labelled with dystrophin and with antibodies to p-spectrin (56A). × 180.
Fig. 5. Serial sections from the same biopsy as Fig. 3 with juvenile dermatomyositis (case 18, SS) immunostained with antibodies to an N-terminal region (60 KD), and a C-terminal region (H12). Labelling for dystrophin is patchy and discontinuous on some fibres (arrows) but the same fibres have normal expression of fl-spectrin (56A). x 240.
patients (58%) by immunostaining. There was not always a direct correlation between the immunoblotting and immunocytochemical results. This lack of correlation emphasises that both techniques should be used but that they should not be equated. Immunocytochemistry detects epitopes on native proteins and gives the cellullar localisation of a protein. Subtle differences in the antibody binding can be seen with immunocytochemistry but it gives no indication of the molecular weight of the detected protein or
of its quantity. Immunoblots, in contrast, detect epitopes on denatured proteins and differences in both the size and total quantity of a protein can be examined. The abnormalities in dystrophin expression cannot be explained on the basis of any consistent correlation with age, sex, steroid treatment, disease type or pathological severity. Hitherto, abnormalities in dystrophin have only been reported in D M D and BMD [6, 9-11], although a preliminary report has suggested abnormalities in some cases of the Fukuyama
338
C.A. SEWRY et al.
60KD
[ Fig. 6. Biopsy from a 6-yr-old boy with juvenile dermatomyositis (case 12, GT) immunostained with antibodies to an N-terminal region of dystrophin (60 KD) showing an extensive area of reduced labelling. Vacuoles in this sample are not labelled with dystrophin antibodies, x 260.
Fig. 7. Biopsy from an adult patient with polymyositis (case 6, FA) immunolabelled with antibodies to an N-terminal region of dystrophin (60 KD) showing staining of a vacuole (arrow) as well as the periphery of the fibres, x 330.
type of congenital muscular dystrophy [22]. It is therefore of considerable interest that alterations in the expression of dystrophin should be found in two inflammatory diseases unrelated to defects in the Xp21 locus. The abnormalities in dystrophin expression in polymyositis and derma-
tomyositis were found in both male and female patients further suggesting that the abnormalities are secondary effects of the disease, rather than genetically determined. The reduced amount of dystrophin in many patients could be the result of general muscle degradation, but there was no evidence of this. The smaller molecular weight form ofdystrophin might be due to a specific proteolytic cleavage resulting from increased protease activity originating from infiltrating cells, but there was no correlation with the degree of inflammation. We also considered the possibility that the presence of the smaller molecular weight protein might relate to regeneration and could be similar to the smaller form ofdystrophin that we have detected in human foetal muscle [23]. There was no correlation with the presence of foetal myosin or the degree of regeneration to support this hypothesis, but until more is known about the isoforms of dystrophin this possibility cannot be excluded. There are no known genetic similarities between the Xp21 dystrophies and the inflammatory myopathies but both are associated with defects in lectin binding glycoproteins [15, 16]. Recently it has been shown that dystrophin is associated with a sarcolemmal glycoprotein complex [14] but no relationship between the glycoproteins in these different studies has yet been established. The localisation ofdystrophin to vacuoles is of interest. Vacuoles are believed to originate from the T-tubule system or the sarcoplasmic reticulum but dystrophin and fl-spectrin are both associated with the sarcolemmal plasma membrane. Although evidence from biochemical fractionation procedures has suggested that dystrophin was localised to the T-tubule system [24, 25], all immunocytochemical data show that it is localised to the cytoplasmic face of the sarcolemma [7, 8]. No connections between the plasma membrane and the vacuoles were observed in the series of sections examined, thus the presence of dystrophin on vacuoles in two of our cases suggests that they may originate from the plasma membrane. Although there is no defect in the dystrophin gene in polymyositis and dermatomyositis, the reasons for the alterations that we have observed are not clear yet. Our studies show that secondary defects in the expression of dystrophin can occur and that abnormalities in the gene product are not confined to the X-linked dystrophies. Our results emphasise the essential need for detailed
Dystrophin in Myositis
clinical assessments for the interpretation of the results from dystrophin analysis and that the latter cannot be used in isolation as a diagnostic marker. work was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland. We thank Miss P. Badiani, Miss A. Mequita and Miss L. Wilson for technical assistance, Mrs K. Davidson for photography and Mr T. Sherratt for the P6 and HI2 dystrophin antibodies. The gifts of antibodies from Drs L. Kunkel, M. Newton and L. Nicholson are also gratefully acknowledged.
l l.
12.
Acknowledgements--This
13. 14.
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