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American Journal of Medical Genetics 71:443–452 (1997)
High Proportions of mtDNA Duplications in
Patients With Kearns-Sayre Syndrome Occur in
the Heart
Bernard Fromenty,1 Rosalba Carrozzo,1 Sara Shanske,1 and Eric A. Schon1,2*
Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York
Department of Genetics and Development, College of Physicians and Surgeons, Columbia University,
New York, New York
Kearns-Sayre syndrome (KSS) is a sporadic
multisystem mitochondrial disorder characterized by progressive external ophthalmoplegia, pigmentary retinopathy, onset
before age 20, and severe cardiac conduction defects that can lead to death. KSS patients harbor partial deletions of mitochondrial DNA (D-mtDNA), sometimes associated
with the corresponding mtDNA duplication
(dup-mtDNA). As reports on the distribution
of dup-mtDNAs among KSS tissues are
scarce, we searched for the presence of dupmtDNAs in different autopsy tissues of two
such patients, one of whom carried the socalled ‘‘common deletion.’’ Using a newly developed long polymerase chain reaction
(PCR) protocol in conjunction with Southern blot analyses, we found dup-mtDNAs
in most of the examined tissues from both
patients. The proportion of dup-mtDNA
in these tissues was much lower than the
proportion of D-mtDNA, with one notable
exception: in both patients, we found an
unusually high level of dup-mtDNA in the
heart. These data suggest that dup-mtDNAs
may be more stable in heart tissue of KSS
patients than in other long-lived postmitotic tissues. Am. J. Med. Genet. 71:443–452,
1997. © 1997 Wiley-Liss, Inc.
Contract grant sponsor: National Institutes of Health; Contract
grant numbers: NS28828, NS11766, AG12131, HD32062; Contract grant sponsor: The Muscular Dystrophy Association; Contract grant sponsor: The Dana Foundation; Contract grant sponsor: The Procter and Gamble Company; Contract grant sponsor:
Ministère Français des Affaires Etrangères.
Bernard Fromenty’s current address is INSERM U24, Hôpital
Beaujon, 92118 Clichy Cedex, France.
*Correspondence to: Eric A. Schon, Department of Neurology,
Room 4-431, Columbia University, 630 West 168th Street, New
York, NY 10032. E-mail: [email protected]
Received 17 October 1996; Accepted 6 March 1997
© 1997 Wiley-Liss, Inc.
KEY WORDS: DNA; KSS; long PCR; mitochondria; mitochondrial disease; progressive external
ophthalmoplegia; polymerase chain reaction
Kearns-Sayre syndrome (KSS) is a sporadic multisystem mitochondrial disorder characterized by the invariant triad of progressive external ophthalmolplegia
(PEO), pigmentary retinopathy, and onset before age
20, with at least one of the following additional manifestations: complete heart block, cerebellar dysfunction, or cerebrospinal fluid protein above 100 mg/dl
[Rowland et al., 1991].
While normal individuals contain a single population
of mtDNAs, KSS patients harbor two populations of
mtDNAs: wild-type mtDNA (wt-mtDNA; 16.6 kb) and
mtDNAs with large-scale rearrangements. The rearrangement is usually a partial deletion of mtDNA (DmtDNA) [Holt et al., 1988; Zeviani et al., 1988; Moraes
et al., 1989]; its size, location, and proportion differs
among patients, and does not appear to be correlated
with the presentation or the severity of the disease
phenotype. One particular species of D-mtDNA, removing 4,977 bp, has been found in about one-third of all
patients studied, and has therefore been called the
‘‘common deletion’’ [Schon et al., 1989; Mita et al.,
1990]. Although it is well known that some KSS patients can harbor both a D-mtDNA and the corresponding partial tandem duplication of mtDNA (dupmtDNA) [Poulton et al., 1989a,b, 1993, 1994; Brockington et al., 1995], there is little information concerning
the tissue distribution of both rearranged species in
such patients.
We recently developed a novel long polymerase chain
reaction (PCR)-based approach that allows for the specific and efficient detection of dup-mtDNAs, even in
tiny amounts of tissue [Fromenty et al., 1996]. We used
this method as a screening tool to search rapidly for the
presence of dup-mtDNAs in different autopsy tissues of
two KSS patients. One of these patients was known to
contain a 26.6-kb species of dup-mtDNA in skeletal
Fromenty et al.
muscle [Fromenty et al., 1996], whereas the second patient was previously thought to harbor only D-mtDNA,
specifically, the common deletion, in the autopsy tissues that were analyzed [Nakase et al., 1990; Shanske
et al., 1990].
