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Marked decrease of mitochondrial DNA with multiple deletions in a patient with familial mitochondrial myopathy
Vol.
167,
March
No.
16,
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
MARKED
DECREASE
OF MITOCHONDFUAL
MULTIPLE WITH
DELETIONS
FAMILIAL
rditsuo Ycshkd.
WITH
IN A PATIENT
MITOCHONDRIAL
b¶ieko Ob~uka~*~,
DNA MYOPATHY#
Kenji Niijima’,
Yoshihuni
Mizun01~~.
Yasuo Kagawa2
and Shlgeo Ohta2**
Departments of lNeurology and 2Biochemistry, Jichi Medical Minamikawachi-machi, Tochigi-ken 329-04, Japan Received
January
25,
680-685
School,
1990
SUMi#MRY: Muscle mitochondrial DNA (mtDNAJ from a patient with mitochomlria.l myopathy was examined by Southern blotting. Her family history suggests autosomal dominant inheritance of this disorder. ln contrast to other cases of this myopathy that are associated with a compensatory increase of mitochondria, in this patient, the content of mtDNA was markedly decreased (15% of controls), whereas protein of the ATP synthase beta-subunit was not decreased appreciably as judged by Western blotting. ln addition, the mitochondrial DNA had multiple deletions, which were located between the replication origins of the heavy strand and light strand. 01990 *cademu Press, Inc. Defects in the mitochondrial mitochondrial
myopathy
mitochondria,
ragged
found
respiratory
or cytopathy
red fibers
in the muscle
of these patients
has been reported.
found
with
in patients
syndrome
dominant other
are sporadic disorder
mtDNA
external
and caused
with
cases of familial
numbers
No decrease
Deletions
multiple
of mitochondrial
in muscle
mtDNA
myopathy
opthalmoplegia
mitochondrial
of mtDNA
myopathy,
DNA (mtDNA) been
as Keams-Sayre
(PEO) (5-7).
was
of
are often
have recently
such
with
proliferation
of mitochondria
by a single large deletion.
deletions
in patients
to compensatory
Owing
increased
(4).
have been identified
some types of mitochondrial
(KSS) and progressive
the deletions
(1. 2).
(3) with
in these patients
chain
Most
cases of
A case of an autosomal
recently
we also found
reported
multiple
(8).
In
deletions
in
(9).
Here, we report with probable
decrease
KSS with an autosomal
#In this paper, the mitochondrial
nucleotides sequence
correspondence
with
multiple
dominant
deletions
of Neurology,
should
of mtDNA
in a patient
family hislory.
of mitochondrial DNA are numbered published in ref 14.
3Present address: Department Bunkyo-ku, Tokyo, 113 Japan. *To whom
of mtDNA
Juntendou
University
according School
to those of Medicme,
be addressed.
The abbrevMi=s used are: mtDNA. mitochondrial DNA: KSS. Keams-Sayre syndrome; PEO, progressive external opthalmoplegia; MELAS. mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes: kbp. kilobase pairs. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
680
of
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Patients. F’atient I. Patient I was a 54-year-old Japanese female with progressive ophthalmoplegia and generalized muscle weakness as reported previously (10). She was diagnosed as probable KSS. With the family’s consent, a muscle specimen was obtained from the quadriceps femoris of the patient after her death. Mitochondria with paracrystalline Inclusion bodies were seen in her muscle fibers by electron micrograph. Patient II. Patient II was a 19-year-old Japanese male diagnosed as mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). The details were reported previously (11). With the family’s consent, a muscle specimen was obtained after his death. Patient III. Patient III was a 30-year-old Japanese male diagnosed as having MELAS without having a family history of this disease. A brain CT scan showed regions of low density area in the right frontal, right temporal, bilateral parietal and bilateral occipital lobes. Slight decrease of the cytochrome c oxidase subunit I was observed by Western blotting. A muscle specimen (right biceps brachii) was obtained with his and his family’s consent. Control specimens were obtained Controls; who were free from muscle diseases.
