Proc. Natl. Acad. Sci. USA Vol. 86, pp. 7154-7158, September 1989 Medical Sciences Dystrophin diagnosis: Comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses (Duchenne muscular dystrophy/Becker musl dystrophy) KIICHI ARAHATA*, ERIC P. HOFFMANt, Louis M. KUNKELt, SHOICHI ISHIURA*, ToSHIFUMI TSUKAHARA*, TADAYUKI ISHIHARA*, NOBUHIKO SUNOHARA*, IKUYA NONAKA*, EuIRo OZAWA*, AND HIDEO SUGITA* *National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187 Japan; and tHoward Hughes Medical Institute, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA 02115 Communicated by Alexander G. Bearn, May 30, 1989 ABSTRACT Immunoblot characterization and immuno- have been reported for 68 Becker patients, no attempts have fluorescence localization of dystrophin are presented for 76 been made to correlate specific dystrophin biochemical ab- human patients with various neuromuscular diseases. Normal normalities with cellular localization abnormalities in Becker dystrophin (shown by immunoblotting) was invariably visual- patients. An additional diagnostic use of dystrophin analysis ized as a continuous, peripheral membrane immunining of has been the delineation of female carriers (heterozygotes) myofibers. Biochemical abnormalities of dystrophin (either for Duchenne dystrophy (12, 13). lower or higher molecular weight dystrophin) resulted in Given the rapidly evolving importance of dystrophin test- patchy, discontinuous immunostaining, suggesting that the ing in the clinical diagnosis of neuromuscular disease, it is abnormal dystrophin proteins are not capable of creating a necessary to evaluate the relative accuracies of immunoblot complete membrane cytoskeleton network. There was a very and immunofluorescence studies of dystrophin and to iden- strong correlation of clinical diagnoses with the type of dys- tify situations in which one type of assay system shows trophin abnormality; all Duchenne muscular dystrophy patient marked advantages over the other type. Finally, it is ofbasic muscle contained no detectable dystrophin, Becker muscular biological interest to determine whether specific types of dystrophy patient muscle had clearly abnormal dystrophin, dystrophin abnormalities correlate with specific immunoflu- and unrelated diseases showed normal dystrophin. However, a orescent staining patterns. To address these questions we single patient of five carrying the diagnosis of Fukuyama have studied dystrophin by both immunoblot and immuno- dystrophy showed no detectable dystrophin and thus appeared fluorescence techniques in 76 patient muscle biopsy speci- to be a Duchenne dystrophy patient by the biochemical assays. mens. We know of no other case of a patient with a disease thought to be unrelated to Duchenne/Becker dystrophy yet demon- MATERIALS AND METHODS strating dystrophin deficiency. Based on the data presented, we Patient Muscle Biopsies. All patient muscle samples were conclude that immunofluorescence is the best technique for the obtained as portions of the diagnostic biopsy specimens. The detection of female carriers of Duchenne dystrophy, whereas same muscle biopsy sample was used for both immunofluo- immunoblotting appears superior for the prognostic diagnosis rescence analysis and immunoblot analysis, and both analy- of Becker muscular dystrophy. ses were performed for each patient. Of the 76 samples studied, all were new, previously unpublished samples, ex- The underlying biochemical defects responsible for Duch- cept for those from 6 Becker dystrophy patients, which were enne and Becker muscular dystrophies are abnormalities of included in a previous report (7). All clinical diagnoses were dystrophin, the protein product of the Duchenne muscular assigned in the National Institutes of Neuroscience, Japan, dystrophy gene (1-3). Dystrophin is thought to be a part ofthe using standard criteria. Five of the 20 patients that were membrane cytoskeleton in all myogenic cells (4-6). In cry- assigned a clinical diagnosis of Becker muscular dystrophy ostat cross sections of both normal muscle and muscle from were under 5 years of age. Such young patients cannot be patients with disorders unrelated to Duchenne/Becker dys- accurately assigned a definitive Becker dystrophy diagnosis trophy, dystrophin is visualized by immunofluorescence as a on purely clinical grounds unless a previous X chromosome- continuous, thin ring of staining at the periphery of every linked history indicative of Becker dystrophy is available. Of muscle fiber (7-9). By immunoblotting, dystrophin is de- these 5 young Becker patients, 2 had such a family history. tected as a large (--400-kDa), low-abundance (<0.01% of The remaining 3 young patients were given a