Dystrophm Analysis in Duchenne and Becker Muscular Dystrophy Carriers: Correlation with Intracellular Calcium and Albumin L. Morandi, MD,’ M. Mora, PhD,” E. Gussoni, PhD,’ S. Tedeschi, PhD,? and F. Cornelio, MD* ~
~~
Immunocytochemical localization and immunoblot analysis of dystrophin in muscle fibers of 11 obligate and probable, and 7 possible carriers of Duchenne and Becker muscular dystrophy revealed an abnormal expression of the protein in 3 of them. Localization of calcium and albumin, as endogenous markers of extracellular fluid penetration, showed the presence of both molecules inside some fibers lacking dystrophin. Our morphological studies show that the initial stages leading to fiber necrosis in Duchenne muscular dystrophy are present in carriers with mosaicism. Comparison of dystrophin studies with restriction fragment length polymorphism analysis and creatine kinase levels showed that neither immunocytochemical nor immunoblot techniques for dystrophin are sensitive enough to provide a basis for genetic counseling. Morandi L, Mora M, Gussoni E, Tedeschi S, Cornelio F. Dystrophin analysis in Duchenne and Becker muscular dystrophy carriers: correlation with intracellular calcium and albumin. Ann Neurol 1990;28:674-679
Dystrophin, a large (427 kd) protein product of the Duchenne muscular dystrophy (DMD) locus, is found localized on the cytoplasmic side of the muscle fiber membrane El-41. It is absent from the muscles of D M D patients and is reduced or has a different molecular weight in muscles of Becker muscular dystrophy (BMD) patients IS}. Immunocytochemical studies with anti-dystrophin antibodies have revealed the presence of two populations of muscle fibers in D M D carriers {G, 71, one with normally expressed dystrophin, the other with the protein missing, the percentage of dystrophin-negative fibers being higher in manifesting carriers. In the present study, we evaluated dystrophin expression in the muscle cells of obligate, probable, and possible D M D and BMD female carriers, both by irnmunocytochemical and immunoblot techniques. We have also localized calcium and albumin as endogenous markers of extracellular fluid penetration to verify whether the pathogenetic events occurring in DMD muscles [S, 91 are also evident in the muscle of carriers and whether they are related to dystrophin absence. The muscle biopsy technique was also investigated for possible use in genetic counseling.
From the “Neuromuscular Diseases Department, Istituto Neurologic0 “C. Besta,” and the Clinical Research Laboratory, Istituti Clinici di Perfezionamento,Milano, Italy.
Materials and Methods Clinical Evaltlation We evaluated 15 D M D carriers ( 5 obligate, 3 probable, and 7 possible) and 3 obligate BMD carriers classified on the basis of pedigree and creatine kinase (CK) plasma levels [lo]. Obligate carriers were defined as mothers of an affected son who also had an affected brother or other male relative in the female line, and the daughters of B M D patients. Probable carriers were mothers of two or more affected sons; possible carriers were women with an affected male relative in the female line. C K plasma determination was done on 3 consecutive samples. If a carrier defined as possible by pedigree analysis had increased CK levels in all of 3 determinations, she was considered a probable carrier. One patient, a probable D M D carrier, showed clinical evidence of proximal muscle weakness of the lower limbs and hypertrophic gastrocnemii. Muscle biopsies were performed by the needle technique after informed consent.
Genetic Studies Genomic D N A extracted from peripheral blood lymphocytes by the phenol-chloroform method was used to study restriction fragment length polymorphisms (RFLPs). Restriction endonuclease digested D N A fragments were separated by electrophoresis, transferred to a nylon membrane, and
Address correspondence to Dr Morandi, Neuromuscular Diseases Department, Istituto Neurologico “C. Besta,” Via Celoria, 1120133 Milano, Italy.
Received Feb 9, 1990, and in revised form May 2 and Jun 7. Accepted for publication Jun 10, 1990.
674 Copyright 0 1990 by the American Neurological Association
probed with the intragenic P20, JBir, pERT87, XJ1.l, and the flanking C7 and 754 fragments [11-13].
Antisera Three bacterial strains (E.Coli RR1, kindly supplied by E. P. Hoffman and L. M. Kunkel, Children’s Hospital, Boston, MA) were cultured; they contained the expression vector pATH2 either alone or associated with one of two distinct DMD gene fragments (700 base pairs (bp) or 1,400 bp) from mouse heart, expressing a 30-kd and a 60-kd peptide, respectively. The bacteria were lysed, and the insoluble proteins were isolated by centrifugation and inoculated into rabbits for antisera production [I].