from the quadriceps
femoris
of patients
Family history of patient I. As shown in Figure 1, one brother of the patient and one of her sons were diagnosed as having mitochondrial myopathy. In addition, her grandmother, one aunt, three uncles, her mother, a brother, one sister and four cousins had symptoms of the same disease; Although their muscles were not histologically and biochemically examined, all of them had ptosis. which is one of the major features for KSS. As the disorder in two cousins was inherited from one of her uncles. the disease was not considered to be inherited as a maternal inheritance but was as an autosomal dominant disorder. Southern blotting. DNA purification and Southern blotting were performed as described previously (12). Quantitative analysis was performed by a reported method (13). A Sac I-Hind III fragment (nucleotide numbers 9,643 to 11,680, probe 2; nucleotides of mtDNA are numbered according to those of the published mtDNA sequence (14)) was subcloned. amplified and digested with the endonucleases. The other fragments used as hybridization probes were obtained by amplifying the mtDNA fragment by the polymerase chain reaction using the partial purified human mtDNA as a Furthermore, the amplified mtDNA fragments were purified by DNA source (15). agarose electrophoresis followed by a second amplification by the polymerase chain reaction. The oligonucleotides used for the polymerase chain reaction were as follows: Probe
1: dGATATGTCTCCAATACCCA?TAC dCGAAGAAGCAG CTTCAAACCTGC Probe 3: dCCACTAAGCCAATCAC’lTTA’ITGA dClTCCCCATGAAAGAACAGAGAA Restriction Figure 2.
map of the human
mtDNA
(nucleotide number (5.791-5.769) (15.701-15.724) (16.039-16.017)
and the position
4.2 14-4235)
of the probes
are illustrated
in
Muscle specimens were soaked in 10% trichloroacetic acid to stop Western blotting. enzymatic degradation and then washed with acetone. The samples were homogenized, neutrised by adding appropriate amount of 2 M Tris and dissolved in 5 % SDS. 1 % 2mercaptoethanol at 100 o C for IO min. Insoluble materials were removed by centrifugation and 100 microgram of protein samples were subjected to SDS polyacrylamide gel electrophoresis followed by Western blotting as described (16). Antiserum against the yeast ATP synthase beta subunit (Fl beta) was prepared as described previously ( 17).
RESULTS AND DISCUSSION Decrease
of
Muscle subjected
mitochondrid
DNA in Patient
I
DNAs from three patients
with mitochondrial
to Southern
These
blotting.
681
specimens
myopathy gave
were extracted
bands
of 16.6
and kbp,
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
n l
: probable
Q
: ptosis
0
Figure 1. Pedigree of Patient I. III-3 is Patient diagnosed as having mitochondrial myopathy her sons (Iv-l) was diagnosed as having the aunt (11-l). three uncle (11-3. 6 and 7). her cousins (111-1. 2. 8 and 9) had symptoms of not biopsied for examination.
corresponding
to human
mtDNA.
(lanes E and F in Figure
BIOPHYSICAL
Kearns-Sayre
RESEARCH
COMMUNICATIONS
syndrome
I (arrow). One of her brothers (111-5) was and died in 1986 at the age of 46. One of same disease. Her grandmother (I-2). one mother (R-2). one sister (111-7) and four the same disease, but their muscles were
of various
intensities;
those
of patients
3) were more than twice those of the controls
II and III
(lanes B, C and
D). However, the intensity of the mtDNA band from Patient I was markedly decreased (lane A). Quantitative analysis showed that the amount of mtDNA in the specimen from Patient
I was less than
15 96 of that in the control
from the muscle
of Patient
VA 0.82
microgram/mg
+ 0.39
I was in the normal wet weight
specimens. range
(0.75 microgram/mg
for control
e
The total DNA extracted
b
muscle],
so the
wet weight amount
,L-strand
ATPase8
piaun 2. Restriction map of human mitochondrial DNA and locations of hybridization probes. ND, CO. ATPase and cyt b. genes for NADH-Co(J reductase, cytochrome c oxidase, ATPase and cytochrome b, respectively; OH and Oh, origins of replication of the H-strand and L-strand. respectively; 16s and 12.9. genes for mitochondrial ribosomal RNA
682
of
Vol.
