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Clonal expansion of mitochondrial DNA with multiple deletions in autosomal dominant progressive external ophthalmoplegia.

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Clonal Expansion of Mitochondrial DNA with
Multiple Deletions in Autosomal Dominant
Progressive External Ophthahnoplegia
Ali-Reza Moslemi, BSc,* Atle Melberg, MD,? Elisabeth Holme, MD, PhD,$ and Anders Oldfors, MD, PhD*
Sporadic progressive external ophthalmoplegia and Kearns-Sayre syndrome are usually associated with single large-scale
mitochondrial DNA deletions in muscle. In progressive external ophthalmoplegia with autosomal dominant inheritance,
multiple mitochondrial DNA deletions have been reported. We studied several members of a Swedish family with
autosomal dominant progressive external ophthalmoplegia and multiple mitochondrial DNA deletions by polymerase
chain reaction analysis of single muscle fibers and by in situ hybridization, combined with enzyme histochemical analysis.
Muscle fiber segments with deficiency of cytochrome c oxidase, which is partially encoded by mitochondrial DNA, had
accumulated mitochondrial DNA with deletions and showed reduced levels of wild-type mitochondrial DNA. The deletions varied between individual muscle fibers. There was one predominant deletion in each cytochrome c oxidasedeficient muscle fiber segment. Sequencing of the deletion breakpoints showed that most but not all of the deletions
were flanked by direct repeats. Young, clinically affected individuals of this family without limb muscle symptoms did
not show mitochondrial DNA deletions or cytochrome c oxidase-deficient muscle fibers. Our results indicate that a
nuclear factor predisposes to the development of somatic multiple mitochondrial DNA deletions. Mitochondrial DNA
with multiple different deletions shows clonal expansion, which leads to mitochondrial myopathy with ragged-red fibers
and muscle weakness.
Moslemi A-R, Melberg A, Holme E, Oldfors A. Clonal expansion of mitochondrial D N A with multiple
deletions in autosomal dominant progressive external ophthalmoplegia. Ann Neurol 1336;40:707-713
Single large-scale mitochondrial DNA (mtDNA) deletions were first described in a report on mitochondrial
niyopathies [I], and were later shown to be especially
associated with syndromes such as progressive external
ophthalmoplegia (PEO), frequently as part of multisystem disorders such as Kearns-Sayre syndrome (KSS)
12, 31. Most cases are sporadic. A typical finding is
mitochondrial myopathy with cytochrorne c oxidase
(COX)-deficient muscle fibers.
In some patients PEO and mitochondrial myopathy
are inherited as an autosomal dominant disease (adPEO). In adPEO there are multiple mtDNA deletions
in muscle tissue [4-81. The disease is not confined to
muscle tissue and multiple deletions have been found
in other tissues as well [3]. Because of the mendelian
inheritance, a nuclear factor has been implicated in the
pathogenesis of these multiple deletions [7, 101.
In this study on a Swedish family with adPEO, we
demonstrated that mtDNA with multiple deletions
shows clonal expansions in muscle fiber segments resulting in COX deficiency. Analysis of the deletion
breakpoints showed that there are deletions flanked by
short direct repeats as well as deletions with no or imperfect repeats at their borders. Our results indicate
that the mtDNA deletions are somatic mutations,
which accumulate with increasing age, eventually leading to mitochondria1 inyopathy and muscle weakness.
Materials and Methods
Patients
T h e family pedigree is shown in Figure 1. In addirion to
PEO, most affected individuals had hypogonadism. There
were also various other neurological signs and symptoms.
Clinical findings in this family were reported previously [ 1 1131. Muscle and peripheral nerve involvement in this family
has been reported elsewhere (141. A summary of the clinical
features of the investigated patients at the age when muscle
biopsy was performed is given in the Table. Muscle biopsy
specimens were obtained from the quadriceps muscle of Patients IV:2, IV:3, IV:4, 1V:5, IV:7, 1V:8, V:l, and V:6. T h e
muscle tissue was immediately frozen in liquid nitrogen, and
stored at -70°C until analyzed.
