association of myopathy with large-scale mitochondrial dna duplications and deletions: which is...

Association of Myopathy with Large-Scale Mitochondria1 DNA Duplications and

Deletions: Which Is Pathogenic? Giovanni Manfredi,*t Tuan Vu,*t Eduardo Bonilla,*l- Eric A. Schon,*tf Salvatore DiMauro,*i

Enrica Arnaudo,*? Lee Zhang,*t Lewis P. Rowland,*? and Michio Hiram*?

We identified large-scale heteroplasmic mitochondrial DNA (mtDNA) rearrangements in a 50-year-old woman with an adult-onset progressive myopathy. The predominant mtDNA abnormality was a 21.2-kb duplicated molecule. In addition, a small population of the corresponding partially deleted 4.6-kb molecule was detected. Skeletal muscle histology revealed fibers that were negative for cytochrome c oxidase (COX) activity and had reduced mtDNA-encoded COX subunits. By single-fiber polymerase chain reaction analysis, COX-negative fibers contained a low number of wild-type or duplicated mtDNA molecules (ie, nondeleted). In situ hybridization demonstrated that the abnormal fibers contained increased amounts of mtDNA compared with normal fibers and that most of the genomes were deleted. We concluded that deleted mtDNA molecules were primarily responsible for the phenotype in this patient.

Manfredi G, Vu T, Bonilla E, Schon EA, DiMauro S, Arnaudo E, Zhang L, Rowland LP, Hirano M. Association of rnyopathy with large-scale rnitochondrial DNA duplications and deletions:

which is pathogenic? Ann Neurol 1997;42: 180-188

Large-scale mitochondrial DNA (mtDNA) rearrangements have been associated with disorders of mitochondrial metabolism affecting muscle and other tissues. Partial mtDNA deletions were first described in 1988 11-31 in patients with Kearns-Sayre syndrome (KSS), a progressive multisystemic disease characterized by onset before age 20, ptosis, ophthalmoplegia, pig- mentary retinopathy, heart block, ataxia, elevated cere- brospinal fluid (CSF) proteins, and diabetes mellitus [4, 51. In 1989, Poulton’s group first reported the association of KSS with heteroplasmic mtDNA tandem duplications [6]. It was later demonstrated that duplications and deletions coexisted in a subset of KSS patients [7, 81. Other investigators reported different clinical phenotypes associated with mtDNA duplications, including renal tubulopathy, cerebellar ataxia, and diabetes mellitus [9]; chronic progressive external ophthalmoplegia (CPEO), myopathy, and diabetes [ 101; and diabetes and deafness [ 1 I , 121. Diabetes is a frequent clinical feature in patients with mtDNA duplications.

Low amounts of a relatively small heteroplasmic (260-bp) duplication located within the D-loop region

of the mtDNA have been reported in patients with KSS who also harbored large-scale deletions [ 131. Higher amounts of the same 260-bp duplication, without the corresponding deletion, were thought to be pathogenic in a patient with myopathy [ 141. Because this mtDNA rearrangement has also been reported to be present in very low abundance in elderly individuals [15] and in normal whites [16], the pathogenicity of this particular mtDNA duplication remains unclear.

Unlike mtDNA deletions, which are generally spo- radic, duplications are frequently maternally transmitted [9-11]; maternal inheritance was also present in some of the pedigrees with the 260-bp duplication [13]. The origin and the pathogenic significance of large-scale mtDNA duplications also remain unclear.

Herein, we report the unusual observation of a large- scale mtDNA tandem duplication and small amounts of the corresponding deletion in a woman with a pure myopathy. She did not show any symptoms or signs of multisystemic involvement and did not have diabetes mellitus. We also attempted to determine the pathogenic significance of the two species of mtDNA rearrangements.

From the *H. Houston Merritt Clinical Research Center for Mus- cular Dystrophy and Related Disorders, and Departments of tNeu- rology and $Generics and Dcvelopment, Columbia University Col- lege of Physicians and Surgeons, New York, NY.