167,
No..
mtDNA/mg
BIOCHEMICAL
2, 1990
wet weight
of muscle
Patients
II and III are consistent
patients
With mitochondrial
mtDNA
in Patient
unlikely,
because
of the ATP synthase Western
blotting
not caused fibers
Thus, mtDNA Multiple
that mtDNA
As reported
of Patient
specifically
of mitochondrial
I. indicating
in Patient
of muscle mtDNA
with those in blood
blood cells was used as a control.
in
our conditions
is
Moreover,
in Patient the decrease
previously
I, as shown by of mtDNA (lo),
accumulation
the content
ragged
to nuclear
from Patient
I are seen in lane A
DNA in Patient
cells from the controls. for longer
I was unusually
the total DNA from the
exposure
C
time are shown
P
-16.6kbp
ABCDEFMr
19.3kbp
P
03
04
Figure 3. Decreased mtDNA in Patient I. Southern blots of mtDNAs from Patient I. controls and patients with other mitochondrial myopathies. Samples of 1 microgram of total DNA were digested with Barn HI and subjected to 1 % agarose electrophoresis After electrophoresis. the DNA was stained with followed by Southern blotting. ethidium bromide and material was detected under UV light (lower panel). A Hind IIISac I fragment of mtDNA was used as a hybridization probe (upper panel). Lanes A. B. C, D. E. F and Mr: samples from Patient I. controls 1. 2 and 3. Patient II. Patient III and a Sty I digest of lambda phage DNA, respectively. FIgure 4. Western blot of the ATF’ synthase beta-subunit. The total muscle specimen was treated as described in MATERIALS AND METHODS. A sample of 100 microgram of soluble protein in SDS was subjected to SDS polyacrylarnide The ATP synthase beta-subunit was stained gel electrophoresis. followed by blotting with antiserum, and detected with 125-I iodinated Protein A. Lanes C and P show samples from a control and Patient I. respectively. The arrow indicates the position of the ATP synthase beta-subunit.
683
red
of mitochondria.
ABCDEF
-
was
I
mtDNA
Autoradiograms
in
I.
DNA from Patient bands
in
to the decrease
under II.
appreciably
also suggests
of mitochondria.
3. Since the ratio of muscle
low but comparable
was degraded
observed
of mitochondria
contrast
in Patient
COMMUNICATIONS
of mtDNA
number
(4). and in marked
4). This observation
Faint and discontinuous of Figure
of increased
was not decreased
in the muscle
RESEARCH
The increases
was found to be increased
was decreased
deletion
reports
myopathies
by degradation
were found
with
beta subunit
(Figure
BIOPHYSICAL
was decreased.
I. The possibility mtDNA
AND
in
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
46.6
lQ2 kbp 9.6 -
COMMUNICATIONS
kbp
=e
ure 5 Southern blot of mitochondrial DNA. Tota DNAs (1 microgram) from the muscle of Patient I (lanes P) and from blood cells of a healthy individual (lanes C were digested with the indicated restriction endonucleases. subjected to Southern blotting and hybridized with probe 1 (PRl) for the Sac I, Dra I and BamH I digests, and with probe 2 (PR2) or probe 3 (PR3) for the Pvu II digests, respectively. Note that the amount of mtDNA is much less in the blood cells than in the muscle cells.
