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Abnormal expression of dystrophin-associated proteins in Fukuyama-type congenital muscular dystrophy
521
1.
REFERENCES Langman MJS. Upper gastrointestinal bleeding: the trials of trials. Gut 1985; 26: 217-20.
Storey DW, Brown SG, Swain CP, Salmon PR, Kirkham JS, Northfield TC. Endoscopic prediction of recurrent bleeding in peptic ulcer. N Engl J Med 1981; 305: 915-16. 3. Harris DC, Heap TR. Significance of signs of recent haemorrhage at endoscopy. Med J Aust 1982; 2: 35-40. 4. Macleod IA, Mills PR. Factors identifying the probability of further haemorrhage after acute upper gastrointestinal haemorrhage. Br J Surg 1982; 69: 256-58. 5. Poller L. Fibrinolysis and gastrointestinal haemorrhage. J Clin Pathol 1979; 14 (suppl): 63-67. 6. Lowe GDO, Prentice CRM. Laboratory investigation of fibrinolysis. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 2nd ed. Edinburgh: Churchill Livingstone, 1980: 222-60. 7. Haverkate F. A simple device for measuring diameters of fibrinolysis zones in fibrin plates. Haemostasis 1974; 1: 55-60.
2.
8. Elms MJ, Bunce IH, Bundesen PG, et al. Measurement of cross-linked fibrin degradation products by immunoassay using monoclonal antibodies. Thromb Haemost 1983; 50: 591-94. 9. Bertaglia E, Belmonte P, Vertolli U, Martines D. Bleeding in cirrhotic
patients: a precipitating factor due to intravascular coagulation or to hepatic failure? Haemostasis 1983; 13: 328-34. 10. Lowe GDO, Rumley A. Laboratory aspects of thrombolytic therapy. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 177-208. 11. Henry DA, O’Connell DL. Effects of fibrinolytic inhibitors on mortality from upper gastrointestinal haemorrhage. BMJ 1989; 298: 1142-46. 12. Booth NA. The laboratory investigation of the fibrinolytic system. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 115-50. 13. Nieuwenhuizen W. The formation, measurement and clinical value of fibrinogen derivatives. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 151-76.
SHORT REPORTS Abnormal
expression of dystrophin-associated proteins in Fukuyama-type congenital muscular dystrophy
The absence of dystrophin causes Duchenne muscular dystrophy. Dystrophin is associated with a large complex of sarcolemmal glycoproteins which provides a linkage to the extracellular matrix component, laminin, and when dystrophin is absent all the dystrophin-associated proteins are much reduced. We report here that dystrophin-associated proteins have abnormally low expression in Fukuyama-type congenital muscular dystrophy (FCMD), despite near-normal expression of dystrophin. An abnormality of dystrophin-associated proteins in the sarcolemma seems to be a common denominator in the pathological processes leading to muscle cell necrosis in three forms of severe muscular
dystrophy (Duchenne, Japanese Fukuyama-type, and
north
African
Duchenne-like
autosomal
recessive). Lancet 1993; 341: 521-22.
Dystrophin, a membrane cytoskeletal protein encoded by the Duchenne muscular dystrophy (DMD) gene, is associated with a large oligomeric complex of sarcolemmal glycoproteins.l One of these dystrophin-associated proteins (DAPs) is dystroglycan (156 DAG), which provides a linkage to the extracellular matrix component, laminin.2 The absence of dystrophin causes a drastic reduction in all DAPs.3 The structure of the dystrophin-glycoprotein complex linking subsarcolemmal cytoskeleton to extracellular matrix suggests that its disruption or dysfunction could lead to severe instability of the sarcolemma and, in turn, muscle cell necrosis.1,3An intriguing possibility is that a primary defect in the DAPs could be the cause of hereditary neuromuscular disease with DMD-like phenotype. We have demonstrated deficiency of
the 50 DAG in one such DMD-like condition-namely, severe childhood autosomal recessive muscular dystrophy (SCARMD), which is common in north Africa.4 We report here on the status of the dystrophin-glycoprotein complex in another severe autosomal recessive muscular dystrophy of childhood, Fukuyama-type congenital muscular dystrophy (FCMD).5 In Japan this is the most common form of childhood myopathy apart from DMD. The phenotype consists of a combination of severe muscular dystrophy and an anomaly of the brain.s Immunohistochemical analysis of muscle biopsy specimens from twelve FCMD patients (seven males, five females; age 4-20 months) were done with nine antibodies: Directed against Antibody
DAG
glycoprotein
=
DAP
dystrophi n -associated
proteinn
have found no abnormality of the components of this in complex limb-girdle, myotonic, facioscapulohumeral, oculopharyngeal, or non-Fukuyama congenital muscular dystrophies, in congenital fibre type disproportion, in spinal muscular atrophy, or in amyotrophic lateral sclerosis (age range 5 months to 70 years).