We report here a detailed analysis of the distribution
of the various rearranged mtDNA species in autopsy
tissues from both patients, using both long PCR and
Southern blot approaches.
Patient KB had typical KSS and was described in
detail previously [Shanske et al., 1990]. Briefly, this
girl developed pigmentary retinopathy at age 8 and
external ophthalmoplegia and neurosensory hearing
loss at age 13, when a pacemaker was implanted because of a worsening cardiac conduction defect. CSF
protein was 132 mg/dl. At age 19 she developed diabetes mellitus and was confined to a wheelchair. She died
at age 22.
Patient KK also had typical KSS, but in addition had
cerebral lesions consistent with Leigh disease. The
boy’s problems began at age 3, when right ptosis was
noted. At age 6 he developed pigmentary degeneration
of the retinas and mild hearing loss, and external
ophthalmoplegia at age 8. At this age the electrocardiogram showed bifascicular block, which rapidly evolved
into complete third-degree block. When hospitalized for
biventricular pacemaker placement he was also diagnosed with juvenile diabetes mellitus. At age 10 he
began to require a hearing aid. At age 14 he reported
worsening hearing, some muscle weakness, fatigue,
and some tremors, and his speech was noted to be
slurred. He required a wheelchair and could no longer
walk at age 17. He died at age 18.
Isolation of DNA
Total DNA from tissues was isolated from 10–20 mg
of frozen tissues taken at autopsy (3 hours postmortem), as described [Shanske et al., 1990]. Total DNA
was also extracted from the blood of normal subjects.
Long PCR
All long PCR reactions were performed with the
Expand™ Long Template PCR system (Boehringer
Mannheim, Indianapolis, IN) using the manufacturer’s
recommendations, except that ‘‘Buffer 3’’ (10× concentrated buffer containing 22.5 mM MgCl2, 20% DMSO,
1% Tween 20) was always used; enzyme concentration
was reduced to 1.2 units (unless otherwise indicated),
and 200 mg/ml of purified bovine serum albumin (BSA;
New England Biolabs, Beverly, MA) was added to all
reactions. The primer concentration was 300 nM
(equivalent to 15 pmol in a 50-ml reaction volume) unless specified otherwise. Unless otherwise indicated,
long PCR reactions were performed with 10–100 ng of
total DNA in a final volume of 50 ml and in GeneAmp™
thin-walled reaction tubes (Perkin Elmer, Oak Brook,
IL) using a thermal cycler (Perkin Elmer). The tem-
plate used for the long PCR reaction was either uncut
total DNA or total DNA previously digested with the
restriction enzyme PmeI (one site in mtDNA, at nt10414 [Anderson et al., 1981]), in which case 200 ng of
total DNA was digested with 1.6 units of PmeI in a final
volume of 20 ml for 3 hr, followed by heat denaturation
at 65°C for 20 min.
Long PCR reactions were electrophoresed through
0.8% agarose gels containing ethidium bromide. Some
long PCR products were digested with the restriction
enzymes HindIII or PvuII (Boehringer Mannheim),
and the products were visualized in agarose gels.
Long PCR for the Amplification of Wild-Type
and Dup-mtDNAs
Primer pairs FP1 (nt 10364–10377)/BP1 (nt 8783–
8764) and FP2 (nt 13949–13972)/BP2 (nt 8020–7994)
were used for patients KB and KK, respectively, in order to amplify simultaneously both wt- and dupmtDNAs (Fig. 1). The PCR conditions were: initial denaturation at 93°C for 2 min, followed by 30 cycles of 30
s at 93°C, 30 s at 62°C, and 30 min at 68°C, with a final
extension at 68°C for 10 min; the extension time was
increased by 6 s/cycle. Primer pair FP3 (nt 13330–
13350)/BP3 (nt 8582–8561) was also used for both patients KB and KK when the DNA samples were previously digested with PmeI (Fig. 1). The PCR conditions
were as described above, except that the annealing
temperature was 60°C.
Long PCR for the Specific Amplification of
To amplify the dup-mtDNAs specifically, we used a
novel approach employing a forward ‘‘breakpoint’’ or
‘‘breakpoint loop-out’’ primer annealing across the abnormal breakpoint junction, and a backward primer
annealing in the non-duplicated region of the dupmtDNA [Fromenty et al., 1996].