5. The discontinuous
Figure
Patient
I (lanes P. BamH
bands
were caused
DNA with
several
fragments
of mtDNA.
with
no smaller
2.047
(nucleotide
respectively,
In the present
Zeviani
origins
disorder
case, most because
as a hybridization
probe
deletions
to examine
deletions
the report
that in cases with
(8). However,
as pointed
at the D-loop
of Patient
I is an autosomal of multiple
disorders with
(8). This
an autosomal
the deletion decrease
is the second dominant
of mtDNA.
of mtDNA
disorder.
However.
was observed
This
dominant
result
of mtDNA
appears start
disorder.
confirming
684
This
for
probe
3,
In Patient
I,
to be contrary
to
at the D-loop
region
that all the deletions Thus,
it is suggested our findings
are
autosomal
dominant
deletions
of mtDNA
in patients
the role of a nuclear I.
As shown
with
in the previously
in Patient
was used
with
origin.
AND METHODS,
in patients
on the multiple
unlike
the
the autosomal
hybridized
all deletions
deletions
report
of the
between
of the deletions.
deletions
in MATERIALS
the report
These
by degradation,
with
fragment
(9). there was no evidence
that the disease with
points
deletions,
consistent
probes).
were located
in patients
(8). A small
randomly.
As described
and from
the two Sac I sites
did not start from the D-loop
out previously
region.
several
2 for the positions
deletions
5. DNAs with
multiple
the
was intact.
the starting
are distributed
with
36 to 9,643
between
5 (see Figure
in mtDNA
origin
that most of the deletions
the multiple
was no deletion
the Sac I fragment
PR3. lane P of Figure
numbers
were not produced
of the multiple
multiple
the digests
these hybridization
fragments
in Figure
start at the D-loop
the Pvu II digest, indicating
from nucleotide
and that there
et al. reported
dominant
and hybridizing
by digesting
5, the Sac I or Dra I digests gave only one band
the smaller
36 and 9,643)
cells from
that the discontinuous
of DNA was excluded
can be seen by using
that
numbers
in Figure
(Fragments
deletions,
two replication
start
As shown
were seen in the muscle
The possibility
degradation
endonucleases
fragments
but by multiple
probes).
by non-specific
indicated
of mtDNA
I and Pvu II digests).
restriction
to 10,415.
observations
short bands
reported
decrease
gene product
in
case (8). marked
of mtDNA
may have
Vol.
167,
No.
resulted respiratory
2, 1990
from
the multiple
BIOCHEMICAL
deletions
AND
BIOPHYSICAL
in the mtDNA.
RESEARCH
which
cause
COMMUNICATIONS
the
defect
chain.
REFERENCES 1. DtMauro, S., Bontlla. E., Zebtani, M.. Nakagawa. M. and Devivo, D.C. (1985) Ann. Neurol. 17, 521-538. 2. Capardi, R.A. (1988) Trends Biochem. Sci. 13. 144-148. 3. Engel. W.K. and Cunntngham. G.G. (1963) Neurology, 13. 919-923. 4. Bril, V., Rewcastle, N.B. and Humphreg. J. (1984) Can. J. Neurol. Sci. 11, 390-394. 5. Holt. I.J.. Harding, A.E. and Morgan-Hughes, J.A. (1988) Nature 331, 717-719. 6. Schon, E.A.. Rizzuto. R, Moraes, C.T.. Nakase. H.. Zeviani. M. and DiMauro, S. (1989) Science., 244, 346-349. 7. Moraes. C.T., DiMauro. S., Zeviani. M.. Lobes, A.. Shanske. 3. Mtranda. A.F.. Nakase, H., Bonilla, E., Wemeck, L.C., Servidei. S.. Nonaka. H.. Koga, Y., Sriro, A., Brrownell, A.K.W., Schmidt, B.. Schotland. D.L., Zupanc, M., Devivo. D., Schon. EA., and Rowland, L.P. (1989) N. Engl. J. Med. 320, 1293- 1299. 