Enzyme Histochemisty
For identification and quantification of COX-deficient muscle fibers, cryostat sections were subjected to enzyme hisco-
From the Departnienrs of 'Parhology and +Clinical Chemistry,
Sahlgrenska Universiry Hospital, Goteborg, and the ?Department
of Neurology, Uppsala Univrrsity Hospiral, Uppsala, Swrden.
Copyright
Received Apr 2, 1996, and in revised form Jut1 5. Accepted for
publication Jun 6, 1996.
Address correspondence to Dr Oldfors, Deparrmrnt of Pathology.
Sahlgrenska Hospiral, S-4 13 45 Gorehorg, Sweden.
0 1996 by the American Neurological Association
707
(0.9 mol/liter of 'rris-hydrochloric acid [HCI] p H 8.3, 0.3
mol/liter of potassium chloride [KCI], and 0.2 mol/liter of
HCI) were then added. Two microliters were used for each
reaction with 10 pmol of each primer, 2 units of Taq polymerase (Boehringer Mannheim, Germany), each deoxynucleoride triphosphate (dNTP) at a final concentration of
200 pnioUliter, and 5 pl of PCR buffer (1 5 nimol/liter of
magnesium chloride [MgCI:], 100 nimol/liter of Tris-HCI
p H 8.3, 500 nimol/liter of KCI, and 0.01% gelatine) at a
final volume of 50 pl. PCR was performed with a thermal
cycler (System 9600, Perkin Elmer Cetus, CA) for 35 cycles:
94°C for 30 seconds. 55°C for 30 seconds, and 72°C for
30 seconds. 'The positions of the primers and the investigated
regions of intDNA are described in the legend to Figure 3.
W e also investigated the occurrence of the mtDNA 260-bp
tandem duplication in the D-loop region described in patients with large-scale mtDNA deletions [18], by PCR analysis using the primer pairs described by Manfredi and coauthors [19]. The amplified fragments were then separated in
a 1.5% agarose gel and stained with ethidium bromide. Deletion breakpoints of nitDNA in isolated single muscle fibers
were analyzed by direct sequencing of the PCR-amplified
nitDNA fragments with a model 377 DNA sequencer (Applied Biosystems, CA) using the Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystenis. CA).
Breakpoints of the multiple deletions of mtDNA detected
by PCR analysis of muscle homogenate were sequenced after
cloning (TA Cloning Kit, Invitrogen, CA) of rhe PCR-amplified fragments.
In situ hybridization was performed to detect nitDNA
transcripts. For probes we used double-stranded "S-labeled
DNA fragments of mtDNA amplifed by PCR as previously described [17]. These were parts of the NADHdehydrogenase (ND) subunit 2 gene (ND 2 probe; nucleotides [nt] 4614-4933), COX subunit I1 gene (COX I1
probe; nt 8000-8160), the N D subunir 4 gene (ND 4
probe; nt 11406-11776), the N D subunit 5 gene (ND
Fig 1. Family pedigree. Filled symbols correspond to clinical!y affected indiividunls. The harched symbol indicates a
probtrb(y afficted individual.
chemical staining for succinate dehydrogenase (SDH) and
COX. Fibers were considered C O X deficient if they showed
low COX activity and high S D H activity [ 151. For comparison of i n situ hybridization results and mitochondria1 enzyme
activities in muscle fibers, the first and last sections of each
series of consecutive sections were stained for COX and S D H
activity to make it possible to identify muscle fibers with
COX deficiency in the entire investigated segment.
Mitochondria1 Analysis
Total DNA was extracted from muscle tissue and Southern
analysis was performed as previously described [ 161.