For patient KB, the forward ‘‘breakpoint loop-out’’
primer FP4 was a 22-mer spanning the breakpoint
junction, and which omitted an 18-nt region containing
a 13-bp perfect repeat (see Fig. 1): 58-TTAAACACAAACTACTTGGCAG-38. The first 15 nt of this
primer are specific for the ATPase 8 gene (nt 8450–
8464), whereas the last 7 nt (bold characters) are specific for the ND5 gene (nt 13460–13466). Long PCR
with the forward ‘‘breakpoint loop-out’’ primer FP4
used a ‘‘hemi-nested’’ PCR approach. For the first PCR,
the initial denaturation at 93°C for 2 min was followed
by five cycles performed with 8 pmol of primer FP4 and
backward primer BP4 (nt 9950–9931) and 0.6 units of
enzyme mixture, with denaturation at 93°C for 30 s
and annealing at 38°C for 20 s, followed by a slow increase in temperature (10°C/min) up to 68°C (the extension step), which was maintained for 9 min. At the
end of this first PCR, tubes were kept in ice (or stored
at −20°C) and 5 ml of the PCR reaction were removed
and used as template for the second PCR, which was
performed in a second reaction tube with 8 pmol of
primer FP4 and backward primer BP1 and the following PCR conditions: initial denaturation at 93°C for 2
min, followed by 35 cycles of 30 s at 93°C, 30 s at 58°C,
mtDNA Duplications in KSS
Fig. 1. Description of the rearranged mtDNAs. Shown are circular maps [notation of Anderson et al., 1981 with counterclockwise gene order] of the
dup-mtDNAs and their corresponding D-mtDNAs, as well as the wt-mtDNAs (genome sizes in boxes, in bp). Only the genes (names [ND5 and ND6,
subunits 5 and 6 of NADH dehydrogenase-CoQ oxidoreductase; COX II, subunit II of cytochrome c oxidase; A8/6, subunits 8 and 6 of ATP synthase] in
italics; solid and open boxes) involved in each rearrangement are shown. The origins of replication are indicated by right-angled arrows located outside
(OH) and inside (OL) the circles. The duplicated portion of the mtDNA (D1 and D2 ‘‘pie sections’’) and its size (in bp), and the non-duplicated region (large
D) and its size (16,569 minus the size of the duplication, which is equal to the size of the region deleted in D-mtDNA [small D]), are indicated. The map
positions at the insertion breakpoint boundaries straddle the pie section boundaries. For each D-mtDNA, the deleted region (triangle), its size, and its
breakpoint coordinates, are indicated. Primers (arrows) are located at approximate map positions (not to scale). The restriction sites used in the RFLP
analyses (PvuII at nt-2650; PmeI at nt-10414) are shown. The breakpoint loop-out primer FP4 is denoted by the ‘‘interrupted’’ and ‘‘broken’’ arrows;
breakpoint primer FP5 is denoted by a straight or ‘‘broken’’ arrow, as appropriate. The deletion-specific regional probe (nt 10354–12413) used in the
Southern blot analyses (Fig. 2) is indicated by the thick arc. The bottom panels show the sequences flanking the abnormal breakpoint junctions (brackets)
and their map positions. The sequences of the upstream gene are in plain text; those of the downstream gene are in bold. The perfect 13-bp direct repeats
in KB are in italics. Sequences of the breakpoint primers are shown below the junction sequences and are derived from the underlined areas.
and 11 min at 68°C, with a final extension at 68°C for
10 min. The extension time was increased by 6 s/cycle.
In preliminary experiments, primer FP4, in conjunction with appropriate backward primers, yielded a specific amplification product from a patient [patient GP
in Baerlocher et al., 1992] known to harbor the common
duplication [Poulton et al., 1995a] (data not shown).
For patient KK, the forward ‘‘breakpoint’’ primer
FP5 was a 23-mer spanning the breakpoint junction
entirely, as described previously [Fromenty et al.,
1). The first 16 nt of this primer are specific for the
COX II gene (nt 7820–7835), whereas the last 7 nt (bold
characters) are specific for the ND6 gene (nt 14331–
14337). Forward ‘‘breakpoint’’ primer FP5 was used
with backward primer BP2 and the following PCR conditions: initial denaturation at 93°C for 2 min, followed
by 30 cycles of 30 s at 93°C, 30 s at 64°C, and 15 min at
68°C, with a final extension at 68°C for 10 min; the
extension time was increased by 6 s/cycle.
Fromenty et al.
Southern Blot Hybridization Analyses
One to 3 mg of total DNA were digested with the
restriction enzyme PvuII (Boehringer Mannheim) or
PmeI (New England Biolabs) according to the manufacturer’s recommendations. Gel electrophoresis and
blotting were performed as described [Shanske et al.,
1990]. The blots were hybridized sequentially with two
random-primed 32P-labelled human mtDNA probes derived either from full-length placental mtDNA or from
a 2,060-bp PCR product spanning nt 10354–12413. The
quantitation of the different mtDNA species was performed on the fragments digested with PmeI and hybridized with the total mtDNA probe (see Fig. 2) using
a GS-363 Molecular Imager System (Bio-Rad, Richmond, CA). The densitometric data were corrected for
the sizes of the relevant mtDNA species [Zeviani et al.,
1988], including presumed deletion dimers.