Received Sep 26, 1996; and in revised form Jan 13, 1997. Accepted for publication Jan 22, 1937.

Address correspondence to Dr Manfredi, Department of Neurology, Room 4-431, Columbia University, 630 West 168th Street, Ncw York, NY 10032.

180 Copyright 0 1997 by the American Neurological Association

Patients and Methods Patient The patient was normal in childhood and through adoles- cence. She was active in games and sports in school. At age 20, she first noted difficulty in climbing stairs, rising from low chairs, and running. She complained of myalgias, which were worse after exercise. These symptoms progressed slowly. Presently, at age 51, she has difficulty getting in and out of a car and reaching for objects above her head. She never complained of dysphagia, dysarthria, ptosis, or double vision. A neurological examination revealed mild facial and neck flexor muscle weakness, decreased bulk and strength against moderate resistance of the proximal arm and leg muscles. There was no ptosis or ophthalmoparesis. Sensory examination was normal. Tendon reflexes were present. Babinski and Hoffman signs were absent. Routine laboratory tests, including creatine kinase, blood glucose, glycohemoglobin, and a glucose tolerance test, were normal. Standard audiologic evaluation was normal. Venous lactate at rest ranged from normal to 3.1 mM (control values, 0.5-2.2 mM). The patient performed a cycle ergometry test. She attained a workload of 25 W for 58 seconds, before premature interruption of the rest due to the onset of dizziness. At the exercise peak, heart rate was 80% of predicted, blood pressure was 184/85 mm Hg, and oxygen consumption was only 37% of predicted. With this mild exercise, arterial lactate rose ninefold above the baseline level. Nerve conduction studies revealed normal motor and sensory amplitudes and conduction velocities. Electromyography did not reveal spontaneous activity, and recruitment patterns were nondiagnostic.

The patient has two sons and a daughter, all of whom are normal in stature and build. They have no evidence of diabetes, hearing loss, myopathy, ptosis, or ophthalmoparesis. The patient's two brothers (age, 54 and 57 years) are healthy. The patient's mother died at age 83 without history of muscle weakness. The patient's maternal aunt developed a waddling gait, ptosis, and dysarthria 2 months before death at age 84. The son of that aunt had a normal neurological examination at age 62 and died 1 year later of a myocardial infarction.

Methods A biopsy was obtained from the deltoid muscle for diagnostic purposes, with the patient's informed consent. All studies were conducted on discarded pathology samples under a Columbia-Presbyterian Medical Center institutional review board protocol.

Mitochondria1 enzyme activities in the patient's whole muscle homogenate were measured as described previously [ 171. Eight-micrometer-thick frozen sections were used for histochemical analyses. Staining for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activities were performed as described [ 18, 191. Indirect immunofluores- cence staining to detect COX subunit I1 (COX 11) and COX subunit IV (COX IV) was performed as described [20].

We used restriction fragment length polymorphism (RFLP) screening of polymerase chain reaction (PCR) products to search for the most commonly encountered pathogenic mtDNA mutations in MELAS (mitochondria1 enceph- alopathy, lactic acidosis, and stroke-like episodes; mutations

in the tRNAL'"'UUK' gene at nucleotide [nt] 3,243 and nt 3,271), M E W (myoclonus epilepsy with ragged-red fibers [RRFs]; mutations in the tRNALYs gene at nt 8,344 and nt 8,356), and NARP (neuropathy, ataxia, and retinitis pigmentosa; mutations in the ATPase 6 gene at nt 8,993). PCR screening for the 260-bp D-loop duplication was performed according to the protocol described previously 11 3, 141.