Immunoblotting Muscle biopsy samples, stored in liquid nitrogen until use, were pulverized, weighed, and dissolved in buffer (18% sodium dodecyl sulfate [SDS], 0.1 M Tris, p H 8.0, 5 mM ethylenediaminetetraacetic acid, 5 0 mM DL-dithiothreitol) to a concentration of 50 mgiml. Solutions were then boiled for 2 minutes, centrifuged to remove insoluble proteins, and loaded (850 p g of total protein per lane) onto SDS-polyacrylamide gradient gels (main gel gradient 12.5-3.5%; stacking gel 3%) { 1, 41. After electrophoresis, fractionated proteins were electroblotted onto nitrocellulose filters at 200 mA for 2 hours at room temperature. The filters were allowed to dry overnight. The blots on nitrocellulose paper were then incubated for 4 hours with anti-dystrophin antiserum diluted 1:400 in Tris-buffered saline (Tween 20 [TBST] [ l o mM Tris-HCI, pH 8.0, 150 mM sodium chloride, 0.05% Tween 201) and, subsequently, with the secondary antibody alkaline phosphatase-conjugated goat anti-rabbit IgG for 1 hour. Western blot development was performed by nitro blue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate.
Morphological Studies Muscle biopsies were frozen in 2-methylbutane (isopentane), cooled to - 183”C, and stored under liquid nitrogen. Consecutive 6- or 10-pm thick freshly prepared cryosections were collected on gelatin-coated slides. Intracellular calcium was localized histochemically by the alizarin red S method {8) on the 10-pm sections. For immunocytochemical analysis, the 6-pm sections were first incubated in avidin (Blocking Kit, Vector Labs, Burlingame, CA), diluted in phosphate-buffered solution (PBS) 1:20 for 15 minutes, washed a few times in PBS, incubated in biotin (Blocking Kit, Vector) 1:20 for 15 minutes, washed again, and then placed in 10% heat-inactivated normal goat s e w (NGS) for 30 minutes. After several washes, either anti-dystrophin antiserum (1 :600 dilution in PBS plus 10% NGS) or rabbit anti-human albumin antibody (Dakopatts, Copenhagen, Denmark) (2 CLgiml in PBS plus NGS) was applied for 120 minutes. Incubation in biotinylated goat antirabbit IgG (Vector) (1.5 pdml in PBS, 60 minutes) was followed by incubation (60 minutes) in either avidin peroxidase-conjugated (undiluted ABC Kit, Vector) or rhodamine-avidin D (Vector) (5 pdml in PBS). Between each incubation, sections were rinsed 10 times for 3 minutes each. After a final rinse, sections with avidin-peroxidase were de-
veloped with 3-3’-diaminobenzidine, and those with rhodamine were mounted in a glycerol medium containing paraphenylenediamine. All incubations were performed at room temperature in humid chambers. As control, on adjacent sections the primary antibody was omitted. Both dystrophin and albumin were localized simultaneously on a few sections. In this case, the primary incubation for 120 minutes was with a mixture of anti-dystrophin 1 :600 and anti-albumin 4 pdml (goat anti-human albumin) (Organon Teknika-Cappel, West Chester, PA) diluted in PBS plus 10% horse serum plus 2% bovine serum albumin. This was followed by incubation for 60 minutes with biotinylated donkey anti-rabbit IgG (Amersham, Amersham International Place, Amersham, UK) 1 pgiml in PBS, for a further 60 minutes, with fluorescein isothiocyanate (F1TC)-labeled streptavidin (Amersham) 1 pdrnl, and finally, by incubation in rhodamine-conjugated rabbit anti-goat IgG (Sera-Lab Ltd, Crawley-Down, UK) 2 p,g/ml for 60 minutes. Between each incubation, sections were rinsed in PBS 10 times for a total of 60 minutes. Because biopsies were performed with the needle technique, many fibers at the periphery of fiber fascicles could have been artifactually positive for calcium and albumin. We therefore counted only fibers found inside the fascicles. For each biopsy, a minimum of 500 fibers was counted. In a few patients (Patients 1-8, Table l), longitudinal sections were analyzed.