The deletion in KSS patient KK is located between
nt-7836 in the COX II gene and nt-14331 in the ND6
gene [Fromenty et al., 1996], and removes 6,495 bp of
mtDNA (i.e., a partially deleted molecule of 10,074 bp);
the deletion is not flanked by direct repeats (i.e., a
‘‘class II’’ deletion [Mita et al., 1990]). Patient KK also
harbored the corresponding dup-mtDNA, which is
26.6-kb-long (i.e., 16,569 bp + 10,074 bp; see Fig. 1), in
skeletal muscle [Fromenty et al., 1996].
The deletion in KSS patient KB is the ‘‘common deletion’’ [Nakase et al., 1990; Shanske et al., 1990]. It is
located between nt-8483 in the ATPase 8 gene and nt13460 in the ND5 gene, and removes 4,977 bp of
mtDNA (i.e., a partially deleted molecule of 11,592 bp);
the deletion is flanked by a perfect direct repeat of 13
bp (i.e., a ‘‘class I’’ deletion [Mita et al., 1990]). The
corresponding species of dup-mtDNA (henceforth referred to as the ‘‘common duplication’’ for convenience)
is 28.2-kb-long (i.e., 16,569 kb + 11,592 bp; see Fig. 1).
Use of Long PCR to Detect Dup-mtDNAs
In an attempt to amplify the dup-mtDNAs in the
various samples, we used two different long PCR-based
approaches. In our first approach, we used two primers
annealing in the non-duplicated region and pointing
towards the putative duplicated region (FP1/BP1 for
patient KB and FP2/BP2 for patient KK; see Fig. 1),
theoretically allowing for the amplification of both dupmtDNA and wt-mtDNA, but not D-mtDNA [Fromenty
et al., 1996]. Using this method, we were unable to
amplify any PCR product corresponding to a dupmtDNA (i.e., a 26.6-kb or a 20.7-kb fragment in patients KB and KK, respectively) from any of the DNA
samples tested (Figs. 3A and 3B, upper gels). On the
other hand, the wt-mtDNA (15.0 kb and 10.6 kb for
patients KB and KK, respectively) was amplified efficiently (Figs. 3A and 3B, upper gels). Efforts to amplify
the dup-mtDNA species, either by varying the time of
extension and/or the template concentrations [Fromenty et al., 1996] or by linearizing the mtDNA prior
to amplification [Linz et al., 1990; Stewart et al., 1995]
were unsuccessful (data not shown).
In order to overcome these difficulties, we employed
a second long PCR-based approach recently developed
by us [Fromenty et al., 1996]. This novel methodology
uses a forward primer designed to anneal specifically
across the abnormal breakpoint junction (‘‘breakpoint
primer’’) and a backward primer annealing in the nonduplicated region of dup-mtDNA (see Fig. 1), thereby
allowing for the specific amplification of the dupmtDNA without interference from wt-mtDNA templates. The design of the forward breakpoint primers is
dictated by the nature of the sequence at the abnormal
breakpoint junction [Fromenty et al., 1996].
For patient KK, who harbored a class II rearrangement, we used forward breakpoint primer FP5 and
backward primer BP2 (located in the deleted region, so
D-mtDNAs cannot be amplified) to amplify specifically
a 10.3-kb PCR product diagnostic for the 26.6-kb dupmtDNA present in the psoas skeletal muscle [Fromenty et al., 1996 and Fig. 3B, lower gel]; note that
wt-mtDNA was not amplified. We were also able to
amplify this dup-mtDNA from heart, liver, cerebellum,
and frontal lobe (Fig. 3B, lower gel). The yield of this
fragment was higher from skeletal muscle, heart, and
TABLE I. Proportion of mtDNA Species in Autopsy Tissues From Two KSS Patients
Present work
Shanske et al. [1990]
Patient KB
Skeletal muscleb
Smooth muscleb
Frontal lobe
Patient KK
Skeletal muscleb
Frontal lobe
Sum of deletion monomer plus dimer.
Average of two Southern blot experiments.