For Southern blot analysis, total DNA was extracted from muscle and blood [3]. Five micrograms of total DNA was digested with the restriction enzymes PvuII, BamHI, or SnaBI. Gel electrophoresis and blotting were performed as described [3]. Two random-primed j2P-labeled DNA fragments corresponding to nt 1,227-2,896 and nt 11,680- 12,570 of the human mtDNA sequence [al l were used as probes 1 and 2, respectively. The relative proportions of the radiolabeled PCR fragments were quantitated by scanning the membrane in a PhosplhorImager Model SF by Molecular Dynamics (Sunnyvale, CA). Quantification of muscle mtDNA on Southern blots was performed by hybridizing PvuII-digesred total DNA with "P-labeled probes of a nuclear-encoded 18s ribosomal DNA fragment and of purified whole human mtDNA, as described [22].

Mapping of the duplication junction point was performed by PCR amplification of the patient's muscle mtDNA. A series of PCR reactions was performed using a single backward primer, located at nt 15,870-15,849 [21], together with four different forward primers (located at nt 3,470- 3,492, 4,185-4,208, 5,2(;0-5,289, and 5,685-5,703). The following PCR conditiom were used in all experiments: 25 cycles at 9 4 T , 1 minute; 55°C 1 minute; 72°C 2 minutes; 10-minute final extension at 72"C, in the presence of [a-"P]dATP.

For DNA sequencing, PCR products were isolated from an agarose gel with a Wizard PCR-preps DNA purification system (Promega Inc, Madison, WI) and direct sequencing was performed using an fmole DNA sequencing kit (Pro- mega Inc), according to the manufacturer's protocol.

Single-Fiber PCR Based on their histochemical phenotype, muscle fibers were classified as COX-positive (COX') or COX-negative (COX-). Dissection and lysis of single muscle fibers were performed as described [23, 241. The PCR amplification was conducted using primers P3 (nt 8,657-8,685) and P4 (nr 9,298-9,276), which yielded a 642-bp fragment from the unduplicatedldeleted mtDNA region (see Fig 4). The amplification products obtained from a single fiber were electro- phoresed through a 6% nondenaturing polyacrylamide gel and subjected to autoradiography.

The amplified fragment was also gel purified from low- melting agarose gel by using a Wizard PCR-preps DNA purification system and was inserted into a pGEM-t phagemid bacterial vector from Promega Inc. Tenfold serial dilutions of the plasmid (in amounts corresponding to 15, 1.5, 0.15, and 0.015 pg of mtDNA), were used to verify the linearity of the PCR amplification in this range of template DNA concen- trations and as standards to estimate the PCR yield. Total DNA extracted from human cells devoid of mtDNA (po cells [25]) was added in a 100:l mass ratio (nuclear DNAlplasniid DNA) to supplement the system with a nuclear genomic

Manfredi et al: Large-Scale Mitochondria1 DNA Rearrangements 181

background. Before PCR amplification, the DNA standards were submitted to the same procedures as those used for the muscle fibers. Amplification and gel electrophorebis of the standards were performed simultaneously and under the same conditions as for the single fibers.