Results DNA RFLP analysis confirmed the heterozygotic condition in the obligate and probable DMD carriers, but only 1 of the possible DMD carriers was shown to be a true heterozygotic carrier (Patient 14, see Table 1). Immunocytochemical studies on transverse sections revealed a normal distribution of dystrophin on the surface of the muscle fibers in all definitely nonheterozygotic women. Of the heterozygotic DMD carriers, 3 (Patients 3,4, and 6, Table 1) had mosaic expression of dystrophin in their muscle, with some fibers totally or partially lacking the protein (4.5-18.7%) and others expressing it normally (Fig 1);the remaining heterozygotic carriers had normal distribution of dystrophin. The single clinically symptomatic DMD carrier (Patient 6) had mosaic expression of dystrophin. Hematoxylin-and-eosin-stained sections showed a greater variability of fiber size and some hypercontracted fibers in all patients with abnormal dystrophin expression and occasional necrotic fibers in 2 of them. Fiber size variability was observed in 6 other patients with normal dystrophin (2 obligate, 1 probable, and 1 possible DMD carrier, and 2 BMD carriers). Irnmunoblot substantially supported the immunocytochemical results; in those patients in whom dystrophin was missing from muscle fibers, qualitative inspection of the immunoblot showed lower amounts of the protein (Fig 2). CK levels (see Table 1) were raised in the 3 women
Morandi et al: Dystrophin, Calcium, and Albumin in Carriers’ Muscle 675
Table 1. Clinical Data and Dytrophin Expression in Muscles of Duchenne and Brcker Muscuhr Dystrophy Carriers" Patient DMD obligate carriers 1 P.M. 2 M.S. '3 C.T. 4 S.A. 5 C.L. DMD probable carriers 6 N.R. 7 S.A. 8 A.W. DMD possible carriers 9 S.L. 10 S.E. 11 S.M. 12 T.M. 13 G.M. 14 M.I. 15 C.L. BMD carriers 16 C.V. 17 C.G. 18 M.A.
Creatin e Kinase N N
t
t t
t t
t
N N N
N N N N
t
t T
Symptoms
Age (yr)
DNA~
Negative Negative Negative Negative Negative
31 27 31 28
100 100 100 100 100
Positive Negative Negative
42 50 29
100 100 100
Negative Negative Negative Negative Negative Negative Negative
22 20 25 34 42 28 23
35.7 35.7 64.2 49 55 79.8 0.6
Negative Negative Negative
16 47 20
100 99.1 99
44
Immunocytochemistry N
h '
Western Blot N N
Mosaic Mosaic
1
N
N
4
Mosaic
i
N N
N N
N
N
N
N
N N N
N N N
N
N
N
N
N
.1
N N
N
N
"Carriers were defined as obligate, probable, or possible from pedigree and creatine kinase levels {lo]. bPercentage of risk of being carrier from restriction fragment length polymorphism analyses combined with pedigree and creatine kinase levels
[161. DMD = Duchenne muscular dystrophy; N
=
normal; BMD
=
Becker muscular dystrophy.
Fig 1. Fluorescence-immunoj tained section showing severalfibers totally or partially lacking dystrophzn. (Original magnification x 250.1 Fzg 2, Reduction of dystrophin amount t n carriers with mosazcism (lane 2, patzent 3: lane 4, patient 6). Lanes I (normal control) and 3 (patient 9) show a normal amount of dystrophin, while in iane 5 (patient 16) the protein is slightly reduced. Results in lane G (patient 12) are considered normal even afthe staining appears reduced as the myosin band (205 K), indicative of reduced amounts of skeletal muscle proteins.
676 Annals of Neurology Vol 28 No 5 November 1990
Fig 3. Consecutive cryosections peroxidase-inimunos~ained for dystrophin (a) and albumin (b). Dystrophin is absentfrom albumin-positiziefibers. (Original magnification x 250.)
with mosaicism. Increased CK levels were also observed in 1 of the obligate and 2 of the probable D M D carriers, and in all the BMD carriers who had otherwise normal immunocytochemical localization of dystrophin. Calcium- and albumin-positive fibers were observed in nonnecrotic muscle fibers of the carriers with mosaicism. Calcium- and albumin-positive fibers were always devoid of dystrophin immunoreactivity on consecutive serial sections (Fig 3); in the same muscles, fibers lacking dystrophin were more numerous than calcium- and albumin-positive fibers (Fig 4). The proportion of calcium-positive fibers among dystrophin-negative fibers varied between 0.8% and 3.496; that of albuminpositive fibers varied between 2.19% and 5.5% (Table 2). In longitudinal sections, dystrophin was absent either along the total fiber surface or, more often, on segments thereof (Fig 5). Sometimes calcium and albumin penetrated only portions of the muscle fibers (Figs 6, 7). Simultaneous immunostaining for albumin and dystrophin on both transverse and longitudinal sections confirmed that on most of the surface of albuminpositive fibers, dystrophin was absent or reduced (Fig 8). ,