Fig. 2. Detection of rearranged mtDNAs in KSS tissues by Southern blot hybridization analysis. Total DNA from the indicated tissues was digested
with the indicated enzymes (U, uncut; Pm, PmeI; Pv, PvuII), electrophoresed through a 0.8% agarose gel, transferred to a membrane, and hybridized with
a regional probe from nt 10354–12413 (upper panels) or with total mtDNA probe (lower panels). Markers (M) are HindIII-digested phage l (sizes at
left, in kb). Assignment of the various hybridizing bands (WT, normal length 16.6-kb molecule; Dup, duplicated mtDNA; D, deleted mtDNA monomeric
circle; DD, presumed deletion dimeric circle; D*, linearized deleted mtDNA species derived from the various rearranged mtDNAs) and their sizes (in kb)
is based on the hybridization patterns and the relative positions of migration in the gel. A: Patient KB. B: Patient KK. Sm, smooth muscle; L, liver; K,
kidney; H, heart; FL, frontal lobe; Sk, skeletal muscle; C, cerebellum.
Fromenty et al.
PCR product appeared to be higher in the heart than in
skeletal muscle or kidney (Fig. 3A, lower gel).
The 11.9-kb PCR product was deemed to be derived
from the putative 28.2-kb dup-mtDNA species by three
criteria. First, the length of the long PCR product corresponded to the distance between FP4 and BP1
(11,893 bp), the primer pair used in the second PCR of
our hemi-nested long PCR protocol. Second, the identity of the PCR product was confirmed by RFLP analysis (data not shown). Third, none of the DNA controls
gave an 11.9-kb PCR product (Fig. 3A lower gel and
data not shown), whereas they gave the expected long
PCR products (15.0 kb) under other long PCR conditions (Fig. 3A, upper gel and data not shown).
In addition, we verified that the 11.9-kb PCR fragment produced by our hemi-nested protocol did not derive from a recombination event [Pääbo et al., 1990]
which could theoretically have occurred between the
D-mtDNA and the wt-mtDNA templates during the
PCR reaction. For this purpose we used as a template
a mixture of two DNA samples, one containing nearly
100% of the common deletion (assessed by Southern
blot) and the other containing only wt-mtDNA (3 different PmeI-digested DNA samples were used in three
independent experiments). We did not observe any long
PCR product corresponding to the 11.9-kb fragment,
indicating that ‘‘jumping PCR’’ did not occur with our
long PCR protocol (data not shown).
Quantitation of Rearranged mtDNA Species by
Southern Blot Hybridization
Fig. 3. Detection of rearranged mtDNAs in KSS tissues by long PCR. A:
Patient KB. Upper gel: Long PCR with two primers annealing in the
non-duplicated region in dup-mtDNA (FP1/BP1) and whose 38 ends point
away from each other (i.e., towards the duplicated area; see Fig. 1). Shown
are PCR products from blood DNA from two controls (C1, C2), and from
DNA from smooth muscle (Sm), liver (L), kidney (K), heart (H), frontal lobe
(FL), and skeletal muscle (Sk). The size of the PCR product, in kb, is at
right, and is consistent with that of wt-mtDNA (WT) only. The 0.8% agarose gel was loaded with 6 ml of the 50-ml PCR reaction. Markers (M) are
HindIII-digested phage l (sizes at left, in kb). Lower gel: Specific detection of dup-mtDNA (Dup) using hemi-nested long PCR with ‘‘breakpoint
loop-out’’ forward primer FP4 (i.e., FP4/BP4 followed by FP4/BP1) on
DNAs digested with PmeI prior to PCR. The 0.8% agarose gel was loaded
with 25 ml of the second 50-ml PCR reaction. B: Patient KK. Upper gel:
Long PCR with primers FP2/BP2. Samples include cerebellum (C). Lower
gel: Long PCR with ‘‘breakpoint’’ forward primer FP5 (i.e., FP5/BP2). All
other notation and amounts loaded as in A.
liver than from cerebellum and frontal lobe (Fig. 3B,
lower gel).
For patient KB, who harbored a class I rearrangement, we used a hemi-nested PCR protocol (forward
primer FP4 and backward primers BP4/BP1, annealing in the non-duplicated region of the putative dupmtDNA) to amplify specifically an 11.9-kb PCR product, diagnostic for the 28.2-kb dup-mtDNA, from heart,
skeletal muscle (psoas), and kidney (Fig. 3A, lower gel).
Amplification of this fragment was found to be more
reproducible on mtDNA templates previously linearized with PmeI (Fig. 3A, lower gel). The amount of the
The presence of dup-mtDNAs in some tissues of our
KSS patients was confirmed by Southern blot hybridization analysis of PvuII- and PmeI-digested total DNA
probed with total or regional mtDNA probes. PmeI cuts
at nt-10414, which is located inside the region deleted
in both patients KB and KK, thus allowing one to distinguish between dup-mtDNA and D-mtDNA species.
