Heteroplasmy in Chronic External Ophthalmoplegia: Clinical and Molecular Observations

O REST HURKO, DO NALD R. JOHNS, S. LANE RUTLEDGE, O. CO LIN STI NE , PATTI L. PETERSON, NE IL R. MILLER, MARGARET E. MARTENS, DANIEL B. DR ACHMAN, ROBERT H. BROWN, AN D C. P. LEE

The Johns Hopkins University School ofM edicine, Baltimore, Maryland 21205 [O.H., D.R.J., SL R., O.CS., N.R.M., D.B.D.]. The University Health Center, Wayne State University, Detroit, Michigan 42801 [PLP., MElv!., C P.L.j, and Massachusetts General Hospital East. Charlestown, Massachusetts 02129 [R.H.B.j

ABSTRACf. Chronic progressive external ophthalmople Although this term has proven useful, definition of a specific gia (CPEO) describes a recognizable clinical syndrome disease state on the basis of a single clinical sign is fraught with frequently associated with variable dysfunction in other hazard . Frequent but variable involvement of nonocular tissues organ systems. Histochemical and biochemical studies sug has added to the complexity of a strictly clinical diagnosis of gested primary dysfunction of oxidative phosphorylation, CPEO . Kiloh and Nevin (I ) considered CPEO to be a distinctive This has recently been confirmed by demonstration of myopath ic syndrome, with relatively isolated myopathy of the partially deleted as well as normal mitochondrial DNA extraocular muscles, and only occasional mild weakness in the heteroplasmy-in some of these patients, most of them extremities. However, it became apparent that many CPEO sporadic. In the six heteroplasmic CPEO patients that we patients had evidence of multisystem disease (2-4). There can have examined to date, the partially deleted species has be pigmentary degeneration of the retina, optic atrophy, cerebel been detected in all tissues tested, albeit in vastly different lar ataxia, sensorineural hearing loss, peripheral neuropathy, proportions. We report here detection of physiologicall y electroencephalographic abnormalities, and/or elevation of pro significant proportions of partially deleted mitochondrial tein content of the cerebrospinal fluid. Less frequentl y, there are DNA in several organs taken at autopsy from a CPEO pyramid al or extrapyram idal signs, seizures, or frank cogniti ve patient with severe multisystem disease. We discuss the impairment. A few path ologic studies of the nervous system have relationship of CPEO to several other clinical phenotypes dem onstrated spongiform poliodystrophy. associated with mitochondrial dysfunction, and discuss the Although the paresis of the extraocular muscles in CPEO is possible implications of heteroplasmy for the development assumed to be a myopathy, their dysfunction may result from of variable phenotypes. (Pediatr Res 28: 542-548, 1990) prim ary failure of the nervous structures that control them (5). In CPEO, frank ophthalmoparesis is preceded by profound slow Abbreviations ing of saccadic eye movements, with relative preservation of pursuit and vestibular eye movements, suggesting involvement CPEO, chronic progressive external ophthalmoplegia of excitatory medium-lead "burst" neurons (6). These neurons MELAS, mitochondrial encephalopathy, lactic acidosis, likely have unusually high energetic demands, inasmuch as they and stroke-like symptoms selectively initiate saccadic eye movements with an extremel y MERRF, myoclonic epilepsy with ragged red fibers rapid volley of electrical discharge. LHOA, Leber's hereditary optic atrophy Cardiac pathology most frequently takes the form of conduc tion defects, typically intraventri cular branch blocks or interven tricular block, which, curiously, can be heralded by a period of enhanced A-V nodal conduction (7,8). We have also observed supraventricular tachycardias and congestive heart failure. Path ologic studies have demonstrated fatty infiltration, fibrosis, and occasional giant mitochondria, both in conducting fibers and CLINICAL FEATURES OF CPEO elsewhere in the myocardium (9). In strict usage, the term CPEO describes only a clinical sign Pathology is not limited to electrically excitable tissues. Renal paresis ofthe muscles that rotate the eyes in the orbit and elevate dysfunction most often involves a proximal tubular defect, with the eyelids, with sparing of internal ocular muscles that control am inoaciduria and phosphaturia-the De Toni-Fanconi-Debre the lens and the iris. However, it has also come to connote a syndrome (3)- but we have also observed glomerulopathy and specific disease in which such paresis is the predominant clinical renal failure. Proportionate short stature, scoliosis, and a high finding. This latter meaning was used interchangeably with the arched palate have in some instances been associated with defec terms "mitochondrial myopath y" or "ragged-red fiber disease," tive release of growth hormone; in others, they have not (3). until recent descriptions of other clinical phenotypes associated Other endocrine dysfunction includes diabetes mellitus, some with similar pathologic and biochemical changes. tim es latent until the patient is stressed by glucocorticoids (10), which had been used by some clinicians to treat these patients. Correspondence: Orest Hurko,M.D., Departments of Neurology and Medicine. Parathyroid dysfunction has also been described (3). The Cent er for Medical Genetics. Meyer 6-119. Th e Johns Hopkins Hospital. 