Discussion Our results confirm that the heterozygotic D M D gene carrier condition can be phenotypically expressed by absence of dystrophin on the sarcolemma of some muscle fibers. Most likely, this abnormal expression is associated with lyonization of the healthy X chromosome and subsequent activation of the affected one 141. Of the women characterized as heterozygotic carriers by RFLP analysis, only 3 had an abnormal im-
r
Morandi et
al:
munocytochemical distribution of dystrophin with corresponding reduced immunoreactivity on Western blot; these women with mosaicism also had increased CK levels. CK was above normal in 2 other obligate DMD carriers and in all the BMD carriers. Our study shows therefore that only in certain patients do immunoblot analysis and immunocytochemical analysis reveal abnormalities in carriers already defined as such by RFLP and CK evaluation. We conclude that neither technique is sensitive enough to provide a basis for genetic counseling. The presence of dystrophin mosaicism is not always related to clinical signs C71. We also found alterations in dystrophin distribution in nonsymptomatic carriers. Our morphological studies show that the initial stages leading to fiber necrosis in D M D are present in carriers with mosaicism; calcium and albumin were found inside some apparently nonnecrotic muscle fibers lacking dystrophin. Dystrophin-negative fibers are more numerous than fibers penetrated by albumin or calcium. It is possible that dystrophin-negative fibers that did not show pathological changes in the plane of the section could have been abnormal in other sites, although in longitudinal sections we observed large segments of fibers lacking dystrophin with no penetration of calcium and albumin. The absence of the protein over a large part of a fiber, however, may be necessary before membrane alterations become observable. It is also possible that dystrophin-negative fibers require time to develop observable pathology; in fact, there are many stages in the process leading to fiber necrosis, the first being the absence of the protein. We suspect that our antibodies against 60-kd and 30-kd nonoverlapping peptides, both of which occur on the N-terminal portion of the dystrophin molecule, may detect an alteration of the protein not essential for its function. The different domains of this large molecule are probably not equally important for the integrity of dystrophin function. This is supported by a recent report by England and colleagues [l5} Dystrophin, Calcium, and Albumin in Carriers’ Muscle 677
Fig 5 . Rhwlamine-immunostained longitudinal section. Dystrophin is absent or reduced (arrows) on segments offiber surface. (Originalmagn6cation x 100.)
F i g 6. Albumin immunojluorescencestaining on a longitudinal section showing the presence of albumin inside portions of muscle fibers (arrows). (Original magnification x 250.)
g 4. (a) Dystrophin, (b) albumin, and (c) calcium. Loww
tgnification of Figure 3, showing dystrophin-negativefibers TOWS)&void of albumin and calcium. Note that som rtrophin-negabivefibers are hypercontracted (asterisks). riginal magnification x 100.)
Table 2. Dystrophin, Calcium, and Albumin in Muscle Fibers
Patient
DystrophinNegative Fibers (%)
AlbuminPositive Fibers (%)
CalciumPositive Fibers (%)
3
4.5
4
18.2
2.1 5.5
0.8 2.6
6
18.7
4.5
3.4
678 Annals of Neurology
Vol 28 No 5
Fig 7. Alizarin red S-stained longitudinalsection. Calcium penetrated only a portion of a fiber. (Originalmagnification x 250.1
November 1990
References
Fig 8. Dystrophin (a)and albumin (bi simultaneozlsly localized on a longitudinal section. Dystrophin is partially missing (arrows) fmm the surface of the fiber containing albumin. (Original magnification X 250.1
demonstrating that, in a family with 46% deletion of dystrophin mainly in the central domain of the molecule, the dystrophic phenotype was very mild. Better knowledge of the precise role played by the different domains of the dystrophin molecule will be needed to clarify the relation between muscle membrane function and the absence, in whole or in part, of the dystrophin protein.
Supported in part by a research grant of the Italian Ministry of Health. We thank Drs L. M. Kunkel and E. P. Hoffman from Children’s Hospital, Boston, MA, for providing the bacterial strains containing the Duchenne muscular dystrophy gene fragments. We thank Mr D. C. Ward for help in preparing the manuscript, and Mr S. Daniel and Miss F. Blasevich for technical assistance.
