14. Domin BA, Serabjit-Singh CJ, Philpot RM. Quantitation of rabbit cytochrome p-450, form 2, in microsomal preparations
bound directly to nitrocellulose paper using a modified peroxidase-immunostainingtechnique. Anal Biochem 1984;136:390396 15. Turnbull DM. Banlett K, Watmough NJ, et al. Defects of fatty acid oxidation in skeletal muscle. J Inherited Metab Dis 1987; lO(supp1 1):105-112 16. Watmough NJ, Bindoff LA, Birch-Machin MA, et al. Impaired mitochondrial P-oxidation in a patient with an abnormahty of the respiratory chain. Studies in skeletal muscle mitochondria.J Clin Invest 1990;85:177-184
Normal DvstroDhin in McLeod MyGpathy Adrian Danek, MD,” Thomas N . Witt, MD,* H. B. A. C. Stockmann, BSc,t Barbara J. Weiss, BA,t Donald L. Schotland, MD,t and Kenneth H. Fischbeck, M D t
Dystrophin and its gene were studied in a patient with McLeod syndrome. This X-linked recessive myopathy has been localized to Xp21, as has the Duchenne muscular dystrophy gene locus, which codes for dystrophin. Histopathological study of the patient’s muscle showed mild subclinical myopathy. Immunological studies of dystrophin in two separate biopsy specimens and analysis of dystrophin gene DNA from a blood sample did not detect an abnormality. This suggests that the Duchenne muscular dystrophy gene, albeit close to the McLeod Locus, is not involved in McLeod myopathy. Danek A, Witt TN, Stockmann HBAC, Weiss BJ, Schotland DL, Fischbeck KH. Normal dystrophin in McLeod myopathy. Ann Neurol 1990;28:720-722
McLeod syndrome is an uncommon, X-linked multisystem disorder [I]. It was discovered in the blood donor Hugh McLeod as a rare phenotype of the Kell erythrocyte antigen system 121. Manifestations include elevation of serum levels of the muscle isoform of creatine kinase (CK) due to an usually subclinical, nonspecific myopathy (1, 31. The McJiod erythrocyte phenotype was also found in some patients with Duchenne muscular dystrophy (DMD). From the “Neurologische Klinik, Unikum Grosshadern, LudwigMaximilians-Universitat, Munich, Federal Republic of Germany, and the ?Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA. Received Dec 13, 1989, and in revised for1 Apr 27, 1990, and bfay 14. Accepted for publication May 14, 19cJ. Address correspondence to Dr Danek, Neurologische Klinik, Postfach 701260, D 8000 Munich 70, Federal Republic of Germany.
Genetic analyses localized the McLeod gene to Xp21 on the short arm of the X chromosome, in the same region as the gene for D M D (4-8). This chromosomal proximity suggested a close etiologic relationship between the myopathies of McLeod syndrome and of D M D [9]. The designation as an “Xp21 myopathy” has been extended to the McLeod syndrome, implying an abnormality of the dystrophin gene in t h s disorder [lo]. To answer the question of whether the D M D gene may be involved in the myopathy of McLeod syndrome, we studied muscle and blood from a patient who had McLeod syndrome with subclinical myopathy .