I% Situ Hybridization In situ hybridization was performed as described (261 with some modifications. In brief, 8-pm-thick muscle sections were fixed in 4% paraformaldehyde for 45 minutes, washed in distilled water, and dehydrated in graded alcohol solu- tions. After a wash in phosphate-bufferrd saline (PBS) containing 5 mM MgCI,, the sections were exposed to protein- ase K (10 pgiml) for 45 minutes, PBS washed, acetylated, and treated with DNase-free RNase from Boehringer- Mannheim (Indianapolis, IN). The sections were prehybrid- ized for 2 hours at 42°C in a solution containing 50%) for- mamide, 0.6 mM NaCI, 20 mM Tris-HC1, pH 7.5, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.12% bovine serum albumin, 1 mM EDTA, 0.5 mgiml herring sperm DNA, 10% dextran sulfate, 0.5 mg/ml total yeast DNA, and 0.01 pg/ml yeast rRNA. Probes were prepared by PCR using two pairs of oligonucleotide primers corresponding to mrDNA positions nt 1,690-1,711 and 2,447-2,426 and to nt 9,175- 9,198 and 9,764-9,740 (probes 3 and 4, respectively, in Fig 4A). Digoxigenin-dUTP from Boehringer-Mannheim was incorporated into the probes during PCK. The labeled PCR products were purified from an agarose gel by using a Wiz- ard PCR-preps DNA purification system and were quantitated by dot blot. Specificity of these probes was confirmed by Southern blot, The probes were added to the sections and denaturated at 92°C for 10 minutes. The sections were then hybridized at 42°C overnight. After washing in 1X saline- sodium citrate (SSC) twice, 0.2X SSC, and then in washing buffer (0.3% Tween 20 in 0.1 M maleic acid buffer, pH 7.5), detection was performed using a Genius Kit from Boehringer-Mannheim, according to the manufacturer's in- structions. Control sections included nondcnatured tissues and nondenatured probes. Images of sections were captured with a Dage-MTI CCD-72 digital camera from MTI (Mich- igan City, IN) and analyzed using an NIH Image software package (version 1.59). All images were analyzed in one ses- sion. Optical density was expressed in arbitrary units, relative to a standardized gray scale. Serial muscle sections, immedi- ately above and below those used for the in situ hybridization, were stained for COX and SDH activities. Fiber type was determined from serial sections treated with ATPase stain at p H 9.4.

Results Muscle Biochemistry, Morphology, and Immunocytochemistry Mitochondria1 respiratory chain enzyme activities on whole muscle homogenate were normal (not shown). However, the muscle biopsy revealed the presence of numerous nonatrophic COX- fibers (Fig lB) , a proportion of which stained very intensely for SDH (Fig lA), indicating abnormal mitochondria1 proliferation (ie, RRFs). We did not observe any COXt RRFs. The overall percentage of COX- fibers was approximately 10% in the muscle sections examined, which is higher than age-matched controls, who have < I % [27]. There was no muscle fiber atrophy, necrosis, or inflam- mation. Besides the mitochondria1 alterations, he- matoxylin-eosin, nicotinamide adenine dinucleotide- tetrazolium reductase, and modified Gomori trichrome stains showed normal cytoarchitecture without a loss of muscle fibers.

Serial muscle sections were stained for COX activity and with anti-COX I1 (mtDNA-encoded) or anti- COX IV (nuclear DNA-encoded) antibodies. All COX- fibers showed a strong signal for COX IV, whereas immunoreactivity for COX I1 was reduced (Fig 2A-C), indicating a selective decrease of the mtDNA-encoded polypeptides.

DNA Studies PCR-RFLP analysis failed to reveal the presence of known pathogenic point mutations at nt 3,243, 3,271, 8,344, 8,356, or 8,993. The 260-bp D-loop duplication was also absent by PCR.

Southern blot analysis of muscle DNA digested with PvuII and probed with a radiolabeled DNA fragment corresponding to mtDNA positions 1,227 to 2,896 (probe 1) revealed two bands, one, 16.6 kb in size, corresponding to wild-type mtDNA, and the other, 4.6 kb in size, corresponding to a mtDNA with an osten- sible 12.0-kb deletion (Fig 3). However, with BamHI (not shown) and SnaBI digestions, the same probe revealed the 16.6-kb band and a 21.2-kb band, whereas the 4.6-kb band was not detectable (see Fig 3) , demonstrating that the rearranged mtDNA was predomi- nantly a 4.6-kb duplication, not a deletion. A band of