1. Hoffman EP, Brown RM Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919-728 2. Zubrzycka-Gaarn EE, Bulmann DE, Karpati G, et al. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988;333:466-469 3. 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;333:861863 4. Bonilla E, Samitt CE, Miranda AF, et al. Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface. Cell 1988;54:447-452 5. Hoffman EP, Fischbeck KH, Brown RH, et al. Characterization of dysrrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N Engl J Med 1988;318:1363-1368 6. Arahata K, Ishihara T, Kamakura K, et al. Mosaic expression of dystrophin in symptomatic carriers of Duchenne’s muscular dystrophy. N Engl J Med 1989;320:138-142 7. Bonilla E, Schmidt B, Samitt CE, et al. Normal and dystrophindeficient muscle fibers in carriers of the gene for Duchenne muscular dystrophy. Am J Pathol 1988;133:440-445 8. Cornelio F, Dones I. Muscle fiber degeneration and necrosis in muscular dystrophy and other muscle diseases: cytochemical and immunocytochemical data. Ann Neurol 1984;16:694-701 9. Bodensteiner JB, Engel AG. Intracellular calcium accumulation in Duchenne dystrophy and other myopathies. Neurology (NY) 1978;28:439-446 10. Gardner-Medwin D, Pennington RJ, Walton JN. The detection of carriers of X-linked muscular dystrophy genes. J Neurol Sci 197 1;13:459-474 11. Hejtmancik JF, Harris SG, Tsao CC, et al. Carrier diagnosis of Duchenne muscular dystrophy using restriction fragment polymorphisms. Neurology (NY) 1986;36:1553- 1562 IL. Darras BT, Harper JF, Franke U.Prenatal diagnosis and detection of carriers with DNA probes in Duchenne’s muscular dystrophy. N Engl J Med 1987;316:985-992 13. Williams H , Sarfarazi M, Brown C, et al. The use of flanking markers in prediction for Duchenne muscular dystrophy. Arch Dis Child 1986;61:218-222 14. Hurko 0, Hoffman EP, McKee L, et al. Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier. Am J Hum Genet 1989;41:820-826 15. England SB, Nicholson LVB, Johnson MA, et al. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 1990;343:180-182 Lathrop GM, Lalouuel JM. Easy calculation of lod scores and genetic risks on small computers. Am J Hum Genet 1984; 36~460-465
Morandi et al: Dystrophin, Calcium, and Albumin in Carriers’ Muscle
679
~~
Immunocytochemical localization and immunoblot analysis of dystrophin in muscle fibers of 11 obligate and probable, and 7 possible carriers of Duchenne and Becker muscular dystrophy revealed an abnormal expression of the protein in 3 of them. Localization of calcium and albumin, as endogenous markers of extracellular fluid penetration, showed the presence of both molecules inside some fibers lacking dystrophin. Our morphological studies show that the initial stages leading to fiber necrosis in Duchenne muscular dystrophy are present in carriers with mosaicism. Comparison of dystrophin studies with restriction fragment length polymorphism analysis and creatine kinase levels showed that neither immunocytochemical nor immunoblot techniques for dystrophin are sensitive enough to provide a basis for genetic counseling. Morandi L, Mora M, Gussoni E, Tedeschi S, Cornelio F. Dystrophin analysis in Duchenne and Becker muscular dystrophy carriers: correlation with intracellular calcium and albumin. Ann Neurol 1990;28:674-679
Dystrophin, a large (427 kd) protein product of the Duchenne muscular dystrophy (DMD) locus, is found localized on the cytoplasmic side of the muscle fiber membrane El-41. It is absent from the muscles of D M D patients and is reduced or has a different molecular weight in muscles of Becker muscular dystrophy (BMD) patients IS}. Immunocytochemical studies with anti-dystrophin antibodies have revealed the presence of two populations of muscle fibers in D M D carriers {G, 71, one with normally expressed dystrophin, the other with the protein missing, the percentage of dystrophin-negative fibers being higher in manifesting carriers. In the present study, we evaluated dystrophin expression in the muscle cells of obligate, probable, and possible D M D and BMD female carriers, both by irnmunocytochemical and immunoblot techniques. We have also localized calcium and albumin as endogenous markers of extracellular fluid penetration to verify whether the pathogenetic events occurring in DMD muscles [S, 91 are also evident in the muscle of carriers and whether they are related to dystrophin absence. The muscle biopsy technique was also investigated for possible use in genetic counseling.
From the “Neuromuscular Diseases Department, Istituto Neurologic0 “C. Besta,” and the Clinical Research Laboratory, Istituti Clinici di Perfezionamento,Milano, Italy.
Materials and Methods Clinical Evaltlation We evaluated 15 D M D carriers ( 5 obligate, 3 probable, and 7 possible) and 3 obligate BMD carriers classified on the basis of pedigree and creatine kinase (CK) plasma levels [lo]. Obligate carriers were defined as mothers of an affected son who also had an affected brother or other male relative in the female line, and the daughters of B M D patients. Probable carriers were mothers of two or more affected sons; possible carriers were women with an affected male relative in the female line. C K plasma determination was done on 3 consecutive samples. If a carrier defined as possible by pedigree analysis had increased CK levels in all of 3 determinations, she was considered a probable carrier. One patient, a probable D M D carrier, showed clinical evidence of proximal muscle weakness of the lower limbs and hypertrophic gastrocnemii. Muscle biopsies were performed by the needle technique after informed consent.
Genetic Studies Genomic D N A extracted from peripheral blood lymphocytes by the phenol-chloroform method was used to study restriction fragment length polymorphisms (RFLPs). Restriction endonuclease digested D N A fragments were separated by electrophoresis, transferred to a nylon membrane, and
Address correspondence to Dr Morandi, Neuromuscular Diseases Department, Istituto Neurologico “C. Besta,” Via Celoria, 1120133 Milano, Italy.