600 N. Wolfe Street. Baltimore, Marvland 2 1205. In summary, CPEO can present with clinical dysfunction of Supp orted by National of Health Gra nt RO I AR 38231 and grants variable severity in a number of highly oxidative organs, reflect from the Muscular Dystroph y Association. ing prim ary pathol ogy of tissues derived from man y cell lineages. 542 HETEROPL ASMY INCPEO 543 Furth ermore, the pattern of organ involvement and severity is consumption, with precise delineation of an anaerobic threshold notoriously variable. Und erstandably, this extreme pleiotropy (24-26). However, such tests are laborious and commonly re gave rise to a plethora of diagnostic labels: in McKusick's cata quire invasive catheterization. logue, Mendelian Inheritance in Man (II ), there are over a dozen A more convenient indirect measure of oxidative phosphoryl listings for CPEO with different patterns of organ involvement. ation, nuclear magnetic resonan ce spectroscopy of high energy Of these, the most enduring has been that of Kearns and Sayre, phosphates before and after exercise, has indicated abnormalities who proposed that the triad of CPEO, retinal pigmentary degen in skeletal muscle of many CPEO patients (27).Recently, we eration, and heart block constitutes a distinctive syndrome (12). have used a similar procedure to detect abnormalities of high Several years later, Rowland and his colleagues (5) proposed that energy phosphates in the frontal lobes of five patients with such a syndrome is indeed distinctive and that the definition be dissimilar mitochondrial encephalomyopathy phenotypes (28), modified to include ophthalmoplegia, pigmenta ry retinopath y, among them two patients with different CPEO syndromes with onset before age 20, and at least one of three other features: no clinically evident dysfunction of the telencephalon. either high cerebrospinal fluid protein , heart block, or ataxia. A Further indirect evidence implicating mitochondrial dysfunc commonly held view is that this expanded definition will distin tion in CPEO and other ragged-red syndromes comes from guish those patients with systemic disease from those who have reports of improvement after treatment with agents that might isolated involvement of the extraocular muscles. correct defective oxidative phosphorylation or its immediate In an earlier clinical review (13, 14), Drachman proposed a consequences (29-32): cofactors for the protein components of unified concept-ophthalmoplegia plus-lumping together all the electron transport chain ; "electron shuttles," substituting for CPEO patients, whatever the pattern of nonocular disease. Some a defective respiratory chain component, or scavengers of toxic authors have even included patients without CPEO but with a free radicals that could accumulate in high ambient oxygen typical constellation of neural and systemic involvement (3). concentrations. Although isolated reports of clinical improve Although this view may seem intellectually unsatisfactory-and ment have been impressive and , in some cases supported by indeed is demonstrably too global-recent genetic and laboratory spectroscopic, electrophysiologic, and/or lactate measurements, studies recommend that aspects of this unitary view be reexam some of us have been disappointed in our ability to intervene ined. successfully in the majority of our patients. More direct assessments of mitochondrial function support further the idea of defective oxidative phosphorylation in CPEO, BIOCH EMICAL AND MOR PHOLOGIC FEATU RES OF CPEO but even these assays can be problematic. Our current preference The idea that many CPEO syndromes might be variations on is for polarographic analysis of intact fresh mitochond ria har the same fundamental theme finds support from laboratory vested from biopsied muscle (33, 34). Unfortunately, this re findings indicating primary dysfunction of mitochondrial oxi quires a large biopsy specimen and , ideally, assay of nonfrozen dative phosphorylation. Most