So far
we
In FCMD patients staining for dystrophin was well preserved in the sarcolemma except in a few muscle fibres with reduced and patchy staining.6,’ In contrast, staining for the DAPs (figure) was greatly reduced in the sarcolemma of most muscle fibres, and this was especially true for 43DAG (figure).* A few muscle fibres had abnormally intense staining in the sarcolemma or had diffuse cytoplasmic staining (figure); these abnormalities were common to all patients but the severity varied and did not correlate with age or sex. The reduction in FCMD was confirmed by the immunoblot analysis of skeletal muscle biopsy extracts (not shown). These findings in FCMD contrast sharply with those in DMD, where the absence of dystrophin causes a *Composite panels showing
36 slides of FCMD and control material are available from K. P. C.
immunostained with various antibodies
522
THE LANCET
K. P. C. is an investigator of the Howard Hughes Medical Institute. This work was also supported by the Muscular Dystrophy Association of America and the Uehara Memorial Foundation. We thank Cynthia J. Leveille and Michael J. Mullinix for technical assistance.
REFERENCES JM, Campbell KP. Membrane organization of the dystrophinglycoprotein complex. Cell 1991; 66: 1121-31. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP, Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature
1. Ervasti 2.
analysis of components of dystrophinglycoprotein complex in skeletal muscle. Transverse cryosections (7 m) of skeletal muscle biopsy material from a patient with non-Fukuyama congenital muscular dystrophy (CMD) and two patients with FCMD were immunostained with antibodies against dystrophin (DYS) and the dystrophin-associated proteins. Bar= 50 11m. Immunohistochemical
reduction in all DAPs,3or in SCARMD where 50 DAG deficiency causes a secondary reduction of 35 DAG.’ This abnormal expression ofDAPs despite near-normal expression of dystrophin is pathognomonic and immunochemical analysis should considerably assist in the diagnosis of FCMD. The finding that dystrophin remained localised to the sarcolemma supports our hypothesis that dystrophin is linked to both the subsarcolemmal cytoskeleton and DAPS.l,2 In the absence of DAPs, dystrophin could still be associated with cytoskeletal components such as y-actin and be properly localised to sarcolemma. An abnormality of DAPs in the sarcolemma seems to be the common denominator to the pathological process that leads to muscle cell necrosis in three major forms of severe childhood muscular dystrophy (DMD, SCARMD, and FCMD). We propose that disruption/dysfunction of the dystrophin-glycoprotein complex interrupts the link between subsarcolemmal cytoskeleton and extracellular matrix, resulting in cascade of events leading to muscle cell necrosis. The cause of the abnormal expression of DAPs in FCMD is unknown. One possibility is a defect in the structure or expression of a gene for a DAP, which would be consistent with the recent observation of a possible interaction between dystrophin and the putative FCMD gene product.8 On clinical grounds the FCMD gene product should be expressed in both muscle and brain. We have identified a single gene which encodes both 156DAG and 43DAG, and the transcript is expressed in these two tissues.2 Characterisation of this 156DAG/43DAG gene from FCMD patients is underway in our laboratory.