Polymerase chain reaction (PCR) analysis was performed
on DNA preparations from isolated single muscle fibers and
from homogenates of 800 to 1,600 muscle fibers from each
patient. Single muscle fibers, both COX-deficient and normal fibers, were dissected as previously described [ 171. The
isolated single muscle fiber segments and 15-pm-thick cryostat sections of muscle tissue were transferred to 10 and 25
PI. respectively, of 0.2 mol/liter of potassium hydroxide
(KOH) with 50 mmol/liter of dithiothreitol for 10 minutes
at 94°C. Ten and 25 PI, respectively, of neutralization buffer
Sronmay of Clinical and Labortrtoy Findings
Patient No.
Gender
Age (yr)
Age at onset of ocular
symptoms (yr)
Ophthalmoplegia
Ptosis
Limb weakness
History of hypogonadisin
Additional clinical features
Electroinyogram of frontalis rnusclc
Serum creatine kinase
(pkat/liter)A
1V:8
v:1
V:6
F
53
23
F
51
21
M
30
20
+
+
+
M
18
Congenital
wabismiis
+
T
y
i
-
-
-
-
4
IV:2
IV:3
IV:4
IV:5
I*
IM
57
27
55
M
53
25
+
+
+
35
+
+
+
t
-
+
IV:7
+
+
+
-
-
+
+
cnat
cna
cnard
cnar
cnat
Myopathic
M yopathic
Myopathic
Myopathic
Myoparhic
M yopathic
Myopathic
2.4
11
4.9
44
28
1.8
34
-
~
"Normal < 3.0 pkadlirer (male) and 2.5 pkat/liter (female).
+ = present:
a
=
ataxia;
~
t =
absent: x = convergence weakness; y = pathological nystagmus and convcrgence weakness; c
tremor; d = mental depression; r = mental retardation.
=
708 Annals of Neurology
Vol 40
No 5
November 1996
=
cataracts; n
=
neuropathy;
1234
A
1 2 3
B
Fig 2. Southern analysis of muscle mtDNA af2er cleavage
with BamHI (A) and Pvu 11 (B). Lane I = Patient IV.7;
lane 2 = Patient IV8; lane 3 = Patient IE3; lane 4 =
normal control subject. In addition to the normal 165-kb
rntDNA fiagment in all lanes, several short f i a p e n t s are
present in lanes 2 and 3.
5 probe; nt 13501-13805), the ND subunit 6 gene (ND
6 probe; nt 14184-14542), and the cytochrome bgene (Cyt
b probe; nt 15340-15736). Nucleotides are numbered according to the Cambridge sequence [20]. Consecutive 8-pmthick cryostat sections of muscle tissue were mounted on
SuperfrostlPlus slides (Gerhard Menzel Glasbearbeitungswerk GmbH, Germany), fixed in 2% glutaraldehyde in 0.1
M sodium cacodylate buffer for 2 minutes, and briefly rinsed
in distilled water and then in phosphate-buffered saline
(PBS) solution for 10 minutes. The sections were then digested with Pronase (protease, Streptornyces griseus, Calbiochem, CA) (100 pg/ml) for 5 minutes, rinsed in distilled
water and incubated in 25 rnM HCI for 10 minutes, and
postfixed again in 2% glutaraldehyde in buffer for 2 minutes.
Hybridization and autoradiography were performed as previously described [ 171.
Results
Morphological examination of muscle biopsy specimens showed mitochondria1 myopathy with frequent
COX-deficient muscle fibers and many ragged-red fibers in Patients IV2, IV3, IV4, IV5, and IV:8
(range, 6- 12% COX-deficient fibers). The muscle
specimens from Patients IV7, V:l, and V:6 were
normal.
Southern analysis of muscle mtDNA after cleavage
with Barn HI or Puu I1 showed multiple mtDNA deletions in clinically affected Patients IV3 and I V 8 but
not in the healthy sibling, Patient IV7 (Fig 2).
PCR analysis of mtDNA from muscle homogenate
including 800 to 1,600 muscle fibers of affected individuals in Generation IV (Patients IV:2, IV:4, IV:5,
and IV8) showed multiple mtDNA deletions (Fig 3a).