studies have been performed on material. The isolation of intact mitochondria from skeletal skeletal muscle, because of both historical tradition and practical muscle-a tough, fibrillar tissue-is a delicate procedu re: artifac considerations. However, similar changes have occasionally been tual uncoupling (i .e. mimicking the biochemical phenotype of observed in other tissues (15). Luft's disease) can occur with minor variations in the homoge Histochemical and electron microscopic observation of skele nization procedure. Furthermore, polarograph y is routinely per tal muscle demonstrates that some, but never all, of type I fibers formed at ambient atmospheric oxygen concentrations, consid contain abnormally large collections of mitochondria, the mor erably higher than those encountered by most mitochondria in phology of which may be altered (16- 18). Similar mitochondrial vivo.Finally, there is the problem of studying a mixture of norm al alterations have been indu ced in experimental animals that were and abnormal mitochondria. exercised excessively or treated with toxins that interfere with Given these considerations, only equivocal alterations in oxi oxidative phosphorylation at any of several biochemical sites. dative phosphorylation have been found in many CPEO patients, These changes can be prevented with chloramphenicol, a specific even those in whom there is other indirect evidence of mito inhibitor of mitochondrial, but not nucleocytoplasmic, protein chondrial dysfunction. However, certain CPEO patients have synthesis (19, 20). This suggests that these cellular changes are a demonstrated dysfunction in many sites along the electron trans nonspecific proliferative response to a mismatch between de port chain. Complex I (NADH-coenzyme Q reductase ) dysfunc mands for oxidative phosphorylation and the ability to meet tion has been found in a typical CPEO syndrome (35, 36) as well these demands. as a case in which paresis of vergence and upgaze only were Some of these mitochondria may contain as yet biochemically minor features of a moderately severe, generalized myopathy uncharacterized inclusions, often with a characteristic paracrys (37); other CPEO patients had partial deficiency of complex IV talline "parking-lot" morphology (16, 17). Curiously, the major (cytochrome oxidase) (21-23), sometimes with increased levels ity of fibers, including those immediately adjacent, have no of cytochrome c (34). Complex III deficiency was observed in a morphologic alterat ions of mitochondria. This indicates heter father and son, only the latter having CPEO (38); a child with ogenous involvement even within the class of type I fibers, all of myopathy and ptosis but no other ophthalmoparesis (39) was which are likely expressing identical nuclear-encoded isozymes. reported to have severe deficiency of complex II (succinate An even more telling observation was reported by John Morgan dehydrogenase), the only respiratory complex that does not Hughes and others (21-23): serial cross-sections revealed that contain subunits encoded by mitochondrial DNA. most ragged-red fibers contain excessive activity of all mitoch on Conversely, dysfunction of a given site along the electron drial enzymes, as would be expected. However, an occasional transport chain has been associated with many seemingly dispar fiber showed increased numbers of ragged-red mitochondrial ate clinical phenotypes (40, 41): some cases of norm oglycemic membranes by the Gomori trichrome stain and the expected congenital lactic acidosis (42- 45); individual cases with Leigh's increase of succinate dehydrogenase activity, but complete ab polioencephalopathy (46- 48); MELAS syndrome (49- 51); sence of cytochrome oxidase activity. MERRF syndrome (26, 52-55); and proximal myopath y with Biochemical observations give further support to the hypoth easy fatiguability (2). Despite their biochemical and histologic esis of defective oxidative phosphorylation. Elevations of lactic similarities, they have been described as distinct clinical syn acid, although and variable within the same patient, are dromes. However, upon following patients for several years, we observed frequently. In several of our CPEO patients, venous have observed several instances of individuals progressing from levels of lactic acid are normal at rest, but rise abnormally on one syndrome to another: a 34-y-old patient with MELAS who graded exercise. Such tests are ideally performed with concomi later developed typical CPEO and retinal pigmentary degenera tant measurement of cardiac output, oxygen extraction, and tion (Fig. 1); another 34-y-old in whom CPEO preceded the 544 H URKO E T AL.