Received Feb 9, 1990, and in revised form May 2 and Jun 7. Accepted for publication Jun 10, 1990.
674 Copyright 0 1990 by the American Neurological Association
probed with the intragenic P20, JBir, pERT87, XJ1.l, and the flanking C7 and 754 fragments [11-13].
Antisera Three bacterial strains (E.Coli RR1, kindly supplied by E. P. Hoffman and L. M. Kunkel, Children’s Hospital, Boston, MA) were cultured; they contained the expression vector pATH2 either alone or associated with one of two distinct DMD gene fragments (700 base pairs (bp) or 1,400 bp) from mouse heart, expressing a 30-kd and a 60-kd peptide, respectively. The bacteria were lysed, and the insoluble proteins were isolated by centrifugation and inoculated into rabbits for antisera production [I].
Immunoblotting Muscle biopsy samples, stored in liquid nitrogen until use, were pulverized, weighed, and dissolved in buffer (18% sodium dodecyl sulfate [SDS], 0.1 M Tris, p H 8.0, 5 mM ethylenediaminetetraacetic acid, 5 0 mM DL-dithiothreitol) to a concentration of 50 mgiml. Solutions were then boiled for 2 minutes, centrifuged to remove insoluble proteins, and loaded (850 p g of total protein per lane) onto SDS-polyacrylamide gradient gels (main gel gradient 12.5-3.5%; stacking gel 3%) { 1, 41. After electrophoresis, fractionated proteins were electroblotted onto nitrocellulose filters at 200 mA for 2 hours at room temperature. The filters were allowed to dry overnight. The blots on nitrocellulose paper were then incubated for 4 hours with anti-dystrophin antiserum diluted 1:400 in Tris-buffered saline (Tween 20 [TBST] [ l o mM Tris-HCI, pH 8.0, 150 mM sodium chloride, 0.05% Tween 201) and, subsequently, with the secondary antibody alkaline phosphatase-conjugated goat anti-rabbit IgG for 1 hour. Western blot development was performed by nitro blue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate.
Morphological Studies Muscle biopsies were frozen in 2-methylbutane (isopentane), cooled to - 183”C, and stored under liquid nitrogen. Consecutive 6- or 10-pm thick freshly prepared cryosections were collected on gelatin-coated slides. Intracellular calcium was localized histochemically by the alizarin red S method {8) on the 10-pm sections. For immunocytochemical analysis, the 6-pm sections were first incubated in avidin (Blocking Kit, Vector Labs, Burlingame, CA), diluted in phosphate-buffered solution (PBS) 1:20 for 15 minutes, washed a few times in PBS, incubated in biotin (Blocking Kit, Vector) 1:20 for 15 minutes, washed again, and then placed in 10% heat-inactivated normal goat s e w (NGS) for 30 minutes. After several washes, either anti-dystrophin antiserum (1 :600 dilution in PBS plus 10% NGS) or rabbit anti-human albumin antibody (Dakopatts, Copenhagen, Denmark) (2 CLgiml in PBS plus NGS) was applied for 120 minutes. Incubation in biotinylated goat antirabbit IgG (Vector) (1.5 pdml in PBS, 60 minutes) was followed by incubation (60 minutes) in either avidin peroxidase-conjugated (undiluted ABC Kit, Vector) or rhodamine-avidin D (Vector) (5 pdml in PBS). Between each incubation, sections were rinsed 10 times for 3 minutes each. After a final rinse, sections with avidin-peroxidase were de-
veloped with 3-3’-diaminobenzidine, and those with rhodamine were mounted in a glycerol medium containing paraphenylenediamine. All incubations were performed at room temperature in humid chambers. As control, on adjacent sections the primary antibody was omitted. Both dystrophin and albumin were localized simultaneously on a few sections. In this case, the primary incubation for 120 minutes was with a mixture of anti-dystrophin 1 :600 and anti-albumin 4 pdml (goat anti-human albumin) (Organon Teknika-Cappel, West Chester, PA) diluted in PBS plus 10% horse serum plus 2% bovine serum albumin. This was followed by incubation for 60 minutes with biotinylated donkey anti-rabbit IgG (Amersham, Amersham International Place, Amersham, UK) 1 pgiml in PBS, for a further 60 minutes, with fluorescein isothiocyanate (F1TC)-labeled streptavidin (Amersham) 1 pdrnl, and finally, by incubation in rhodamine-conjugated rabbit anti-goat IgG (Sera-Lab Ltd, Crawley-Down, UK) 2 p,g/ml for 60 minutes. Between each incubation, sections were rinsed in PBS 10 times for a total of 60 minutes. Because biopsies were performed with the needle technique, many fibers at the periphery of fiber fascicles could have been artifactually positive for calcium and albumin. We therefore counted only fibers found inside the fascicles. For each biopsy, a minimum of 500 fibers was counted. In a few patients (Patients 1-8, Table l), longitudinal sections were analyzed.