W e did not detect mtDNA deletions in the clinically
affected patients of Generation V (Patients V:l and
V:6) by Southern or PCR analysis. These patients did
not have limb muscle weakness. We did not find in
any of our patients the mtDNA 260-bp tandem duplication in the D-loop region described in patients with
large-scale mtDNA deletions [ 181.
To study the deletions in single cells, muscle fiber
segments from Patients IV:2, IV:4, IV:5, and IV:8
were dissected and analyzed by PCR. In several COXdeficient muscle fibers, mtDNA with large deletions
was amplifed. Only one type of deletion was detected
in each muscle fiber segment (Figs 3b-3d). The 854bp fragment, which was amplified by primer pair B,
was not detected in some COX-deficient fibers (see Fig
3b). This fragment is part of a deletion-prone region
and absence of this fragment indicates very low levels
of wild-type mtDNA in the corresponding COX-deficient fiber segments. O n the other hand, the 780-bp
fragment amplified by primer pair A was always present, and served as a control of the PCR. The finding of
unique deletions in each COX-deficient fiber segment
was also visualized by in situ hybridization using different mtDNA probes on consecutive sections from
Patients IV2, IV:4, IV5, and IV:8 (Fig 4). The
COX-deficient fibers usually showed accumulation of
mtDNA transcripts with deletions. The probes identifying transcripts of wild-type mtDNA showed reduced
hybridization in the COX-deficient fibers compared to
surrounding muscle fibers, indicating reduced levels of
wild-type mtDNA in these fibers. In situ hybridization
in Patients V l and V:6 did not show any mtDNA
deletions.
Deletion breakpoints were analyzed by direct sequencing of PCR-amplified fragments of mtDNA from
single muscle fibers and by sequencing after cloning of
PCR fragments of mtDNA from muscle homogenate.
The identified deletion breakpoints are illustrated in
Figure 5. Some of the deletions were found in several
of the affected patients and in several fibers of an individual. All analyzed deletion breakpoints were distributed in the large arc between the origins of replication
of the light and heavy strands. The 4,977-bp deletion
with flanking 13-bp repeats at nt 8470 to 8482 and
nt 13447 to 13459 was observed in many fibers, and
was present in all patients. The size of the deletions,
which were identified by sequencing of the breakpoints, varied from 4,405 to 8,128 bp. A cluster of
breakpoints was observed in the end of the D-loop
region between nt 16069 and nt 16078. The 3' breakpoints of all deletions identified by PCR with primer
pairs L and M (see Fig 3) were located in this region,
Moslemi et al: Clonal Expansion of mtDNA in Autosomal Dominant PEO
709
0
1,
1
1.
2
1.
3
1.
4
1.
5
1.
6
1
7
1
8
1
9
1
ATP.3.
L1AH780
L- 1
C-
1 0 1 1 1 2 1 3 14 15 1 6 k b
1
1
1
1
1
1
1
NO
L 8397C%H 9050
A
H 4933
~5461+--------D
L 8197L8197v-FF-
- - - - - A H
13640
4H
---
L 8901-
16150
E -+H
G -------+H
15260
13640
~890l~-H----4~i484014840
4 H 16150
L
L 7901
L 7901
M
I H 16560
Fig 3. Poolymerase chain reaction (PCR) nnalysis of mtDNA in
Patient IE8. The upper part of thejgure is a linearized map of
mtDNA showing the genes,fir NADH-dehydrogenase (ND) subunits I to 6, qtochrome c oxiduse (COX) rubunits I to [ I , A T P
synthase (ATPase) subunits ciand 8, and cytochromeb (Cyr b).