8. Deviant. M.. Servtdei, S.. Cellera, C.. Bertini, E., DiMauro. S. and DiDonato, S. (1989) Nature 339, 309-3 11. 9. Yuzaki, M., Ohkoshi, N., Kanazawa, I., Kagawa, Y. and Ohta, S. (1989) Biochem. Biophys. Res. Commun. 164. 1352-1357. 10 Otsuka, M., Ohta. 8. Kaneko. J.. Nttjima. K.. Mizuno. Y.. Yoshida. M. and Kagawa, Y. (1988) EUomed. Res. (Tokyo) 9, 533-538. 11 Shimoizumi. H., Momoi, Y.M.. Ohta. S., Kagawa. Y., Momoi. T. and Yanagisawa, M. (1989) Ann. Neurol. 25. 615-621. 12 Davis, L.C.. Dibner, M.D. and Battey, J.F. (eds.) (1986) in :Basic Methods in Molecular Biology. (Elsevier, New York) ~~47-50. 13 Suissa, M. (1983) Anal. Biochem. 133. 511-514. 14 Anderson, S.. Bankter, AT.. Barrel, B.G.. DeBruijin. M.H.L., Coulson. A.R.. Drouin, J., Eperon. I.C.. Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R. and Young, E.G. (1981) Nature 290. 457-465. 15 Saiki, R.K.. Gelfand. D.H.. Stoffel. S., Schare, S.J., Higuchi. R., Horn, G.T., Mullis, K.B., and Erlich. H.A. (1988) Science. 239, 487-49 1. 16 Towbin, H., Staehelin, T. and Gord0n.J. (1979) Proc, Natl. Acad. Sci. USA., 76, 4350-4354. 17. Ohta, S. and Schatz, G. (1984) EMBO J. 3. 651-657.
685
in
167,
March
No.
16,
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
MARKED
DECREASE
OF MITOCHONDFUAL
MULTIPLE WITH
DELETIONS
FAMILIAL
rditsuo Ycshkd.
WITH
IN A PATIENT
MITOCHONDRIAL
b¶ieko Ob~uka~*~,
DNA MYOPATHY#
Kenji Niijima’,
Yoshihuni
Mizun01~~.
Yasuo Kagawa2
and Shlgeo Ohta2**
Departments of lNeurology and 2Biochemistry, Jichi Medical Minamikawachi-machi, Tochigi-ken 329-04, Japan Received
January
25,
680-685
School,
1990
SUMi#MRY: Muscle mitochondrial DNA (mtDNAJ from a patient with mitochomlria.l myopathy was examined by Southern blotting. Her family history suggests autosomal dominant inheritance of this disorder. ln contrast to other cases of this myopathy that are associated with a compensatory increase of mitochondria, in this patient, the content of mtDNA was markedly decreased (15% of controls), whereas protein of the ATP synthase beta-subunit was not decreased appreciably as judged by Western blotting. ln addition, the mitochondrial DNA had multiple deletions, which were located between the replication origins of the heavy strand and light strand. 01990 *cademu Press, Inc. Defects in the mitochondrial mitochondrial
myopathy
mitochondria,
ragged
found
respiratory
or cytopathy
red fibers
in the muscle
of these patients
has been reported.
found
with
in patients
syndrome
dominant other
are sporadic disorder
mtDNA
external
and caused
with
cases of familial
numbers
No decrease
Deletions
multiple
of mitochondrial
in muscle
mtDNA
myopathy
opthalmoplegia
mitochondrial
of mtDNA
myopathy,
DNA (mtDNA) been
as Keams-Sayre
(PEO) (5-7).
was
of
are often
have recently
such
with
proliferation
of mitochondria
by a single large deletion.
deletions
in patients
to compensatory
Owing
increased
(4).
have been identified
some types of mitochondrial
(KSS) and progressive
the deletions
(1. 2).
(3) with
in these patients
chain
Most
cases of
A case of an autosomal
recently
we also found
reported
multiple
(8).
In
deletions
in
(9).