Becker dystro- total muscle protein) protein in normal muscle and in muscle phy diagnosis based primarily on the dystrophin immunoblot from patients with unrelated disorders (1, 10). Both tech- analyses, which were done "blind" and have been shown to niques have shown the specific absence of dystrophin in be very accurate in detecting Becker dystrophy patients at muscle from all patients with dystrophin dystrophy. The young ages (11). All 5 cases were also confirmed as Becker majority of patients with the clinically milder Becker dystro- dystrophy by immunofluorescence analysis. Eleven ofthe 20 phy have been shown to have dystrophin of abnormal mo- Becker patients studied had an X-linked family history. lecular weight (quality) and/or lower relative cellular abun- Antisera. Each biopsy sample was tested with four differ- dance (quantity) compared to normal muscle (10, 11). Im- ent anti-dystrophin polyclonal antisera, two of which were munofluorescence studies of dystrophin localization in raised in rabbits against synthetic peptides (immunofluores- Becker dystrophy patients have found a disease-specific .cence) and two of which were raised in sheep against fusion "patchy" immunofluorescent pattern (7, 8). Though dystro- proteins (immunoblotting). For immunoblotting, affinity- phin biochemical and immunofluorescence abnormalities purified sheep anti-30-kDa and anti-60-kDa mouse cardiac dystrophin antisera were used simultaneously (1). The mouse The publication costs of this article were defrayed in part by page charge peptides correspond to amino acids 407-815 (60 kDa) and payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CK, creatine kinase. 7154 Downloaded by guest on October 1, 2021 Medical Sciences: Arahata et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7155 1181-1388 (30 kDa) of the human dystrophin sequence (5). The protocols used for 60-kDa and 30-kDa antigen production and purification, antisera production in sheep, and affinity purification of anti-dystrophin antibodies have been de- scribed (1). For immunofluorescence, two rabbit polyclonal anti- human dystrophin antisera were used individually. One of these antisera (anti-DMDP) was raised against a 50-amino acid peptide corresponding to amino acids 440-489 of the human fetal skeletal muscle dystrophin sequence and has been described in detail (4). Here we refer to this antiserum as anti-DMDP II, due to its location towards the carboxyl B terminus relative to the peptide described below. The peptide sequence to which anti-DMDP II is directed is contained A C *a within the 60-kDa dystrophin antigen described above. The second antiserum used for immunofluorescence has FIG. 2. Dystrophin in an asymptomatic, obligate carrier for Duchenne muscular dystrophy. Dystrophin immunofluorescence not been described previously and was raised against a with anti-DMDP 11 (A) shows that both dystrophin-positive and synthetic peptide representing residues 215-264 ofthe human dystrophin-negative fibers are present. Immunoblot detection of amino acid sequence (5): PEDVDTTYPDKKSILMYITS- dystrophin in the same biopsy sample (B) shows no difference from LFQTLPQQVSIEAIQEVEMLPRPPKVTKEE. Rabbits the adjacent controls, after correction for the muscle protein content were immunized with this peptide and immune serum was of the samples (C). (Bar in A = 25 ,um.) produced as described (4). This antiserum is referred to as anti-DMDP I. itive fibers adjacent to completely negative fibers (Fig. 2). Immunofluorescence. Muscle biopsy specimens were pro- Each of these carriers showed apparently normal dystrophin cessed for cryosectioning as described (14). Each coverslip by immunoblot, with no detectable reduction in dystrophin processed for immunofluorescence contained three to seven quantity. experimental samples along with three controls: a normal Becker Muscular Dystrophy Patients. Twenty patients, all muscle biopsy sample, a sample from a known Duchenne male, who had a clinical phenotype consistent with Becker dystrophy patient, and a sample from a known Becker muscular dystrophy were tested. Dystrophin immunofluo- dystrophy patient. Each sample was tested with anti-DMDP rescence invariably consisted of a patchy or discontinuous I and anti-DMDP II (both diluted 1:300) on separate cover- staining pattern around most fibers. The staining intensities slips. in general were considerably fainter than those seen in Immunoblotting. Immunoblot detection of dystrophin was normal muscle biopsy specimens. The dystrophin staining as described (11). was quite variable between individual fibers within the same sample, with some completely negative fibers in all of the RESULTS Becker samples tested. A