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Dystrophin analysis in duchenne and becker muscular dystrophy carriers Correlation with intracellular calcium and albumin.

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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.
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Morandi et al: Dystrophin, Calcium, and Albumin in Carriers’ Muscle
679
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