1992; 355: 696-702. 3. Ohlendieck K, Matsumura K, Ionasescu VV, Towbin JA, Bosch EP, Weinstein SL, Northrup SW, Campbell KP. Duchenne muscular dystrophy: deficiency of dystrophin-associated proteins in the sarcolemma. Neurology (in press). 4. Matsumura K, Tome FMS, Collin H, Azibi K, Chaouch M, Kaplan JC, Fardeau M, Campbell KP. Deficiency of the 50K dystrophinassociated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature 1992; 359: 320-22. 5. Nonaka I, Chou SM. Congenital muscular dystrophy. In: Vinken PJ, Bruyn GW, eds. Handbook of clinical neurology vol 41: Amsterdam: North-Holland, 1979: 27-50. 6. Arahata K, Hoffman EP, Kunkel LM, Ishiura S, Tsukahara T, Ishihara T, Sunohara N, Nonaka I, Ozawa E, Sugita H. Dystrophin diagnosis: comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses. Proc Natl Acad Sci USA 1989; 86: 7154-58. 7. Arikawa E, Ishihara T, Nonaka I, Sugita H, Arahata K. Immunocytochemical analysis of dystrophin in congenital muscular dystrophy. J Neurol Sci 1991; 105: 79-87. 8. Beggs AH, Neumann PE, Arahata K, Arikawa E, Nonaka I, Anderson MS, Kunkel LM. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy. Proc Natl Acad Sci USA 1992; 89: 623-27. ADDRESSES: Howard Hughes Medical Institute and Department of Physiology and Biophysics, Univeristy of Iowa College of Medicine, Iowa City, IA 52242, USA (K. Matsumura, MD, Prof K. P Campbell, PhD); and National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Tokyo, Japan (I. Nonaka, MD). Correspondence to Prof Kevin P. Campbell.
Association of human papillomavirus with nasal
neoplasia
Since HPV-57b has been identified by two different techniques in benign, premalignant, and malignant lesions of the nasal cavity, but not in cases of chronic sinusitis, H PV-57 should be recognised as at least a co-factor in the aetiology of nasal neoplasia. Paraffin sections of 22 histologically confirmed nasal tumours were screened by in-situ hybridisation with riboprobes specific for HPV-57b. Virus was demonstrated in 6 of 7 fungiform papillomas, 6 of 8 inverted papillomas, 1 of 3 inverted papillomas with dysplasia, and 2 of 4 inverted papillomas with carcinoma. The presence of HPV-57b was confirmed with the polymerase chain reaction, which identified an additional 4 positive samples, bringing the total to 86% positive specimens. The results underscore the importance of HPVs in the aetiology of cancers at extragenital sites.
1.
REFERENCES Langman MJS. Upper gastrointestinal bleeding: the trials of trials. Gut 1985; 26: 217-20.
Storey DW, Brown SG, Swain CP, Salmon PR, Kirkham JS, Northfield TC. Endoscopic prediction of recurrent bleeding in peptic ulcer. N Engl J Med 1981; 305: 915-16. 3. Harris DC, Heap TR. Significance of signs of recent haemorrhage at endoscopy. Med J Aust 1982; 2: 35-40. 4. Macleod IA, Mills PR. Factors identifying the probability of further haemorrhage after acute upper gastrointestinal haemorrhage. Br J Surg 1982; 69: 256-58. 5. Poller L. Fibrinolysis and gastrointestinal haemorrhage. J Clin Pathol 1979; 14 (suppl): 63-67. 6. Lowe GDO, Prentice CRM. Laboratory investigation of fibrinolysis. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 2nd ed. Edinburgh: Churchill Livingstone, 1980: 222-60. 7. Haverkate F. A simple device for measuring diameters of fibrinolysis zones in fibrin plates. Haemostasis 1974; 1: 55-60.
2.
8. Elms MJ, Bunce IH, Bundesen PG, et al. Measurement of cross-linked fibrin degradation products by immunoassay using monoclonal antibodies. Thromb Haemost 1983; 50: 591-94. 9. Bertaglia E, Belmonte P, Vertolli U, Martines D. Bleeding in cirrhotic
patients: a precipitating factor due to intravascular coagulation or to hepatic failure? Haemostasis 1983; 13: 328-34. 10. Lowe GDO, Rumley A. Laboratory aspects of thrombolytic therapy. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 177-208. 11. Henry DA, O’Connell DL. Effects of fibrinolytic inhibitors on mortality from upper gastrointestinal haemorrhage. BMJ 1989; 298: 1142-46. 12. Booth NA. The laboratory investigation of the fibrinolytic system. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 115-50. 13. Nieuwenhuizen W. The formation, measurement and clinical value of fibrinogen derivatives. In: Thomson JM, ed. Blood coagulation and haemostasis: a practical guide, 4th ed. Edinburgh: Churchill Livingstone, 1991: 151-76.