Fig I . Examples of histochemical and in situ hybridization studies on muscle serial sectionr. (A) Succinate dehydrogenase (SOH) staining illustrates three raged-red fibers (RRFs) (labeled with numbers 1-3). (B) Cytochrome c oxidase (COX) staining shows that these RRFs are COX-negative (COX--). (C) In situ hybridization to detect mtDNA using probe 3 (I6S, nucleotide [nt] 1,690- 2,443, corresponding to a nondeleted region of mtDNA. (0) In situ hybridization to detect mtDNA using probe 4 (COX Ill, nt 9,175-9,764), corresponding to a deleted region o f mtDNA. (E) Optical densities o f the numberedfibers and of a sample of normal jbers (with standard errors), expressed as arbitraiy units relative to a standard gray scale. (F) Example of polymerase chain reaction (PCR) amplifcation of wild-ype rind duplicated mtDNA (nondeleted mtDNA) fiom COXt and COX- single fibers. Serial dilutions of a plasmid containing tbt, 642-bp fiagment o f interest were used as standards. C, control PCR without template. M, molecular weight marker of Haefll-digested X I 73 (sizes in bp at I&).

182 Annals of Neurology Vol 42 No 2 August 1997

* r

E Fibers

DNA standards, pg M 15 1.5 .15 .015 C COX+ fibers COX- fibers


probe 1 probe 2 Normal Patient Patient

Fig 3. Southern blot analysis of total DNA isolated fiom a normal individual; muscle and from the patient j muscle, digested with Pvu I1 and SnaBI, and probed with a radiolabeled DNA fiagment corresponding to nucleotide (nt) 1,227- 2,896 (probe I ) and to nt 11,680-12,570 (probe 2) of the human mtDNA sequence. U, uncut DNA. M, molecular weight marker o f HindIII-digested phage h fiizes at left). Sizes of wild-type and rearranged mtDNAs are indicated at right, in kb. 166 kb = wild-type mtDNA; 21.2 kb = duplicated mtDNA; 4.6 kb = fragment derivedfiom PvuII digestion o f both duplicated and deleted mtDNA species. A band migrating above the wild-type mtDNA in the patient; U and SnaBI lanes is indicated (7. This band is detectable after hybridization with probe 1, but not with probe 2, indicating that it corresponds to uncut deleted mtDNA monomers or dimers.

Fig 2. Serial sections from the patient? muscle. (A) Cyto- chrome c oxidase (COX) staining (B) Imnzunostaining with anti-COX IV subunit antibodies. (C) Imwcunostaining with anti-COX II subunit antibodies. The indicated COX-negative fibers (stars) reacted intensely with anti-COX IV antibodies, but they were negative with anti-COX II antibodies.

weak intensity was observed above the wild-type and the duplicated bands, Probably corresponding to an uncut molecule (see Fig 3). The membrane was subse- quently stripped and reprobed with a radiolabeled DNA fragment corresponding to nt 11,680-12,570 (probe 2). This probe revealed the 16.6-kb wild-type and the 21.2-kb duplicated molecules, whereas the more slowly migrating bands were not detectable (see

184 Annals of Neurology Vol 42 No 2 August 1997

Fig 3), indicating that they were deleted molecules, possibly nicked circles or deletion dimers. Therefore, the 4.6-kb bands, which was revealed after PvuII digestion and hybridization with probe 1, represented mainly the portion of the duplicated region between the two PvuII sites and corresponded to the size of the duplication (Fig 4A). In addition, a small proportion of this 4.6-kb band was derived from the linearized deleted molecules.

Of the patient’s muscle mtDNA, 40% was duplicated and the deleted molecules were approximately 1%. In the patient’s blood, the duplication was too low for accurate quantitation and deletions were undetect- able by Southern blot analysis (not shown). The total mtDNA content in the patient’s muscle was normal compared with aged-matched controls (not shown). The proportion of the duplication was 78% in the patient’s son’s blood, but no deletion was detectable (not shown). Blood mtDNA from the patient’s other two offspring, her two brothers, and her maternal uncle did not reveal any large-scale rearrangements by Southern blot. We were unable to procure skeletal muscle from these relatives.