Here, we report with probable
decrease
KSS with an autosomal
#In this paper, the mitochondrial
nucleotides sequence
correspondence
with
multiple
dominant
deletions
of Neurology,
should
of mtDNA
in a patient
family hislory.
of mitochondrial DNA are numbered published in ref 14.
3Present address: Department Bunkyo-ku, Tokyo, 113 Japan. *To whom
of mtDNA
Juntendou
University
according School
to those of Medicme,
be addressed.
The abbrevMi=s used are: mtDNA. mitochondrial DNA: KSS. Keams-Sayre syndrome; PEO, progressive external opthalmoplegia; MELAS. mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes: kbp. kilobase pairs. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
680
of
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Patients. F’atient I. Patient I was a 54-year-old Japanese female with progressive ophthalmoplegia and generalized muscle weakness as reported previously (10). She was diagnosed as probable KSS. With the family’s consent, a muscle specimen was obtained from the quadriceps femoris of the patient after her death. Mitochondria with paracrystalline Inclusion bodies were seen in her muscle fibers by electron micrograph. Patient II. Patient II was a 19-year-old Japanese male diagnosed as mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). The details were reported previously (11). With the family’s consent, a muscle specimen was obtained after his death. Patient III. Patient III was a 30-year-old Japanese male diagnosed as having MELAS without having a family history of this disease. A brain CT scan showed regions of low density area in the right frontal, right temporal, bilateral parietal and bilateral occipital lobes. Slight decrease of the cytochrome c oxidase subunit I was observed by Western blotting. A muscle specimen (right biceps brachii) was obtained with his and his family’s consent. Control specimens were obtained Controls; who were free from muscle diseases.
from the quadriceps
femoris
of patients
Family history of patient I. As shown in Figure 1, one brother of the patient and one of her sons were diagnosed as having mitochondrial myopathy. In addition, her grandmother, one aunt, three uncles, her mother, a brother, one sister and four cousins had symptoms of the same disease; Although their muscles were not histologically and biochemically examined, all of them had ptosis. which is one of the major features for KSS. As the disorder in two cousins was inherited from one of her uncles. the disease was not considered to be inherited as a maternal inheritance but was as an autosomal dominant disorder. Southern blotting. DNA purification and Southern blotting were performed as described previously (12). Quantitative analysis was performed by a reported method (13). A Sac I-Hind III fragment (nucleotide numbers 9,643 to 11,680, probe 2; nucleotides of mtDNA are numbered according to those of the published mtDNA sequence (14)) was subcloned. amplified and digested with the endonucleases. The other fragments used as hybridization probes were obtained by amplifying the mtDNA fragment by the polymerase chain reaction using the partial purified human mtDNA as a Furthermore, the amplified mtDNA fragments were purified by DNA source (15). agarose electrophoresis followed by a second amplification by the polymerase chain reaction. The oligonucleotides used for the polymerase chain reaction were as follows: Probe
1: dGATATGTCTCCAATACCCA?TAC dCGAAGAAGCAG CTTCAAACCTGC Probe 3: dCCACTAAGCCAATCAC’lTTA’ITGA dClTCCCCATGAAAGAACAGAGAA Restriction Figure 2.
map of the human
mtDNA
(nucleotide number (5.791-5.769) (15.701-15.724) (16.039-16.017)
and the position
4.2 14-4235)
of the probes
are illustrated
in
Muscle specimens were soaked in 10% trichloroacetic acid to stop Western blotting. enzymatic degradation and then washed with acetone. The samples were homogenized, neutrised by adding appropriate amount of 2 M Tris and dissolved in 5 % SDS. 1 % 2mercaptoethanol at 100 o C for IO min. Insoluble materials were removed by centrifugation and 100 microgram of protein samples were subjected to SDS polyacrylamide gel electrophoresis followed by Western blotting as described (16). Antiserum against the yeast ATP synthase beta subunit (Fl beta) was prepared as described previously ( 17).