In patient KB, we found that 4 out of 6 tissues, skeletal
muscle, kidney, frontal lobe, and heart, contained, in
addition to the common deletion, significant amounts
of the corresponding dup-mtDNA, whose proportion of
total mtDNA species ranged from 5% in skeletal
muscle and kidney to 21% in heart (Fig. 2A; Tables I
and II). In patient KK, dup-mtDNA was found in all
tissues studied, ranging from 2% in frontal lobe to 14%
in heart (Fig. 2B, Tables I and II).
It had been thought that dup-mtDNAs are present
infrequently in patients with sporadic KSS [Poulton et
al., 1989a,b], but it has become increasingly clear that
this is not the case, as a significant number of KSS
patients harbor both a D-mtDNA and the corresponding dup-mtDNA [Poulton et al., 1993, 1994, 1995a,b;
Brockington et al., 1995; our unpublished data]. However, few reports exist on the tissue distribution of dupmtDNAs in KSS patients, present either alone or together with the corresponding D-mtDNAs. We therefore examined tissues from two KSS autopsies for the
presence and relative amounts of these two rearranged
species. We note that both of these patients had diabe-
mtDNA Duplications in KSS
TABLE II. Distribution of Rearranged mtDNA Species in Autopsy Tissues From Four KSS Patients
Patient KB
Skeletal muscle
Smooth muscle
Patient KK
Poulton et al. [1995b]
Brockington et al. [1995]
Sum of deletion monomer plus dimer.
Percentage of dup-mtDNAs among all rearranged species (i.e., Dup/[Dup + Del] × 100).
Frontal lobe in patients KB and KK.
tes mellitus, which has also been associated specifically
with the presence of maternally inherited dupmtDNAs [Rötig et al., 1992; Dunbar et al., 1993; Ballinger et al., 1994; Poulton et al., 1995b].
One patient (KK) had recently been found by us to
harbor dup-mtDNAs in skeletal muscle [Fromenty et
al., 1996], but the other tissues had not been evaluated.
We now show that dup-mtDNAs were present in all
examined tissues from this subject (Table I). The other
patient (KB) had been analyzed by us previously
[Shanske et al., 1990], but the analysis had been done
ambiguously: although all KB tissues had been assayed by Southern blot analysis of total DNA digested
with a battery of enzymes (EcoRI, HindIII, PstI, PvuII,
XbaI), none of them were diagnostic for the detection of
a duplicated species. The reassessment of tissues from
subject KB performed here has shown that patient KB
also harbored dup-mtDNAs in significant quantities, in
4 of 6 tissues examined (Table I).
High Levels of Dup-mtDNAs Are Present
in Heart
We made two surprising observations regarding dupmtDNAs in the heart. First, there were high absolute
amounts of dup-mtDNA in this tissue in both patients
(14% in KK, 21% in KB), higher than in any other
tissue examined (Table I). Second, dup-mtDNA represented a significant fraction (47% in KK, 91% in KB) of
all rearranged species in heart, whereas in the other
tissues the dup-mtDNAs were in the minority (ranging
from 0% in smooth muscle and liver in KB to 39%
in cerebellum in KK [see Table II]). Only two other
papers report the proportion of dup-mtDNA in heart
tissue from KSS patients harboring duplications
[Brockington et al., 1995; Poulton et al., 1995b]. In
those patients, the absolute amount of total rearranged
mtDNAs (i.e., dup- plus D-mtDNAs) was lower (17%
[Brockington et al., 1995] and 14% [Poulton et al.,
1995b]) than the levels found in KB and KK (23% and
30%, respectively), but the proportion of rearranged
molecules that were dup-mtDNAs was still higher in
the heart (41% [Brockington et al., 1995] and 43%
[Poulton et al., 1995b]; see Table II) than in all other
tissues examined, save one.
The pathogenic significance of dup-mtDNAs remains
uncertain. The deleterious effects of D-mtDNAs are
probably due to the lack of the indispensible tRNAs
which are required for translation [Nakase et al., 1990;
Hayashi et al., 1991], but dup-mtDNAs contain all 37
mtDNA-encoded genes, albeit with some in excess.
Thus, dup-mtDNAs might be pathogenic in an indirect
way. For example, they might generate the corresponding D-mtDNA species via a recombination event, at
least in some tissues [Poulton et al., 1994, 1995a]. Alternatively, transcription and/or translation of the fusion gene formed at the duplication breakpoint might
interfere with normal mitochondrial function (note
that the possibility of formation of aberrant translation
products also applies to the corresponding D-mtDNA,
but only in those situations where other mtDNAs
within the organelle, either wt- or dup-mtDNAs can
provide the requisite tRNAs). It may even be that duplications are not pathogenic at all, or are somehow
‘‘less pathogenic’’ than deleted mtDNAs, on a permolecule basis.