Results DNA RFLP analysis confirmed the heterozygotic condition in the obligate and probable DMD carriers, but only 1 of the possible DMD carriers was shown to be a true heterozygotic carrier (Patient 14, see Table 1). Immunocytochemical studies on transverse sections revealed a normal distribution of dystrophin on the surface of the muscle fibers in all definitely nonheterozygotic women. Of the heterozygotic DMD carriers, 3 (Patients 3,4, and 6, Table 1) had mosaic expression of dystrophin in their muscle, with some fibers totally or partially lacking the protein (4.5-18.7%) and others expressing it normally (Fig 1);the remaining heterozygotic carriers had normal distribution of dystrophin. The single clinically symptomatic DMD carrier (Patient 6) had mosaic expression of dystrophin. Hematoxylin-and-eosin-stained sections showed a greater variability of fiber size and some hypercontracted fibers in all patients with abnormal dystrophin expression and occasional necrotic fibers in 2 of them. Fiber size variability was observed in 6 other patients with normal dystrophin (2 obligate, 1 probable, and 1 possible DMD carrier, and 2 BMD carriers). Irnmunoblot substantially supported the immunocytochemical results; in those patients in whom dystrophin was missing from muscle fibers, qualitative inspection of the immunoblot showed lower amounts of the protein (Fig 2). CK levels (see Table 1) were raised in the 3 women
Morandi et al: Dystrophin, Calcium, and Albumin in Carriers’ Muscle 675
Table 1. Clinical Data and Dytrophin Expression in Muscles of Duchenne and Brcker Muscuhr Dystrophy Carriers" Patient DMD obligate carriers 1 P.M. 2 M.S. '3 C.T. 4 S.A. 5 C.L. DMD probable carriers 6 N.R. 7 S.A. 8 A.W. DMD possible carriers 9 S.L. 10 S.E. 11 S.M. 12 T.M. 13 G.M. 14 M.I. 15 C.L. BMD carriers 16 C.V. 17 C.G. 18 M.A.
Creatin e Kinase N N
t
t t
t t
t
N N N
N N N N
t
t T
Symptoms
Age (yr)
DNA~
Negative Negative Negative Negative Negative
31 27 31 28
100 100 100 100 100
Positive Negative Negative
42 50 29
100 100 100
Negative Negative Negative Negative Negative Negative Negative
22 20 25 34 42 28 23
35.7 35.7 64.2 49 55 79.8 0.6
Negative Negative Negative
16 47 20
100 99.1 99
44
Immunocytochemistry N
h '
Western Blot N N
Mosaic Mosaic
1
N
N
4
Mosaic
i
N N
N N
N
N
N
N
N N N
N N N
N
N
N
N
N
.1
N N
N
N
"Carriers were defined as obligate, probable, or possible from pedigree and creatine kinase levels {lo]. bPercentage of risk of being carrier from restriction fragment length polymorphism analyses combined with pedigree and creatine kinase levels
[161. DMD = Duchenne muscular dystrophy; N
=
normal; BMD
=
Becker muscular dystrophy.
Fig 1. Fluorescence-immunoj tained section showing severalfibers totally or partially lacking dystrophzn. (Original magnification x 250.1 Fzg 2, Reduction of dystrophin amount t n carriers with mosazcism (lane 2, patzent 3: lane 4, patient 6). Lanes I (normal control) and 3 (patient 9) show a normal amount of dystrophin, while in iane 5 (patient 16) the protein is slightly reduced. Results in lane G (patient 12) are considered normal even afthe staining appears reduced as the myosin band (205 K), indicative of reduced amounts of skeletal muscle proteins.
676 Annals of Neurology Vol 28 No 5 November 1990
Fig 3. Consecutive cryosections peroxidase-inimunos~ained for dystrophin (a) and albumin (b). Dystrophin is absentfrom albumin-positiziefibers. (Original magnification x 250.)