Patient and Methods An economist presented with atrial fibrillation at the age of 40 years and was found to suffer from dilated cardiomyopathy. He was followed for 11 years. Serum levels of the muscle isoform of CK fluctuated between 101 unitsiliter and 1,232 unitdbter (normal, < 80 unitsiliter), which led to neuromuscular evaluation. On examination, there was no muscle weakness or wasting, and electromyographic findings were normal. McLeod syndrome was diagnosed on the basis of Kell serology with weak erythrocyte reactions against K2, K4, K5, K7, K9, K l l , and K17 antigens (Transfusionzentrum Klinikum Grosshadern and MRC Blood Group Unit London). Muscle specimens were taken by open biopsy at the ages of 41 and 45 years from the left and right biceps muscles, respectively. The specimens were immediately deep frozen in liquid nitrogen and stored at - 80°C. Cryostat sections 8 pm thick were stained by standard techniques. Tissue from both biopsies was analyzed with antibodies against dystrophin. Western blots were done by the technique described elsewhere C11, 12), using affinity-purified sheep and rabbit antibodies raised against 30-kDa and 60kDa dystrophin fusion proteins, as provided by Dr E. Hoffman { 13). On one blot done with anti-60-kDa antibody the signals for dystrophin and a cross-reacting protein migrating near myosin were quantified with a soft laser scanning densitometer (Biomed Instruments, Jnc, Fullerton, CA). Immunocytochemical srudies on muscle samples from the patient with McLeod syndrome were carried out with affinity-purified sheep polyclonal antibodies raised against a fusion protein derived from complementary DNA (cDNA) 10 near the 3’ end of the open reading frame and with the sheep anti-60-kDa antibody utilized for Western blot (Dr E. Hoffman). Cryostat sections 6 p m thick were preincubated with 10% normal rabbit serum diluted in phosphatebuffered saline (PBS) solution and then incubated with antidystrophin antibodies diluted 1:250 in 10% normal rabbit serum for 2 hours. After washing in PBS solution, the sections were incubated for 1 hour with rhodamine-conjugated rabbit anti-sheep antibodies diluted 1: 50. They were viewed in a Zeiss fluorescence microscope after further washing in PBS solution. DNA was isolated from a blood sample for analysis by Southern blot. The blood cells were lysed, digested with rihonuclease and proteinase, and extracted with phenol and chloroform; then the D N A was precipitated with ethanol.
720 Copyright 0 1990 by the American Neurological Association
Results of Immunoblot Quantitation Area under Curve (Arbirrary Units) Sample
Dystrophin
Myosin
Dy strophidMyosin Ratio
Human control (lane A) McLeod sample (lane B)
2.8
12.8
0.22
3.2
12.6
0.25
Fig 1. Western blot of muscle stained with anti-dystrophin antibody (sheep anti-60 kDaj. Lane A is control human muscle, lanes B and C are two separate biopsy sgecimem from the patient with McLeod syndrome, lane D is normal rat muscle, and lane E is musclefrom a patient with Duchenne muscular dystrophy. Quantitation was done on lanes A and B, comparing the dystrophin signal with the signal for a cro.rs-reacting protein migrating near myosin.
Ten-microgram D N A samples were digested with Hind111 and separated by agarose gel electrophoresis, denatured, transferred to nylon membranes, and hybridized with radioactivity labeled cDNA probes for the 5’ end of the dystrophin gene. The probes used were 9-7 (1-2a) (Dr 1., Kunkel) and JK1.4 (Dr R. Worton).
Results When analyzed by Western blot with sheep antibody directed against the 60-kDa fusion protein [13}, the samples from both muscle biopsy specimens of the patient with McLeod syndrome were normal (Fig 1). Also, the use of different affinity-purified polyclonal anti-dystrophin antibodies-rabbit antibody directed against the 30-kDa fusion protein [13]-resulted in normal signal with normal band position and normal signal intensity. There were no abnormal signals. Immunoblot quantitation showed no difference between the McLeod sample and control human muscle (Table). lmmunocytochemical studies disclosed a uniformly intense fluorescent labeling of the sarcolemma of all McLeod muscle fibers with both anti-dystrophin antisections with nonimmune bodies (Fig 2A)’ serum showed virtually no staining. histolo@cal demonstrated an appearante compatible with a mild mYoPathY in both muscle biopsy specimens. It showed scattered necrotic fibers, occasionally in small clusters (Fig 2B), some atrophic