The filled boxes represent traizsfPr RNA genes. 12s and I6.S represent the genesjor ribosomal RNA. The origin of replication of
the heavy (OH) and the light (01,) strands and thepromoters
fir transcription @the heavy (PH) and light (PL) strands are
indicated by arrows. The lines flanked by arrows illustrate the
primer pairs usedfir PCR analysis. Primer pairs A and B were
used to ampl%j wild-qpe mtDNA. The widely separatedprimer
pairs C to M were used to amp115 mtDNA with deletions. The L
primers constituteparts of the light strand and the Hprimers constituteparts of the hea y strand. The sequences of the primers are
numbered according to the Cambridge mtDNA sequence (201,
with rrucleotide sequences (5'-3') given within parentheses: L I
( I -20), L5461(5461-5480), L7901(7901-7920). L8197
(8197-8216), L890l (8901-8920), H780 (780-761),
H4933 (4933-491 4), H9050 (9050-90-3 I), !113640
(J3640-13621), HI4840 (14840-14821), HI5260
(1.5260-15241), HI6150 (16150-161.~1),and HI6560
(16560- 16541). (a) PCR analysis ofmtDNAfrom muscle
homogenate ofPntient IV.8 shows many arnpliJieclfiagmeats
indic&ng multiple rntDNA deletions. (b-d) PCR amrlysis of
three dzfereiit COX-dejciemt single musclefibers of Patient JV.8
shoim oke deletion in eachfibel: The weak ainpltjication of'
intDNA with primer pair B in OtiEfiber (b) is probably due to
ve91 low levels of wildtype mtDNA and a deletion includiag the
part of mtDNA that correspond to primer H9050.
710
Annals of Neurology
Vol 40
No 5
while the 5' deletion breakpoints of these deletions
were distributed from nt 7943 to nt 8394. In most
cases the deletions were flanked by perfect direct repeats, but this was not a consistent finding, although
repeats could frequently be identified in the vicinity of
the breakpoint regions.
November 1996
Discussion
In this study we demonstrated multiple mtDNA deletions in muscle tissue of a family with adPEO. The
nitDNA with deletions was not randomly distributed
but there were clonal expansions of different deleted
mtDNA species but with only one type in each COXdeficient fiber segment.
Since many signs and symptoms in adPEO, for example, ophthalmoplegia and muscle weakness, are typical manifestations in diseases due to nitDNA mutations, it is possible that there is a direct relationship
between the mtDNA deletions and the clinical manifestations in this family. Multiorgan involvement has
been described also in other families with multiple
nitDNA deletions [5, 21, 221 as well as in diseases
due to single mtDNA deletions [23, 241. However,
the pathogenetic importance of multiple deletions of
mtDNA has been questioned. In one family, multiple
deletions of mtDNA were present in 1 patient affected
with progressive encephalomyopathy, and also in blood
leukocytes of clinically healthy family members [21].
Uncini and coauthors [22] stated that it remains to be
established whether the multiple mtDNA deletions
cause the disease or are merely epiphenomena. O u r results support the concept that multiple deletions are,
at least in muscle, of pathogenetic importance in adPEO. In this study, for the first time, we demonstrated
in a family with adPEO and multiple mtDNA deletions, that mtDNA with multiple deletions is not
randomly distributed. Within a single COX-deficient
muscle fiber segment, only one single deletion was
identified, but different deletions were identified in different muscle fiber segments. This indicates clonal
expansion of mtDNA with a single deletion in each
COX-deficient muscle fiber segment. The nonrandom
distribution of rntDNA with deletions explains the myopathy with frequent COX-deficient muscle fibers.
Since the deletions were not found in muscle tissue of
the young affected individuals, who had not yet developed limb muscle weakness, they probably represent
somatic mutations of mtDNA.
Because of the inheritance in adPEO, a nuclear gene
defect has been proposed to predispose to the development of mtDNA deletions [6]. In a Finnish family
with adPEO, an amosomal locus was identified on
chromosome IOq 23.3-24.3 by linkage analysis [ 101.