GENET IC AN DMOLECULAR OBSER VATIONS ON CPEO The majority of CPEO patients are sporadic. Many CPEO patients with severe multisystem involvement are too ill to have children. However, one of our CPEO patients [a 52-y-old woman with partial deletions of mitochondrial DNA detected in skeletal muscle and peripheral blood (56)] has three healthy children in college. Small pedigrees with multiple individuals affected with CPEO have also been encountered (20, 57- 59). In most of these pedigrees, including our own, CPEO has been transmitted along the maternal lineage. There have also been cases of paternal transmission of mitochondrial CPEO with systemic involvement to a son (3, 38, 59), suggesting involvement of an autosomal nuclear gene in those kindreds. Speculation about the possible role of mitochondrial DNA in the pathogenesis of some ragged red syndromes was initially based on consideration of biochemical and morphol ogic abnor malities (60). More compelling, albeit inconclusive, evidence came from transmission genetics. Small pedigrees compatible with mitochondrial inheritance were published in a seminal paper by Egger and Wilson (59). In several of these pedigrees some affected individuals had ophthalmoparesis, but others presumably affected by the same mutati on-only had clinically apparent disease in other organ systems. Egger and Wilson presciently speculated that such variable organ involvement within a given family might reflect random segregation of normal and mutant mitochondria within a given individual. We have encountered such dramatic pleiotropy in one kindred that is segregating cytochrome oxidase deficiency (49). Although mutations of either mitochondrial or nuclear genes may result in a CPEO or related phenotype, until recently the evidence for involvement of mitochondrial DNA in CPEO was inconclusive. In small pedigrees, one would expect occasional chance occurrence of strictly maternal transm ission; many her itable diseases that exhibit pleiotropy and phenotypic variability within the same pedigree have impeccable credentials as auto somal dominant traits. The earliest direct evidence that a mito chondrial mutation was responsible for a case of CPEO came from Byrne et al. (61), who observed an altered electrophoretic mobility of a mitochondrial translation product in skeletal mus cle. More direct evidence for mitochondrial DNA mutati ons in some instances of CPEO was provided by Holt, Harding, and Morgan-Hughes (62, 63), who demonstrated partial deletions in a subpopulation of mitochondrial DNA extracted from skeletal muscle. To their initial reports have been added others, including six of our own (56,64- 70). Most reported cases of mitochondrial DNA deletions, including all of ours, have come from patients with a sporadic CPEO syndrome, some of them fulfilling diag nostic criteria for the Kearns-Sayre syndrom e, others not. No correlation between the size or molecular location of the deletion and the pattern of organ involvement is apparent. Indeed, this is not unexpected as most reported deletions associated with CPEO remove genes encoding components of the electron tran sport chain and also several transfer RNA genes. All but two of our Fig. I. Studies of a middle-aged man with MELAS and CPEO. a, patients have different deletions and all but one involve directly Computerized tomogram of brai n after his first stro ke-like episode that repeated sequences at the deletion jun ctions, the consensus of left him with a perm anent left hom onym ous hem ianopi a. b, Capillary which resembles putative recombination signals (69, 70). phase of carotid arteriogram with early-draining vein, take n 2 y later In all reported CPEO patients with partial deletions of mito when he developed pure word deafness and a lucenc y of his superior chondrial DNA detected by Southern analysis, the partially temporal lobe. c. Retinogram showing atypical pigmentary retinal degen deleted species were not found in blood cells. However, using a erat ion and mild pallor of the optic nerve. more sensitive assay (58, 68, 69) we and others have observed some partially deleted mitochondrial DNA molecules in all development of the MERRF syndrome by 6 y; and a patient who succumbed at age 23 after developing sequentially CPEO tissues tested from each such patient, albeit in vastly different and