A
__
B
Fig 2. Histologicalfindings in mujcle biopsy Jpecirnens from the patient with McLeod syndrome. (A)Immunocytochemicallocalization ddystrophin: Sarcolemma in all musclefibers is unifomzly labeled (antibody directed against fusion protein deerived from cDNA 10 near the 3’ end of the open readingframe). (B) Hematoxylin and eosin stain shows small cluster of necrotic Jibers. {Scale bars = 50 ~m../
Brief Communication: Danek et al: Dystrophin in McLeod Myopathy 721
basophilic fibers, and an increased number of central nuclei. With DNA analysis, we found normal hybridization patterns on Southern blot with the two cDNA probes for the 5‘ end of the dystrophin gene, indicating that no deletion or other major rearrangement had occurred in the first 1 1 exons of the gene. Discussion The results show that myopathic muscle from a patient with McLeod syndrome contains immunologically intact aod normally distributed dystrophin. Its gene was intact in the first 11 exons. It is possible that Western analysis was not sensitive enough to identify a mild abnormality. In DMD carriers, for example, abnormalities of dystrophin sufficient to increase serum CK levels and cause mild histopathologic changes in muscle may go undetected by Western blot [8]. On the other hand, some patients with minimal symptoms due to dystrophin gene defects may have clear abnormalities on Western blot r121. The genetic data favor our conclusion that the dystrophin gene is not usually involved in McLeod syndrome. Large Xp21 deletions can cause both McLeod syndrome and DMD [4}. However, smaller deletions causing McLeod syndrome do not overlap with deletions causing DMD: Therefore, within Xp2 1 the McLeod locus must be physically separated from the DMD locus [5, 77. In keeping with this, no major abnormality at this locus could be detected in our patient. In summary, the myopathic features of McLeod syndrome are not likely to be caused by a defective dystrophin gene. Therefore, the hypothesis that “any myopathy due to mutation at Xp21 is a variant of D M D seems unwarranted, as seems the assumption that McLeod syndrome represents “minimal expression of the Duchenne gene” [lo}. Our findings support the alternative concept of a physically close, yet distinct genetic locus at Xp21 that can also cause myopathic changes, i.e., the McLeod myopathy. In this syndrome, the abnormalities of erythrocytes are thought due to their lack of Kx antigen, marker of a 37-kDa membrane protein [14}. Study of this Kx protein in normal and McLeod muscle may lead to a better understanding of an intriguing multisystem disorder, and perhaps also of normal muscle structure and function.
This work was supported in part by grants from the Muscular Dystrophy Association, the March of Dimes Birth Defects Foundation, and the National Institutes of Health grant NS 08075. We gratefully acknowledge the technical support of Mrs Franziska Anneser (histology), Ms Marion Oronzi Scott (protein analysis), and Mr William Fieles (immunohistochemistry). Kell serology data were provided by Dr M. U. Heim, Munich, and Drs P. Tippett and G. L. Daniels, London.
References 1. Marsh WL, Redman MC. Recent developments in the Kell blood group system. Trans Med Rev 1987;1:4-20 2. Allen FH, Krabbe SMR, Corcoran PA. A new phenotype (McLeod) in the Kell blood-group system. Vox Sang 1961;6: 555-560 3. Marsh WL,Marsh N J, Moore A, et al. Elevated serum creatine phosphokinase in subjects with McLeod syndrome. Vox Sang 1981;40:403-411 4. Francke U, Ochs HD, de Martinville B, et al. Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet 1985;37:250-267 5 . Bertelson C J, Pogo AO, Chaudhuri A, et al. Localization of the McLeod locus (Xk) within Xp21 by deletion analysis. Am J Hum Genet 1988;42:703-711 6. DeSaint-Basile G, Bohler MC, Fisher A, et al. Xp2l DNA microdeletion in a patient with chronic granulomatous disease, retinitis pigmentosa, and McLeod phenotype. Hum Genet 1988;80:85-89 7. Frey D, Machler M, Seger R, et al. Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: fine mapping of the Xk gene locus. Blood 1988;71:252-255 8. Gutmann DH, Fischbeck KH. Molecular biology of Duchenne and Becker’s muscular dystrophy: clinical applications. Ann Neurol 1989;26: 189-194 9. Swash M, Schwartz MS, Carter ND, et al. Benign X-linked myopathy with acanthocytes (McLeod syndrome), its relationship to X-linked muscular dystrophy. Brain 1983;106:717-733 10. Rowland LP. Clinical concepts of Duchenne muscular dystrophy. The impact of molecular genetics. Brain 1988;111:479-
495 11. Hoffman EP, Fischbeck KH, Brown RH, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N End J Med 1988;318:1363-1368 12. Gospe SM, Lazar0 RP,Lava NS, et al. Familial X-linked myalgia and cramps: a nonprogressive myopathy associated with a deletion in the dystrophin gene. Neurology 1989;39:1277-1280 13. Hoffman EP, Brown RH, Kunkel LM.Dystrophin: the protein product of the Duchcnne muscular dystrophy locus. Cell 1987; 5 1:9 19-928 14. Redman CM, Marsh WL, Scarborough A, et al. Biochemical studies on McLeod phenotype red cells and isolation of Kx antigen. Br J Haematol 1988;68:131-136
722 Annals of Neurology Vol 28 No 5 N o v e m b e r 1990
bound directly to nitrocellulose paper using a modified peroxidase-immunostainingtechnique. Anal Biochem 1984;136:390396 15. Turnbull DM. Banlett K, Watmough NJ, et al. Defects of fatty acid oxidation in skeletal muscle. J Inherited Metab Dis 1987; lO(supp1 1):105-112 16. Watmough NJ, Bindoff LA, Birch-Machin MA, et al. Impaired mitochondrial P-oxidation in a patient with an abnormahty of the respiratory chain. Studies in skeletal muscle mitochondria.J Clin Invest 1990;85:177-184
Normal DvstroDhin in McLeod MyGpathy Adrian Danek, MD,” Thomas N . Witt, MD,* H. B. A. C. Stockmann, BSc,t Barbara J. Weiss, BA,t Donald L. Schotland, MD,t and Kenneth H. Fischbeck, M D t
Dystrophin and its gene were studied in a patient with McLeod syndrome. This X-linked recessive myopathy has been localized to Xp21, as has the Duchenne muscular dystrophy gene locus, which codes for dystrophin. Histopathological study of the patient’s muscle showed mild subclinical myopathy. Immunological studies of dystrophin in two separate biopsy specimens and analysis of dystrophin gene DNA from a blood sample did not detect an abnormality. This suggests that the Duchenne muscular dystrophy gene, albeit close to the McLeod Locus, is not involved in McLeod myopathy. Danek A, Witt TN, Stockmann HBAC, Weiss BJ, Schotland DL, Fischbeck KH. Normal dystrophin in McLeod myopathy. Ann Neurol 1990;28:720-722
McLeod syndrome is an uncommon, X-linked multisystem disorder [I]. It was discovered in the blood donor Hugh McLeod as a rare phenotype of the Kell erythrocyte antigen system 121. Manifestations include elevation of serum levels of the muscle isoform of creatine kinase (CK) due to an usually subclinical, nonspecific myopathy (1, 31. The McJiod erythrocyte phenotype was also found in some patients with Duchenne muscular dystrophy (DMD). From the “Neurologische Klinik, Unikum Grosshadern, LudwigMaximilians-Universitat, Munich, Federal Republic of Germany, and the ?Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA. Received Dec 13, 1989, and in revised for1 Apr 27, 1990, and bfay 14. Accepted for publication May 14, 19cJ. Address correspondence to Dr Danek, Neurologische Klinik, Postfach 701260, D 8000 Munich 70, Federal Republic of Germany.
Genetic analyses localized the McLeod gene to Xp21 on the short arm of the X chromosome, in the same region as the gene for D M D (4-8). This chromosomal proximity suggested a close etiologic relationship between the myopathies of McLeod syndrome and of D M D [9]. The designation as an “Xp21 myopathy” has been extended to the McLeod syndrome, implying an abnormality of the dystrophin gene in t h s disorder [lo]. To answer the question of whether the D M D gene may be involved in the myopathy of McLeod syndrome, we studied muscle and blood from a patient who had McLeod syndrome with subclinical myopathy .