SHORT REPORTS Abnormal
expression of dystrophin-associated proteins in Fukuyama-type congenital muscular dystrophy
The absence of dystrophin causes Duchenne muscular dystrophy. Dystrophin is associated with a large complex of sarcolemmal glycoproteins which provides a linkage to the extracellular matrix component, laminin, and when dystrophin is absent all the dystrophin-associated proteins are much reduced. We report here that dystrophin-associated proteins have abnormally low expression in Fukuyama-type congenital muscular dystrophy (FCMD), despite near-normal expression of dystrophin. An abnormality of dystrophin-associated proteins in the sarcolemma seems to be a common denominator in the pathological processes leading to muscle cell necrosis in three forms of severe muscular
dystrophy (Duchenne, Japanese Fukuyama-type, and
north
African
Duchenne-like
autosomal
recessive). Lancet 1993; 341: 521-22.
Dystrophin, a membrane cytoskeletal protein encoded by the Duchenne muscular dystrophy (DMD) gene, is associated with a large oligomeric complex of sarcolemmal glycoproteins.l One of these dystrophin-associated proteins (DAPs) is dystroglycan (156 DAG), which provides a linkage to the extracellular matrix component, laminin.2 The absence of dystrophin causes a drastic reduction in all DAPs.3 The structure of the dystrophin-glycoprotein complex linking subsarcolemmal cytoskeleton to extracellular matrix suggests that its disruption or dysfunction could lead to severe instability of the sarcolemma and, in turn, muscle cell necrosis.1,3An intriguing possibility is that a primary defect in the DAPs could be the cause of hereditary neuromuscular disease with DMD-like phenotype. We have demonstrated deficiency of
the 50 DAG in one such DMD-like condition-namely, severe childhood autosomal recessive muscular dystrophy (SCARMD), which is common in north Africa.4 We report here on the status of the dystrophin-glycoprotein complex in another severe autosomal recessive muscular dystrophy of childhood, Fukuyama-type congenital muscular dystrophy (FCMD).5 In Japan this is the most common form of childhood myopathy apart from DMD. The phenotype consists of a combination of severe muscular dystrophy and an anomaly of the brain.s Immunohistochemical analysis of muscle biopsy specimens from twelve FCMD patients (seven males, five females; age 4-20 months) were done with nine antibodies: Directed against Antibody
DAG
glycoprotein
=
DAP
dystrophi n -associated
proteinn
have found no abnormality of the components of this in complex limb-girdle, myotonic, facioscapulohumeral, oculopharyngeal, or non-Fukuyama congenital muscular dystrophies, in congenital fibre type disproportion, in spinal muscular atrophy, or in amyotrophic lateral sclerosis (age range 5 months to 70 years).
So far
we
In FCMD patients staining for dystrophin was well preserved in the sarcolemma except in a few muscle fibres with reduced and patchy staining.6,’ In contrast, staining for the DAPs (figure) was greatly reduced in the sarcolemma of most muscle fibres, and this was especially true for 43DAG (figure).* A few muscle fibres had abnormally intense staining in the sarcolemma or had diffuse cytoplasmic staining (figure); these abnormalities were common to all patients but the severity varied and did not correlate with age or sex. The reduction in FCMD was confirmed by the immunoblot analysis of skeletal muscle biopsy extracts (not shown). These findings in FCMD contrast sharply with those in DMD, where the absence of dystrophin causes a *Composite panels showing
36 slides of FCMD and control material are available from K. P. C.
immunostained with various antibodies
522
THE LANCET
K. P. C. is an investigator of the Howard Hughes Medical Institute. This work was also supported by the Muscular Dystrophy Association of America and the Uehara Memorial Foundation. We thank Cynthia J. Leveille and Michael J. Mullinix for technical assistance.