Patients KB and KK presented with severe cardiac
conduction defects, necessitating implantation of a
pacemaker. The patients described by Brockington et
al. [1995] and Poulton et al. [1995b] suffered from
milder heart block and had lower proportions of rearranged mtDNA species in their hearts than did KB or
KK (Table II). These data suggest that the overall mutant load correlates with the severity of the cardiac
conduction defect in KSS patients. Alternatively, it is
possible that the conduction defects are due to the focal
presence of D-mtDNAs in the conduction system, as
hypothesized previously [Remes et al., 1992]. It is noteworthy in this regard that in skeletal muscle from a
patient with a late-onset myopathy who harbored dupmtDNAs as the major rearranged mtDNA species in
this tissue, the few respiratory-deficient fibers that
were present contained almost exclusively D-mtDNAs
[Manfredi et al., 1997].
Taken together, our data extend previous reports
[Brockington et al., 1995; Poulton et al., 1995b] that the
proportion of dup-mtDNAs can vary greatly among tissues. Furthermore, it seems that the fraction of total
rearranged mtDNAs that are dup-mtDNAs is consistently high in heart, whereas this fraction is low in
other examined tissues (Table II).
Factors Controlling the Steady-State Levels of
Rearranged mtDNAs
The above observation suggests that dup-mtDNAs
may be more stable in the heart than in other longlived postmitotic tissues, such as skeletal muscle and
Fromenty et al.
brain. For example, it had been shown that the proportion of dup-mtDNAs in skeletal muscle in KSS patients
fell over time [Poulton et al., 1993, 1995a], resulting in
low proportions of dup-mtDNAs and high levels of
D-mtDNAs in this tissue at the time of death (Table II).
Unfortunately, the factors controlling the generation,
stability, and turnover of dup-mtDNAs are not known.
At least four possibilities come to mind.
First, the size of the molecule itself may affect stability, with large dup-mtDNAs being less stable than
smaller ones [Poulton et al., 1994, 1995a]. However,
the 28.2-kb common duplication is one of the largest
dup-mtDNAs described to date, but was present at
high levels in the heart of patient KB (Table I).
Second, the cell type may affect stability and turnover of dup- and D-mtDNAs. Cardiac cells, unlike skeletal muscle fibers, are not syncytia, and they also have
greater oxidative energy requirements. For these two
reasons, it is reasonable to assume that the time period
necessary for D-mtDNAs to accumulate and reach levels causing cellular dysfunction in cardiac cells is
shorter than in skeletal muscle, and that the threshold
level of D-mtDNAs required to cause such dysfunction
is lower in heart than in skeletal muscle. Cells appear
to sense the total number of mitochondrial genomes
they contain and to regulate their numbers based on
their particular energetic requirements [e.g., see King
and Attardi, 1988; Tritschler et al., 1992], although the
mechanisms by which this occurs are obscure. Thus, as
the level of dup-mtDNAs falls, the level of D-mtDNAs
plus wt-mtDNAs should rise, and vice versa. If cardiac
tissue can indeed tolerate fewer D-mtDNAs than can
skeletal muscle, this ‘‘lever principle’’ could then explain why dup-mtDNAs are elevated in this tissue.
Third, it is possible that dup-mtDNAs are generated
relatively more frequently in heart than are DmtDNAs, perhaps because cardiomyocytes contain extremely high numbers of mitochondria and mtDNAs
[Shoffner and Wallace, 1995]; this environment might
favor organellar fusion and/or intermolecular recombination events in which duplicated molecules are generated. There are no data in support of such a scenario,
and no such advantage of duplicated molecules over
deleted molecules was observed in a prokaryotic model
of the generation of deletions and duplications between
direct repeats [Trinh and Sinden, 1993, and references
Finally, the rate of cell division, together with the
respective rates of replication of the different rearranged species present in the cell, may also affect stability of the various mtDNA species [but see Moraes
and Schon, 1995]. Replication of wild-type mtDNA requires two origins of replication [Clayton, 1992], one,
near nt-200, priming heavy-strand synthesis (OH) in
one direction and the other, near nt-5750, priming
light-strand synthesis (OL) in the opposite direction. A
dup-mtDNA usually contains not two, but four, replication origins (2 OHs and 2 OLs); note that the dupmtDNAs in patients KB and KK fall into this category
(see Fig. 1). The proportion of such a ‘‘4-origin’’ dupmtDNA fell rapidly in both uncloned and cloned fibroblasts [Poulton et al., 1993], implying that a dupmtDNA containing two pairs of replication origins
might have difficulty in replicating. On the other hand,
the proportion of a dup-mtDNA containing two OHs but
only one OL (i.e., the corresponding D-mtDNA contained OH but lacked OL) increased during passage in
transformed lymphoblasts [Ballinger et al., 1994].