This locus was excluded in three Italian families, where
a second locus for adPEO was described on chromosome 3p 14.1-2 1.2, iIlustrating genetic heterogeneity
O H PH
ATPase
m
ND 2
+
OL
I
cox I1
ND
I
ND 4
I I
ND5 ND6
m
Cytb
Fig 4. In situ hybridization using six different mtDNA probes. The probes are illustrated as bars below the linearized map o f
mtDNA in the upper part of the figure. For an explanation of the abbreviations, see Figure 3. The first and last of the consecutive series of sections show enzyme activity of succinate dehydrogenase (SDH) (a, j ) and COX (b, 9. Eight COX-deficient fibers
are indicated and numbered I to 8. These muscle fibers show accumulation of mtDNA transcripts using the N D 2 probe fc), but
due to deletions there is no hybridization with some of the other probes (d-h). The size of the deletions differ between individual
fibers. Fibers 1 and 6: deletion fiom the COX 11 to the N D 4 genes. Fibers 2 and 4: deletion of the COX II gene. Fiber 3: deletion ?om the COX 11 to the N D 6 genes. Fiber 5: deletion from the N D 5 to the N D 6 genes. Fiber 7: deletion fiom the N D 4
to the N D 6genes. Fiber 8: deletion fiom the COX 11 to the Cyt b genes.
in adPEO [25].Our finding of different mtDNA deletions in different fiber segments may indicate that these
deletions are an indirect consequence of a putative nuclear defect, although the mechanism behind the somatic mtDNA deletions remains obscure. Clonal
expansions of mtDNA with multiple deletions resulting in a partial COX deficiency, similar to our
findings in adPEO, are also present in inclusion body
myositis (IBM), which is a sporadic inflammatory myopathy [17, 261. As in adPEO, the multiple mtDNA
deletions in IBM could be a consequence of a putative
nuclear gene defect. Because of abnormal expression of
several proteins in muscle fibers and nuclear structural
changes, a nuclear factor has been proposed to be involved in the pathogenesis of IBM [27].It has been
suggested that clonal expansion of deleted mtDNA
may occur during muscle fiber regeneration in IBM
[17]. The combination of a nuclear factor, which
somehow predisposes to mtDNA deletions, and expansion of clones of mtDNA with deletions during muscle
fiber regeneration could be of importance in both IBM
and adPEO. mtDNA with deletions may also propagate as a result of a replicative advantage over wildtype mtDNA [28]. Because of the reduced function of
mitochondria with deleted mtDNA, a compensatory
proliferation of mitochondria may occur, leading to the
development of ragged-red fibers. Apart from frequent
ragged-red fibers, the pathology of IBM is quite different from that of adPEO since in IBM there is infiltration of inflammatory cells and characteristic inclusions
in muscle fibers. IBM patients usually do not show
PEO. Multiple mtDNA deletions are also observed at
Moslemi ec al: Clonal Expansion of mtDNA in Autosomal Dominant PEO 711
ACTACCACCT (ACCTCCCTCACCA
8469
TTCAACCAAT ( m G G C C G T A C
8981
GATTGAAGCC ( B T T C G T A T A
8029
CCGTATGGCC (WCATAATTAC
8394
CCCCCATACT (CCTTACACTATTCCTC
8416
TACACGACCG ( m A T A C T A C G
8152
CTCTCACTTCA) ACCTCCCTCACCATT
1347
CAAACGCCTG) KQXXATCTATTAC
13557
4977 bp
AWTGACTCA) WCAACAACCGC
16071
8041 bp
CCAAGTATTGACT) W C A T C A A C A C C G
16069
7674 bp
4569bp
ATAGCAACAG) CCTTCATi -3CTATG
15126
6709 bp
TGTTCTTTCAT) GGGGAAGCAGATTT
161u3
7880 bp
CG ATTGAAGC ( m C A T T C m A
AGTUGACTCA) QXATCAACAACCGC
16071
TTGAAGCCCC (CATTCGTATA
A m T G ACT) aCCCATCAACAACC
16069
8031
ACCCTAGCAA (UTCAACCATTAACCTTC . CCTAGCAT) UGCAGGAATACGTT
9069
13475
CCTCTAGAGC (EACTGTAAAGCTAA
AGTATTGACTCA) ECATCAACAACCGC
8293
16071
8042 bp
ma
ATCTTCAACT (KTACATACTTCCC
GTATTGACTCAC) EATCAACAACCGCT
7943
16072
ATTAATTCCC ( C T A A A A A T C T m A A
ATTGACTCA) CCCATCAACAACCGC
8230
16071
ATTGAAGCCC ( W T C m A