RESULTS AND DISCUSSION Decrease
of
Muscle subjected
mitochondrid
DNA in Patient
I
DNAs from three patients
with mitochondrial
to Southern
These
blotting.
681
specimens
myopathy gave
were extracted
bands
of 16.6
and kbp,
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
n l
: probable
Q
: ptosis
0
Figure 1. Pedigree of Patient I. III-3 is Patient diagnosed as having mitochondrial myopathy her sons (Iv-l) was diagnosed as having the aunt (11-l). three uncle (11-3. 6 and 7). her cousins (111-1. 2. 8 and 9) had symptoms of not biopsied for examination.
corresponding
to human
mtDNA.
(lanes E and F in Figure
BIOPHYSICAL
Kearns-Sayre
RESEARCH
COMMUNICATIONS
syndrome
I (arrow). One of her brothers (111-5) was and died in 1986 at the age of 46. One of same disease. Her grandmother (I-2). one mother (R-2). one sister (111-7) and four the same disease, but their muscles were
of various
intensities;
those
of patients
3) were more than twice those of the controls
II and III
(lanes B, C and
D). However, the intensity of the mtDNA band from Patient I was markedly decreased (lane A). Quantitative analysis showed that the amount of mtDNA in the specimen from Patient
I was less than
15 96 of that in the control
from the muscle
of Patient
VA 0.82
microgram/mg
+ 0.39
I was in the normal wet weight
specimens. range
(0.75 microgram/mg
for control
e
The total DNA extracted
b
muscle],
so the
wet weight amount
,L-strand
ATPase8
piaun 2. Restriction map of human mitochondrial DNA and locations of hybridization probes. ND, CO. ATPase and cyt b. genes for NADH-Co(J reductase, cytochrome c oxidase, ATPase and cytochrome b, respectively; OH and Oh, origins of replication of the H-strand and L-strand. respectively; 16s and 12.9. genes for mitochondrial ribosomal RNA
682
of
Vol.
167,
No..
mtDNA/mg
BIOCHEMICAL
2, 1990
wet weight
of muscle
Patients
II and III are consistent
patients
With mitochondrial
mtDNA
in Patient
unlikely,
because
of the ATP synthase Western
blotting
not caused fibers
Thus, mtDNA Multiple
that mtDNA
As reported
of Patient
specifically
of mitochondrial
I. indicating
in Patient
of muscle mtDNA
with those in blood
blood cells was used as a control.
in
our conditions
is
Moreover,
in Patient the decrease
previously
I, as shown by of mtDNA (lo),
accumulation
the content
ragged
to nuclear
from Patient
I are seen in lane A
DNA in Patient
cells from the controls. for longer
I was unusually
the total DNA from the
exposure
C
time are shown
P
-16.6kbp
ABCDEFMr
19.3kbp
P
03
04
Figure 3. Decreased mtDNA in Patient I. Southern blots of mtDNAs from Patient I. controls and patients with other mitochondrial myopathies. Samples of 1 microgram of total DNA were digested with Barn HI and subjected to 1 % agarose electrophoresis After electrophoresis. the DNA was stained with followed by Southern blotting. ethidium bromide and material was detected under UV light (lower panel). A Hind IIISac I fragment of mtDNA was used as a hybridization probe (upper panel). Lanes A. B. C, D. E. F and Mr: samples from Patient I. controls 1. 2 and 3. Patient II. Patient III and a Sty I digest of lambda phage DNA, respectively. FIgure 4. Western blot of the ATF’ synthase beta-subunit. The total muscle specimen was treated as described in MATERIALS AND METHODS. A sample of 100 microgram of soluble protein in SDS was subjected to SDS polyacrylarnide The ATP synthase beta-subunit was stained gel electrophoresis. followed by blotting with antiserum, and detected with 125-I iodinated Protein A. Lanes C and P show samples from a control and Patient I. respectively. The arrow indicates the position of the ATP synthase beta-subunit.
683
red
of mitochondria.