Patient and Methods An economist presented with atrial fibrillation at the age of 40 years and was found to suffer from dilated cardiomyopathy. He was followed for 11 years. Serum levels of the muscle isoform of CK fluctuated between 101 unitsiliter and 1,232 unitdbter (normal, < 80 unitsiliter), which led to neuromuscular evaluation. On examination, there was no muscle weakness or wasting, and electromyographic findings were normal. McLeod syndrome was diagnosed on the basis of Kell serology with weak erythrocyte reactions against K2, K4, K5, K7, K9, K l l , and K17 antigens (Transfusionzentrum Klinikum Grosshadern and MRC Blood Group Unit London). Muscle specimens were taken by open biopsy at the ages of 41 and 45 years from the left and right biceps muscles, respectively. The specimens were immediately deep frozen in liquid nitrogen and stored at - 80°C. Cryostat sections 8 pm thick were stained by standard techniques. Tissue from both biopsies was analyzed with antibodies against dystrophin. Western blots were done by the technique described elsewhere C11, 12), using affinity-purified sheep and rabbit antibodies raised against 30-kDa and 60kDa dystrophin fusion proteins, as provided by Dr E. Hoffman { 13). On one blot done with anti-60-kDa antibody the signals for dystrophin and a cross-reacting protein migrating near myosin were quantified with a soft laser scanning densitometer (Biomed Instruments, Jnc, Fullerton, CA). Immunocytochemical srudies on muscle samples from the patient with McLeod syndrome were carried out with affinity-purified sheep polyclonal antibodies raised against a fusion protein derived from complementary DNA (cDNA) 10 near the 3’ end of the open reading frame and with the sheep anti-60-kDa antibody utilized for Western blot (Dr E. Hoffman). Cryostat sections 6 p m thick were preincubated with 10% normal rabbit serum diluted in phosphatebuffered saline (PBS) solution and then incubated with antidystrophin antibodies diluted 1:250 in 10% normal rabbit serum for 2 hours. After washing in PBS solution, the sections were incubated for 1 hour with rhodamine-conjugated rabbit anti-sheep antibodies diluted 1: 50. They were viewed in a Zeiss fluorescence microscope after further washing in PBS solution. DNA was isolated from a blood sample for analysis by Southern blot. The blood cells were lysed, digested with rihonuclease and proteinase, and extracted with phenol and chloroform; then the D N A was precipitated with ethanol.
720 Copyright 0 1990 by the American Neurological Association
Results of Immunoblot Quantitation Area under Curve (Arbirrary Units) Sample
Dystrophin
Myosin
Dy strophidMyosin Ratio
Human control (lane A) McLeod sample (lane B)
2.8
12.8
0.22
3.2
12.6
0.25
Fig 1. Western blot of muscle stained with anti-dystrophin antibody (sheep anti-60 kDaj. Lane A is control human muscle, lanes B and C are two separate biopsy sgecimem from the patient with McLeod syndrome, lane D is normal rat muscle, and lane E is musclefrom a patient with Duchenne muscular dystrophy. Quantitation was done on lanes A and B, comparing the dystrophin signal with the signal for a cro.rs-reacting protein migrating near myosin.
Ten-microgram D N A samples were digested with Hind111 and separated by agarose gel electrophoresis, denatured, transferred to nylon membranes, and hybridized with radioactivity labeled cDNA probes for the 5’ end of the dystrophin gene. The probes used were 9-7 (1-2a) (Dr 1., Kunkel) and JK1.4 (Dr R. Worton).
Results When analyzed by Western blot with sheep antibody directed against the 60-kDa fusion protein [13}, the samples from both muscle biopsy specimens of the patient with McLeod syndrome were normal (Fig 1). Also, the use of different affinity-purified polyclonal anti-dystrophin antibodies-rabbit antibody directed against the 30-kDa fusion protein [13]-resulted in normal signal with normal band position and normal signal intensity. There were no abnormal signals. Immunoblot quantitation showed no difference between the McLeod sample and control human muscle (Table). lmmunocytochemical studies disclosed a uniformly intense fluorescent labeling of the sarcolemma of all McLeod muscle fibers with both anti-dystrophin antisections with nonimmune bodies (Fig 2A)’ serum showed virtually no staining. histolo@cal demonstrated an appearante compatible with a mild mYoPathY in both muscle biopsy specimens. It showed scattered necrotic fibers, occasionally in small clusters (Fig 2B), some atrophic
A
__
B
Fig 2. Histologicalfindings in mujcle biopsy Jpecirnens from the patient with McLeod syndrome. (A)Immunocytochemicallocalization ddystrophin: Sarcolemma in all musclefibers is unifomzly labeled (antibody directed against fusion protein deerived from cDNA 10 near the 3’ end of the open readingframe). (B) Hematoxylin and eosin stain shows small cluster of necrotic Jibers. {Scale bars = 50 ~m../