PCR analysis using the backward primer at nt 15,870-15,849 (primer 2 [P2]) and three forward primers located at nt 4,185-4,208, 5,260-5,289, and 5,685-5,709 failed to generate an amplification product, because the DNA segment between the forward and the backward primers was too large to amplify (1 1,685, 10,610, and 10,185 bp, respectively); how-

located at nt 3,470-3,492 (primer 1 [Pl]), a 431-bp DNA fragment was amplified, presumably from a rearranged mtDNA molecule (see Fig 4A). PCR did not yield any product from the wild-type mtDNA, because

WT-mtDNA

ever, using P2 in conjunction with a forward primer P3 *

h Fig 4. (A) Diagrams o f the wild-iype and of the rearranged mtDNA molecules. Shown are circular maps o f the duplicated mtDNA (Dup-mtDNA) and its corresponding deleted mtDNA (A-mtDNA) a~ well as the wild-type mtDNA (WT-mtDNA). The A-mtDNA harbors a 11,969-bp deletion ftviangle) that encompasses the origin of light-strand replication (OJ. The 3568-3577 pie sections” in the Dup-mtDNA represent the 4,GOO-bp re- C

c gions that are duplicated in tandem. Only the genes involved in the rearrangement junctions (ie, subunit 1 of NADH- dehydrogenase-coenzyme Q-reductase (NDl] and cytochrome b (Cyt b]) are shown. Poiymerase chain reaction primers (PI, nucleotide [nt] 3,470-3,492; P2, nt 15,870-15,849; P3, nt 8,657-8,685; P4, nt 9,298-9,276) are indicated. Pvuh’, BamHI, and SnaBI restktion sites are shown. The probes used for the Southern blots and in sitn hybridizations (num-

(B) DNA sequence (G, A, T, C) of the duplication junction region. The 10-bp sequence common to N D l and Cyt b is boxed; it corresponds to nt 3,568-3,577 in the N D l gene and to nt 15,537-15546 in the Cyt b gene.

C - 3567

bers in boldfice) are also indicated. Map is not to scale. 3


the DNA segment between P1 and P2 was too large (1 2,130 bp) to amplify.

DNA sequencing of the 431-bp fragment showed normal sequence until nt 3,577, at which point the ND1 gene sequence was followed by Cyt b gene sequences commencing at nt 15,547 (see Fig 4 B ) . The rearrangement junction contained one of two copies of a 10-bp perfect direct repeat located, in wild-type mtDNA, at nt 3,568-3,577 in the ND1 gene and at nt 15,537-15,546 in the Cyt b gene (see Fig 4B).

PCR screening using primers P1 t- P2 (see Fig 4A), which were specific for the rearranged region, amplified the abnormal 431-bp fragment only from the muscle and blood of the patient and from the blood of her youngest son, but not from blood of the other two offspring, two brothers, or maternal uncle of the patient.

Single-fiber PCR analysis was used to determine whether there was any difference in the content of wild-type plus duplicated mtDNA (nondeleted mtDNA) between COX’ and COX- fibers. Results of a typical single-fiber PCR experiment are shown in Figure 1F. In the experiment shown in Figure 1, the standard amplification curve obtained from plasmid DNA containing the fragment of interest showed that, for DNA amounts between 0.0 15 and 15 pg, the yield of the PCR amplification was linearly correlated with the template amount. All of the COXt fibers had between 0.15 and 1.5 pg of nondeleted mtDNA, whereas the content of nondeleted mtDNA in the COX- fibers was approximately 10-fold lower, between 0.0 15 and 0.15 pg. In most COX- fibers amplified (25 in total), the content of nondeleted mtDNA was severalfold lower than in the COXt fibers (total 16). In three COX- fibers, the content o f nondeleted mtDNA was similar to that in COXt fibers (not shown). In these single-fiber analyses, the amount of deleted molecules was not measured.