A W T G A C T C A ) CCCATCAACAACCGC
8030
la71
AGTTTCATGC (CCATCGTCCT
AGTATTGACT) CACCCAEAACAACC
8207
16068
AAAGCTAACT ( T A G C A T T U T T T T
TCACCCATCA) ACWCGCTATGTAT
8310
1Mml
TAATTCCCCT ( A A A A A T C T I U A A
8232
TGCGACTCCT (TGACGTTGACAAT
7992
A A G T A m C T C A ) CCCATCAACAACCGC
16071
AGTATTGACTCAC) CCATCAACAACCGCT
16072
8037 bp
4405 bp
T7-fbP
8128 bp
7840 bp
8040 bp
7861 bp
7767 bp
7838 bp
8079 bp
Fig 5. List of identifed rntDNA deletions with seqihenres surrounding the breakpoints of the deletions, which are denoted
by parentheses. Some of' the rtnalyzed deletions uiere janked
by direct repem (boldface nnd underlined). One of these
reprats u m included in the ddetiori, arid in the case of pel.fict direct repeats t h e.wct breakpoints could not be determined. Repents in the uirinity of the breakpoints are
underlined. T h size of emh deletiori is given to the right of
each deleted region.
we identified only deletions in the large arc between
the origins of replication. Several deletion breakpoints
were flanked by perfect direct repeats. These findings
are similar to what is generally found in diseases due
to single large-scale m t D N A deletions such as KSS and
PEO, where the deletions have been suggested to be
formed by slipped mispairing during mtDNA replication [33, 3 4 ) . Some deletions in our family showed
repeats, which were present i n the vicinity of but not
exactly at the breakpoints. Such deletions have been
described also in patients with sporadic PEO and KSS
due to single deletions of mtDNA [35-371. We found
a cluster of deletion breakpoints between nt 16069 and
nt 16078. This hot spot for deletion breakpoints in
PEO with multiple mtDNA deletions was also previously reported in adPEO [6] and sporadic P E O with
multiple mtDNA deletions [38], but the pathogenetic
importance of this observation is not clear. We did not
identify the heteroplasinic 260-bp tandem duplication
in the D-loop region described in patients with largescale m t D N A deletions [ 181.
There is evidence from our study that multiple deletions of mtDNA are pathogenic somatic mutations in
adPEO. Potential nuclear genes involved in the formation of secondary multiple mtDNA deletions have been
outlined [lo, 331. These genes are candidates in further
research on diseases for which multiple mtDNA deletions are of pathogenetic importance.
~~
This study was supported by grants from the Swedish Medical Research Council (Projrct no. 07122 and 10823) and the Swedlsh
Asociation for the Neurologically Disablcd (NHR).
The skilled rechnical assistance of Monica Jacobson and llermengild Barrlind is gratefully acknowledged.
low levels during normal aging [29, 301, but COXdeficient muscle fibers are very sparse in normal individuals of old age [ 171.
By in situ hybridization we found accumulation of
transcripts of deleted mtDNA a n d low levels of transcripts of wild-type mtDNA in COX-deficient muscle
fiber segments. This is similar to what has been reported in sporadic P E O and KSS due to single largescale nitDNA deletions [IS, 31, 321. PCR analysis of
mtDNA in single COX-deficient fiber segments
showed a mixture of deleted and wild-type mtDNA i n
many of these fiber segments. O u r PCR analysis did
not allow us to compare the relative amount of wildtype and mutant mtDNA. As in PEO and KSS due
to single nitDNA deletions the occurrence of COX
deficiency did not depend on deletions of COX genes,
and is probably caused by impaired synthesis of all
mtDNA-encoded proteins due to deficiency of transfer
RNAs included in the deletions [28]. By PCR analysis
712
Annals of Neurology Vol 40
No 5
November 1996
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Zeviani M, Servidei S, Gellera C . er al. An autosonial donii-
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