REFERENCES JM, Campbell KP. Membrane organization of the dystrophinglycoprotein complex. Cell 1991; 66: 1121-31. Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP, Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature
1. Ervasti 2.
analysis of components of dystrophinglycoprotein complex in skeletal muscle. Transverse cryosections (7 m) of skeletal muscle biopsy material from a patient with non-Fukuyama congenital muscular dystrophy (CMD) and two patients with FCMD were immunostained with antibodies against dystrophin (DYS) and the dystrophin-associated proteins. Bar= 50 11m. Immunohistochemical
reduction in all DAPs,3or in SCARMD where 50 DAG deficiency causes a secondary reduction of 35 DAG.’ This abnormal expression ofDAPs despite near-normal expression of dystrophin is pathognomonic and immunochemical analysis should considerably assist in the diagnosis of FCMD. The finding that dystrophin remained localised to the sarcolemma supports our hypothesis that dystrophin is linked to both the subsarcolemmal cytoskeleton and DAPS.l,2 In the absence of DAPs, dystrophin could still be associated with cytoskeletal components such as y-actin and be properly localised to sarcolemma. An abnormality of DAPs in the sarcolemma seems to be the common denominator to the pathological process that leads to muscle cell necrosis in three major forms of severe childhood muscular dystrophy (DMD, SCARMD, and FCMD). We propose that disruption/dysfunction of the dystrophin-glycoprotein complex interrupts the link between subsarcolemmal cytoskeleton and extracellular matrix, resulting in cascade of events leading to muscle cell necrosis. The cause of the abnormal expression of DAPs in FCMD is unknown. One possibility is a defect in the structure or expression of a gene for a DAP, which would be consistent with the recent observation of a possible interaction between dystrophin and the putative FCMD gene product.8 On clinical grounds the FCMD gene product should be expressed in both muscle and brain. We have identified a single gene which encodes both 156DAG and 43DAG, and the transcript is expressed in these two tissues.2 Characterisation of this 156DAG/43DAG gene from FCMD patients is underway in our laboratory.
1992; 355: 696-702. 3. Ohlendieck K, Matsumura K, Ionasescu VV, Towbin JA, Bosch EP, Weinstein SL, Northrup SW, Campbell KP. Duchenne muscular dystrophy: deficiency of dystrophin-associated proteins in the sarcolemma. Neurology (in press). 4. Matsumura K, Tome FMS, Collin H, Azibi K, Chaouch M, Kaplan JC, Fardeau M, Campbell KP. Deficiency of the 50K dystrophinassociated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature 1992; 359: 320-22. 5. Nonaka I, Chou SM. Congenital muscular dystrophy. In: Vinken PJ, Bruyn GW, eds. Handbook of clinical neurology vol 41: Amsterdam: North-Holland, 1979: 27-50. 6. Arahata K, Hoffman EP, Kunkel LM, Ishiura S, Tsukahara T, Ishihara T, Sunohara N, Nonaka I, Ozawa E, Sugita H. Dystrophin diagnosis: comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses. Proc Natl Acad Sci USA 1989; 86: 7154-58. 7. Arikawa E, Ishihara T, Nonaka I, Sugita H, Arahata K. Immunocytochemical analysis of dystrophin in congenital muscular dystrophy. J Neurol Sci 1991; 105: 79-87. 8. Beggs AH, Neumann PE, Arahata K, Arikawa E, Nonaka I, Anderson MS, Kunkel LM. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy. Proc Natl Acad Sci USA 1992; 89: 623-27. ADDRESSES: Howard Hughes Medical Institute and Department of Physiology and Biophysics, Univeristy of Iowa College of Medicine, Iowa City, IA 52242, USA (K. Matsumura, MD, Prof K. P Campbell, PhD); and National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Tokyo, Japan (I. Nonaka, MD). Correspondence to Prof Kevin P. Campbell.
Association of human papillomavirus with nasal
neoplasia
Since HPV-57b has been identified by two different techniques in benign, premalignant, and malignant lesions of the nasal cavity, but not in cases of chronic sinusitis, H PV-57 should be recognised as at least a co-factor in the aetiology of nasal neoplasia. Paraffin sections of 22 histologically confirmed nasal tumours were screened by in-situ hybridisation with riboprobes specific for HPV-57b. Virus was demonstrated in 6 of 7 fungiform papillomas, 6 of 8 inverted papillomas, 1 of 3 inverted papillomas with dysplasia, and 2 of 4 inverted papillomas with carcinoma. The presence of HPV-57b was confirmed with the polymerase chain reaction, which identified an additional 4 positive samples, bringing the total to 86% positive specimens. The results underscore the importance of HPVs in the aetiology of cancers at extragenital sites.