Hybridization of muscle sections with probe 3, which recognized all mtDNA species (see Fig 4A), showed an intense signal in all COX- fibers examined (example shown in Fig 1C). The same fibers showed a very weak hybridization signal with probe 4 (see Fig ID), which corresponded to a deleted region of the mtDNA (see Fig 4A). Normal COX’ fibers did not show any significant hybridization difference with the two probes. Densitometric analysis revealed that the intensity of the hybridization with probe 3 was two- to threefold mote intense in rhe COX- fibers than in both type 1 and type 2 normal fibers. Probe 4 showed an inverse ratio; ie, COX- fibers weie two- to threefold less intense than in both type 1 and type 2 normal fibers (see Fig 1E). These results indicated that the total amount of mtDNA was increased in the COX- fibers and that most mtDNA molecules in these fibers were deleted.

Discussion We identified a patient with clinical, biochemical, and histological features of a mitochondrial myopathy who harbored heteroplasmic mtDNA duplications and deletions.

This patient has unique clinical features compared with others with mitochondrial disorders in which the coexistence of duplicated mtDNA and its corresponding deletion has been reported [6-8, 11, 121. She has a pure myopathy and does not have diabetes mellitus, deafness, or other multisystemic manifestations, nor does she have KSS. Diabetes mellitus was reported in 15 of 20 patients wirh mtDNA duplications and deletions [6, 9, 10-131, suggesting that pancreatic islets might be especially vulnerable to the metabolic impair- ment associated with these mtDNA rearrangements. The absence of diabetes in our patient could be explained by skewed mitotic segregation of the rearranged molecules during embryogenesis (ie, the pancreatic islets might have received an amount of abnormal genomes insufficient to cause dysfunction).

An unbalanced distribution of the rearrangement in different tissues of the proband was also suggested by the very low abundance of duplicated genomes in blood compared with muscle. This was another unusual finding in this patient, because most reported patients contained higher levels of duplicated genomes in blood than in muscle [9, 10, 12, 131. We could not assess whether the mtDNA rearrangement was inherited or arose spontaneously in our patient, because her mother was already deceased at the time of this study. Nevertheless, the duplication was found in the blood of the 20-year-old son of the proband, demonstrating that this mtDNA rearrangement can be transmitted through the germline. Maternal inheritance of a mitochondrial disorder associated with mtDNA duplications has been described in two unrelated families [ lo- 121. In contrast to these reports, our patient’s son was asymptomatic, despite high levels of mtDNA duplication (78%) in blood. We could not establish whether his lack of muscular symptoms was due to the absence of deleted genomes in muscle, because that tissue was not available. It is also possible that he is presymptom- atic, because his mother had been free of symptoms until age 20.

What is the relative contribution of these two genetic abnormalities (ie, deletions and duplications) in the pathogenesis of mitochondrial disorders? The the- ory that large-scale deletions can be pathogenic in hu- mans has been supported extensively both in vivo, by in situ hybridization [28, 291 and single-fiber PCR experiments [24] in muscle, and in vitro, by experiments on somatic cell hybrids [3O] and transmitochondrial hybrids [31] derived from tissues from affected patients. I t has been postulated that mtDNA deletions impair mitochondria] protein synthesis due to the loss

186 Annals of Neurology Vol 42 No 2 A u g h t 1997

of tRNA genes [32]. By contrast, the pathogenic significance of mtDNA duplications is still uncertain, and the tRNA hypothesis does not apply to duplications, as there is no loss of mtDNA genes and presumably no mutation of tRNA sequences [33].