pigmentary retinopathy, MELAS, and terminally, the proportions (56,69, 70). This procedure is a modification of the MERRF syndrome. standard polymerase chain reaction, using oligonucleotide These observations suggest that the clinical phenotype reflects primers with recognition sites widely spaced on normal mito more accurately the degree of impairment of flux throu gh the chondrial DNA but closely bracketing the deleted region. It entire electron transport chain and the anatomic distribut ion of selectively amplifies deletion junctions even when normal, un abnormal mitochond ria, rather than the precise biochemical site deleted mitochondrial DNA is present in 1000-fold excess (69). at which such flux is impaired. As currently performed, it does not permit precise quantitation; HETEROP LASMY INCPEO 545 we can only infer that partially deleted mitochondrial DNA is a most in these patients is not which region of the mitochondrial very minor species in the blood and urinary epithelial cells of DNA is deleted, but how man y normal mitochondria remain in those CPEO patients tested to date, because we can detect it by a given cell. Indeed, several patients with Pearson's syndrome the polymerase chain reaction but not by Southern anal ysis. (infantile aplastic anemia and pancreatic failure) have a subpop Although the small amounts of partially deleted species in blood ulation of mitochondrial DNA with deletions identical at the and epithelium seem well below the threshold necessary for molecular level to three of our adult CPEO patients, but with clinically significant disruption of oxidative phosphorylation in differing tissue distributions (7 1, 72). those cells, their presence clearly indicates that the origin of the An interesting contrast can be made between the ragged-red mutation in these patients anteceded divergence of the muscle fiber syndromes described above, and another heritable disorder lineage. Thus , physiologically significant replacement of normal affecting oxidative phospho rylation , LHOA, discussed earlier in by mutant mitochondrial DNA is possible in tissues derived from this symposium by Dr. Douglas Wallace. Although biochemical any of the three primary cell layers. and histologic evidence of mitochondrial dysfunction in the Our only direct evidence on this point comes from Southern ragged-red syndromes is strong, evidence from transmission ge analysis on multiple tissues from an autopsied case, whose clin netics is indirect and inconclusive. In contrast, the majo r evi ical and biochemical features were reported elsewhere (34, case dence of mitochondrial involvement in LHOA comes from I). We report here that different proportions of partially deleted compelling data in transmission genetics (73, 74), whereas bio and normal mitochondrial DNA were readily detectable by chemical and histochemical evidence of mitochondrial dysfunc Southern analysis of skeletal muscle, brain, heart, liver, and tion is weak or lacking: lactate levels are normal, as are most kidney (Fig. 2). The decrease in oxidative activity measured polarographic and enzymatic studies of isolated mitochondria, polarographically in these tissues paralleled the rank order of and equivocal alterations of mitochondrial morphology are only increasing proportion of partially deleted mitochondrial DNA in evident in some electron microscopic studies (75). Definite proof all tissues but the liver, which was presumed to be autolyzed by of a minor oxidative phosphorylation defect in LHOA comes the time of polarography. There was clinically evident dysfunc from Wallace's elegant association of a point mutation in a tion of skeletal muscle, heart , and brain, but liver and kidney conserved region ofa mitochondrial gene encoding a subunit of were judged normal in vivo. In all tissues, the partially deleted respiratory complex I (76). Furthermore, unlike those individuals species were identical, indicating common descent from a single with a ragged-red syndrome associated with a mutation in mi mutational event. These observation s, albeit limited, suggest that tochondrial DNA, man y LHOA patients appear homoplasmic different patterns of organ involvement in CPEO patients may for the mutant mitochondrial DNA species in all tissues tested be, in part, quantitative rather than qualitative. What may matter to date .