with mosaicism. Increased CK levels were also observed in 1 of the obligate and 2 of the probable D M D carriers, and in all the BMD carriers who had otherwise normal immunocytochemical localization of dystrophin. Calcium- and albumin-positive fibers were observed in nonnecrotic muscle fibers of the carriers with mosaicism. Calcium- and albumin-positive fibers were always devoid of dystrophin immunoreactivity on consecutive serial sections (Fig 3); in the same muscles, fibers lacking dystrophin were more numerous than calcium- and albumin-positive fibers (Fig 4). The proportion of calcium-positive fibers among dystrophin-negative fibers varied between 0.8% and 3.496; that of albuminpositive fibers varied between 2.19% and 5.5% (Table 2). In longitudinal sections, dystrophin was absent either along the total fiber surface or, more often, on segments thereof (Fig 5). Sometimes calcium and albumin penetrated only portions of the muscle fibers (Figs 6, 7). Simultaneous immunostaining for albumin and dystrophin on both transverse and longitudinal sections confirmed that on most of the surface of albuminpositive fibers, dystrophin was absent or reduced (Fig 8). ,
Discussion Our results confirm that the heterozygotic D M D gene carrier condition can be phenotypically expressed by absence of dystrophin on the sarcolemma of some muscle fibers. Most likely, this abnormal expression is associated with lyonization of the healthy X chromosome and subsequent activation of the affected one 141. Of the women characterized as heterozygotic carriers by RFLP analysis, only 3 had an abnormal im-
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Morandi et
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munocytochemical distribution of dystrophin with corresponding reduced immunoreactivity on Western blot; these women with mosaicism also had increased CK levels. CK was above normal in 2 other obligate DMD carriers and in all the BMD carriers. Our study shows therefore that only in certain patients do immunoblot analysis and immunocytochemical analysis reveal abnormalities in carriers already defined as such by RFLP and CK evaluation. We conclude that neither technique is sensitive enough to provide a basis for genetic counseling. The presence of dystrophin mosaicism is not always related to clinical signs C71. We also found alterations in dystrophin distribution in nonsymptomatic carriers. Our morphological studies show that the initial stages leading to fiber necrosis in D M D are present in carriers with mosaicism; calcium and albumin were found inside some apparently nonnecrotic muscle fibers lacking dystrophin. Dystrophin-negative fibers are more numerous than fibers penetrated by albumin or calcium. It is possible that dystrophin-negative fibers that did not show pathological changes in the plane of the section could have been abnormal in other sites, although in longitudinal sections we observed large segments of fibers lacking dystrophin with no penetration of calcium and albumin. The absence of the protein over a large part of a fiber, however, may be necessary before membrane alterations become observable. It is also possible that dystrophin-negative fibers require time to develop observable pathology; in fact, there are many stages in the process leading to fiber necrosis, the first being the absence of the protein. We suspect that our antibodies against 60-kd and 30-kd nonoverlapping peptides, both of which occur on the N-terminal portion of the dystrophin molecule, may detect an alteration of the protein not essential for its function. The different domains of this large molecule are probably not equally important for the integrity of dystrophin function. This is supported by a recent report by England and colleagues [l5} Dystrophin, Calcium, and Albumin in Carriers’ Muscle 677
Fig 5 . Rhwlamine-immunostained longitudinal section. Dystrophin is absent or reduced (arrows) on segments offiber surface. (Originalmagn6cation x 100.)
F i g 6. Albumin immunojluorescencestaining on a longitudinal section showing the presence of albumin inside portions of muscle fibers (arrows). (Original magnification x 250.)
g 4. (a) Dystrophin, (b) albumin, and (c) calcium. Loww
tgnification of Figure 3, showing dystrophin-negativefibers TOWS)&void of albumin and calcium. Note that som rtrophin-negabivefibers are hypercontracted (asterisks). riginal magnification x 100.)
Table 2. Dystrophin, Calcium, and Albumin in Muscle Fibers
Patient
DystrophinNegative Fibers (%)
AlbuminPositive Fibers (%)
CalciumPositive Fibers (%)
3
4.5
4
18.2
2.1 5.5
0.8 2.6
6
18.7
4.5
3.4
678 Annals of Neurology
Vol 28 No 5
Fig 7. Alizarin red S-stained longitudinalsection. Calcium penetrated only a portion of a fiber. (Originalmagnification x 250.1
November 1990
References
Fig 8. Dystrophin (a)and albumin (bi simultaneozlsly localized on a longitudinal section. Dystrophin is partially missing (arrows) fmm the surface of the fiber containing albumin. (Original magnification X 250.1
demonstrating that, in a family with 46% deletion of dystrophin mainly in the central domain of the molecule, the dystrophic phenotype was very mild. Better knowledge of the precise role played by the different domains of the dystrophin molecule will be needed to clarify the relation between muscle membrane function and the absence, in whole or in part, of the dystrophin protein.
Supported in part by a research grant of the Italian Ministry of Health. We thank Drs L. M. Kunkel and E. P. Hoffman from Children’s Hospital, Boston, MA, for providing the bacterial strains containing the Duchenne muscular dystrophy gene fragments. We thank Mr D. C. Ward for help in preparing the manuscript, and Mr S. Daniel and Miss F. Blasevich for technical assistance.
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