To help understand the relative pathogenic roles of mtDNA duplications and deletions in our patient, we explored the relationship between the distribution of the rearranged molecules and the histochemical phenotype in muscle fibers. We studied COX activity and COX subunit immunoreactivities in combination with single-fiber PCR analyses and in situ hybridization. Single-fiber PCR analyses demonstrated that the content of wild-type and duplicated (nondeleted) mtDNA was lower in the COX- fibers than in the COXf fibers. This result could be explained either by depletion of mtDNA or by accumulation of deleted genomes in the COX- fibers. However, by in situ hybridization, we demonstrated that the absolute mtDNA content in the histochemically abnormal fibers was increased and that most mitochondrial genomes in these fibers were deleted. Thus, it appears that the deletions were the primary cause of the mitochondrial myopathy in this patient. It was therefore surprising that the duplication, which was, by far, the predominant mtDNA rearrangement found in the patient’s muscle (comprising 40% of the total mtDNA) was apparently unrelated to the phenotype, whereas the deleted genomes, which only comprised 1% of the total, could apparently cause the disease. Given the uneven distribution of the rearranged mtDNA molecules in different tissues and in cells within a single tissue, as demonstrated by in situ hybridization studies, it is likely that different muscles or even different portions of a single muscle harbor varying amounts of deleted and duplicated mtDNA. Moreover, the segmentary nature of the morphological alterations in skeletal muscle may underestimate the overall number of fibers affected. In other words, the proportion of histologically abnormal muscle fibers and the low percentage of deleted mtDNA molecules found in the muscle biopsy might not be fully representative of the extent of the muscle involvement in this patient.

A hypothetical pathogenic role of the duplications might be the generation of deleted molecules; because the minimal size of a mtDNA molecule containing both the origin of heavy-strand replication (0,) and the origin of light-strand replication (0,) must be at least 5.7 kb, a 4.6-kb deleted mtDNA, lacking 0,, may be unable to replicate. This is supported by two lines of evidence. First, the family described by us and the one described by Ballinger and colleagues [I 1, 121 are the only ones in whom deleted mtDNA molecules lacking 0, were detected, suggesting that this kind of mtDNA rearrangement is extremely rare. Second, Ball- inger and colleagues [I21 observed that Epstein-Barr virus-transformed lymphoblastoid cells, which origi-

nally contained both deletions and duplications, lost the deleted mtDNA during subsequent passages [ 121. This observation reinforces the hypothesis that replication of deleted mtDNA molecules lacking 0, may be severely disadvantaged, at least in rapidly dividing cells. However, the deleted molecules detected in these patients might have arisen from the duplicated mtDNAs, via further recombination events [7]. In this way, duplicated genomes might provide a continuing source of deletions, which could slowly accumulate in nonrepli- cating tissues, such as mature skeletal muscle. Thus, a low rate of accumulation of deleted molecules could explain the late onset and the relatively mild but progressive course of the muscle disorder in our patient.

In conclusion, we describe a patient with pure mitochondrial myopathy associated with a heteroplasmic population of tandem duplications and deletions of mtDNA. The proportion of the molecular rearrangements was higher in muscle than in blood, suggesting that mitotic segregation occurred at an early stage during embryogenesis, sparing other tissues, such as the pancreatic islets. An alternative explanation would be a clonal selection of rapidly dividing stem cells harboring normal mtDNA in the patient’s bone marrow. Al- though representing a small fraction of the rearrangements, the deletions appeared to have a disproportion- ate impact on the phenotype in this patient. To our knowledge, this is the first investigation of the relative pathogenic roles of duplicated and deleted mtDNAs. Further studies are necessary to assess if the duplicated mtDNA molecules play a more subtle role in the pathogenic process, perhaps by contributing to the genesis of the deletions.

This study was supported by grants from the Muscular Dystro- phy Association, the National Institutes of Health (HD32062, NS28828, NS11766, AG12131, HD32062, and NSO1617), Tele- thon Italy, the Myoclonus Research Foundation, the Procter & Gamble Company, and the Dana Foundation. The Irving Clinical Research Center also supported this project.

We thank Drs Filippo M. Santorelli, Bernard Fromenty, and Francesco Pallotti for their expert advice. We appreciate Dr Ro- chelle Goldsmith’s cardiopulmonary exercise evaluation.

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188 Annals of Neurology Vol 42 ’No 2 August 1997

association of myopathy with large-scale mitochondrial dna duplications and deletions: which is...

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