Brief Communication: Danek et al: Dystrophin in McLeod Myopathy 721
basophilic fibers, and an increased number of central nuclei. With DNA analysis, we found normal hybridization patterns on Southern blot with the two cDNA probes for the 5‘ end of the dystrophin gene, indicating that no deletion or other major rearrangement had occurred in the first 1 1 exons of the gene. Discussion The results show that myopathic muscle from a patient with McLeod syndrome contains immunologically intact aod normally distributed dystrophin. Its gene was intact in the first 11 exons. It is possible that Western analysis was not sensitive enough to identify a mild abnormality. In DMD carriers, for example, abnormalities of dystrophin sufficient to increase serum CK levels and cause mild histopathologic changes in muscle may go undetected by Western blot [8]. On the other hand, some patients with minimal symptoms due to dystrophin gene defects may have clear abnormalities on Western blot r121. The genetic data favor our conclusion that the dystrophin gene is not usually involved in McLeod syndrome. Large Xp21 deletions can cause both McLeod syndrome and DMD [4}. However, smaller deletions causing McLeod syndrome do not overlap with deletions causing DMD: Therefore, within Xp2 1 the McLeod locus must be physically separated from the DMD locus [5, 77. In keeping with this, no major abnormality at this locus could be detected in our patient. In summary, the myopathic features of McLeod syndrome are not likely to be caused by a defective dystrophin gene. Therefore, the hypothesis that “any myopathy due to mutation at Xp21 is a variant of D M D seems unwarranted, as seems the assumption that McLeod syndrome represents “minimal expression of the Duchenne gene” [lo}. Our findings support the alternative concept of a physically close, yet distinct genetic locus at Xp21 that can also cause myopathic changes, i.e., the McLeod myopathy. In this syndrome, the abnormalities of erythrocytes are thought due to their lack of Kx antigen, marker of a 37-kDa membrane protein [14}. Study of this Kx protein in normal and McLeod muscle may lead to a better understanding of an intriguing multisystem disorder, and perhaps also of normal muscle structure and function.
This work was supported in part by grants from the Muscular Dystrophy Association, the March of Dimes Birth Defects Foundation, and the National Institutes of Health grant NS 08075. We gratefully acknowledge the technical support of Mrs Franziska Anneser (histology), Ms Marion Oronzi Scott (protein analysis), and Mr William Fieles (immunohistochemistry). Kell serology data were provided by Dr M. U. Heim, Munich, and Drs P. Tippett and G. L. Daniels, London.
References 1. Marsh WL, Redman MC. Recent developments in the Kell blood group system. Trans Med Rev 1987;1:4-20 2. Allen FH, Krabbe SMR, Corcoran PA. A new phenotype (McLeod) in the Kell blood-group system. Vox Sang 1961;6: 555-560 3. Marsh WL,Marsh N J, Moore A, et al. Elevated serum creatine phosphokinase in subjects with McLeod syndrome. Vox Sang 1981;40:403-411 4. Francke U, Ochs HD, de Martinville B, et al. Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet 1985;37:250-267 5 . Bertelson C J, Pogo AO, Chaudhuri A, et al. Localization of the McLeod locus (Xk) within Xp21 by deletion analysis. Am J Hum Genet 1988;42:703-711 6. DeSaint-Basile G, Bohler MC, Fisher A, et al. Xp2l DNA microdeletion in a patient with chronic granulomatous disease, retinitis pigmentosa, and McLeod phenotype. Hum Genet 1988;80:85-89 7. Frey D, Machler M, Seger R, et al. Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: fine mapping of the Xk gene locus. Blood 1988;71:252-255 8. Gutmann DH, Fischbeck KH. Molecular biology of Duchenne and Becker’s muscular dystrophy: clinical applications. Ann Neurol 1989;26: 189-194 9. Swash M, Schwartz MS, Carter ND, et al. Benign X-linked myopathy with acanthocytes (McLeod syndrome), its relationship to X-linked muscular dystrophy. Brain 1983;106:717-733 10. Rowland LP. Clinical concepts of Duchenne muscular dystrophy. The impact of molecular genetics. Brain 1988;111:479-
495 11. Hoffman EP, Fischbeck KH, Brown RH, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N End J Med 1988;318:1363-1368 12. Gospe SM, Lazar0 RP,Lava NS, et al. Familial X-linked myalgia and cramps: a nonprogressive myopathy associated with a deletion in the dystrophin gene. Neurology 1989;39:1277-1280 13. Hoffman EP, Brown RH, Kunkel LM.Dystrophin: the protein product of the Duchcnne muscular dystrophy locus. Cell 1987; 5 1:9 19-928 14. Redman CM, Marsh WL, Scarborough A, et al. Biochemical studies on McLeod phenotype red cells and isolation of Kx antigen. Br J Haematol 1988;68:131-136
722 Annals of Neurology Vol 28 No 5 N o v e m b e r 1990