While the absence of OL in a D-mtDNA should impair
or even prevent the ability of such a molecule to replicate, the presence of a single functional OL in a ‘‘3origin’’ dup-mtDNA might actually compensate for any
potential impairment of this molecule’s ability to replicate. In support of this idea, another patient harboring a different ‘‘3-origin’’ dup-mtDNA with only one OL
[patient CH in Fromenty et al., 1996] contained an unusually high proportion of dup-mtDNA and a very low
amount of the corresponding D-mtDNA (with no OL) in
skeletal muscle [Manfredi et al., 1997].
Long PCR Can Amplify
Dup-mtDNAs Specifically
The long PCR results reported here were confirmed
by Southern blot analysis. However, we consistently
had problems trying to amplify dup-mtDNA in the
frontal lobe of patient KB, even though Southern blot
analysis demonstrated the presence of dup-mtDNA in
this tissue. While we cannot omit the possibility that
contaminating elements inhibited PCR amplifcation, it
may also be that brain tissue is particularly difficult to
analyze. Indeed, long PCR amplified dup-mtDNAs efficiently from other tissues containing similar proportions of this rearrangement (e.g., 5% in kidney and
skeletal muscle in patient KB; Fig. 3A, lower gel; Table
I). Similarly, although dup-mtDNA-derived long PCR
products were obtained from the cerebellum and the
frontal lobe DNA of patient KK, the yields were lower
in those tissues than in other tissues harboring essentially similar proportions of dup-mtDNAs, such as
liver and skeletal muscle (Fig. 3B, lower gel; Table I).
We note that dup-mtDNAs are very long molecules
which may be more prone to damage (e.g., oxidation,
depurination, chain scission) than are shorter wt- or
D-mtDNA templates, either in vivo or in vitro (e.g.,
following preservation of the brain tissue or during the
isolation of the DNA). Such damage could lead in turn
to a dramatic decrease in the efficiency of long PCR,
which requires high template integrity [Barnes, 1994;
Cheng et al., 1994]. It is therefore conceivable that
brain tissues or brain-derived DNA contained a high
proportion of ‘‘Southern blot-observable’’ dup-mtDNA
templates, and yet contained so few intact full-length
molecules that long PCR could not amplify them. Although we do not know the lower limit of detection of
dup-mtDNAs by long PCR, we note that we were able
to use the ‘‘breakpoint loop-out primer’’ method to amplify a short 5.5-kb fragment derived from a 21.2-kb
class I mtDNA duplication in a sample of blood DNA
containing less than 1% dup-mtDNA [Fromenty et al.,
Since about one-third of all KSS/PEO patients harbor the common deletion [Schon et al., 1994], a long
PCR method for the specific amplification of the corresponding 28.2-kb common duplication should be useful
in detecting this prevalent rearrangement, especially
mtDNA Duplications in KSS
when the amount of DNA available is a limiting factor
(e.g., analysis on endomyocardial biopsy specimens).
The common deletion is also found at low levels in tissues (mainly brain, skeletal muscle, and heart) of aged
individuals [Cortopassi et al., 1992; Schon et al., 1996;
Pallotti et al., 1996]. However, the real nature of this
mtDNA rearrangement in aged tissues is not known,
because the standard PCR methodologies previously
used for their detection cannot differentiate a
D-mtDNA from its corresponding dup-mtDNA [Fromenty et al., 1996]. Our novel approach to amplify dupmtDNAs in general, and the 28.2-kb common duplication in particular, should help clarify this important
In conclusion, we have used long PCR and classic
Southern blotting to detect dup-mtDNA species in different tissues of two KSS patients. Our data confirm
that the relative amounts of D- and dup-mtDNAs vary
widely among tissues, making the relative contribution
of each species of rearranged mtDNA to the overall
clinical picture even more difficult to sort out than had
been appreciated previously, as heteroplasmy for both
rearranged mtDNA species raises obvious questions regarding the relative pathogenicity of each molecule.
Furthermore, it seems that dup-mtDNAs are more
stable in the heart of KSS patients than in other longlived postmitotic tissues. Our long PCR methodology
for the specific amplification of dup-mtDNAs could be a
convenient and powerful tool to help attack these problems.
We thank Drs. E. Bonilla, S. DiMauro, M. Hirano,
and A. Rötig for providing tissues, and Drs. M. P. King
and J. Poulton for helpful comments. B.F. was supported by the Lavoisier Fellowship from the Ministère
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