ABCDEF
-
was
I
mtDNA
Autoradiograms
in
I.
DNA from Patient bands
in
to the decrease
under II.
appreciably
also suggests
of mitochondria.
3. Since the ratio of muscle
low but comparable
was degraded
observed
of mitochondria
contrast
in Patient
COMMUNICATIONS
of mtDNA
number
(4). and in marked
4). This observation
Faint and discontinuous of Figure
of increased
was not decreased
in the muscle
RESEARCH
The increases
was found to be increased
was decreased
deletion
reports
myopathies
by degradation
were found
with
beta subunit
(Figure
BIOPHYSICAL
was decreased.
I. The possibility mtDNA
AND
in
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
46.6
lQ2 kbp 9.6 -
COMMUNICATIONS
kbp
=e
ure 5 Southern blot of mitochondrial DNA. Tota DNAs (1 microgram) from the muscle of Patient I (lanes P) and from blood cells of a healthy individual (lanes C were digested with the indicated restriction endonucleases. subjected to Southern blotting and hybridized with probe 1 (PRl) for the Sac I, Dra I and BamH I digests, and with probe 2 (PR2) or probe 3 (PR3) for the Pvu II digests, respectively. Note that the amount of mtDNA is much less in the blood cells than in the muscle cells.
5. The discontinuous
Figure
Patient
I (lanes P. BamH
bands
were caused
DNA with
several
fragments
of mtDNA.
with
no smaller
2.047
(nucleotide
respectively,
In the present
Zeviani
origins
disorder
case, most because
as a hybridization
probe
deletions
to examine
deletions
the report
that in cases with
(8). However,
as pointed
at the D-loop
of Patient
I is an autosomal of multiple
disorders with
(8). This
an autosomal
the deletion decrease
is the second dominant
of mtDNA.
of mtDNA
disorder.
However.
was observed
This
dominant
result
of mtDNA
appears start
disorder.
confirming
684
This
for
probe
3,
In Patient
I,
to be contrary
to
at the D-loop
region
that all the deletions Thus,
it is suggested our findings
are
autosomal
dominant
deletions
of mtDNA
in patients
the role of a nuclear I.
As shown
with
in the previously
in Patient
was used
with
origin.
AND METHODS,
in patients
on the multiple
unlike
the
the autosomal
hybridized
all deletions
deletions
report
of the
between
of the deletions.
deletions
in MATERIALS
the report
These
by degradation,
with
fragment
(9). there was no evidence
that the disease with
points
deletions,
consistent
probes).
were located
in patients
(8). A small
randomly.
As described
and from
the two Sac I sites
did not start from the D-loop
out previously
region.
several
2 for the positions
deletions
5. DNAs with
multiple
the
was intact.
the starting
are distributed
with
36 to 9,643
between
5 (see Figure
in mtDNA
origin
that most of the deletions
the multiple
was no deletion
the Sac I fragment
PR3. lane P of Figure
numbers
were not produced
of the multiple
multiple
the digests
these hybridization
fragments
in Figure
start at the D-loop
the Pvu II digest, indicating
from nucleotide
and that there
et al. reported
dominant
and hybridizing
by digesting
5, the Sac I or Dra I digests gave only one band
the smaller
36 and 9,643)
cells from
that the discontinuous
of DNA was excluded
can be seen by using
that
numbers
in Figure
(Fragments
deletions,
two replication
start
As shown
were seen in the muscle
The possibility
degradation
endonucleases
fragments
but by multiple
probes).
by non-specific
indicated
of mtDNA
I and Pvu II digests).
restriction
to 10,415.
observations
short bands
reported
decrease
gene product
in
case (8). marked
of mtDNA
may have
Vol.
167,
No.
resulted respiratory
2, 1990
from
the multiple
BIOCHEMICAL
deletions
AND
BIOPHYSICAL
in the mtDNA.
RESEARCH
which
cause
COMMUNICATIONS
the
defect
chain.
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