A T HEORETICAL CO NSIDERATIONS

l&J J >- ... Oxidative phosphorylation is such a fundamentally important Z 0: U ILl IX: w (/) z -< :> clI: process that it appears unlikely that a mutation completely Q :::> E 1&01 CD ::::i :f x :E: disrupting the electron transport chain in every cell in the bod y will permit extrauterine life. Therefore, patients harboring mu IG 6 · tations that affect oxidative phosphorylation must fall into one 11 .6 - of two categories. 1) Those patients with mutations that com pletely disrupt oxidat ive phosphorylation must also contain a complement of normally functioning mitochondria to sustain life. Tissues with a large complement of dysfunctional mitochon dria would be am enable to biochemical diagnosis. This seems true in those CPEO patients with large partial deletions of mitochondrial DNA. 2) Those mutations expressed in every 100 B C mitochondrion in the body must disrupt oxidative metabolism W only minimally. Therefore, direct biochemical verification of the t- 80 abnormality may prove difficult. This appears true in those cases W ofLHOA in which a pathogen ic point mutation has been deter ...J W mined . C 60 In both situations, clinical involvement of some tissues but t- not others could result from a number of possible mechanisms. Z These include: 1) differing requirements for oxidative metabo W 40 lism in individual tissue types, which may change with activity 0 and development; 2) involvement of nuclear-encoded compo a: 20 nents of the electron transport chain or other regulatory proteins, W some of which exist as tissue-specific isoforms (77); and 3) D. interaction with variable, nongenetic environmental factors. Our 0 /" discussion will be limited to the possible relationship of another KIDNEY LIVER HEART BRAIN MUSCLE factor-heteroplasmy, the presence of more than one type of mitochondrial DNA in an individual organism-to the clinical TISSUE heterogeneity evident in CPEO and other ragged red syndromes. Fig. 2. Wild type and parti ally deleted mitochondrial DNA species In somatic cells of mammals, each mitochondrion contains in various organs harvested at autopsy from a child with CPEO and about six loops of mitochondrial DNA (78), each a separate multi system disease. a. Southern blot of total cellular DNA restricted genetic unit; a mutation in one such mitoc hondrial genome will with BamHI, subject to electrophoresis on 1.1% agarose, transferred to affect directly only itself and its daughter molecules. Because nit rocellulose, hybridized with a cloned probe (prepared from HeLa each somatic cell contains thousands of such molecules, it ap mitochondrial DNA, nucle otides 16453 to 3245) that had been made pears unlikely that such a mutation would significantly affect radioactive by random-primer labeling , and visualized by autoradiogra cellular metabolism unless it came to represent a major popu la phy. b, Proportions of partiall y deleted species, as estimated by laser tion. Such threshold effects of mitochondrial mutants conferring densitom etry and expressed as a percentage of total mitochondrial circles. resistance to chloramphenicol have been demonstrated (79, 80), 546 HURKO ET AL and it appears likely that other mitochondrial mutants will possibly death of the cell. Cells that remain mitotically active beha ve similarly. throughout life, such as blood and epithelium, are more likely to How daughters of a single mutant molecule can become a generate, through random drift , populations with skewed repre physiologically significant population in certain cell lineages may sentation of either normal or mutant species, and on whom such be und erstood by consid eration of early embryonic development, selection may be exaggerated. However, above a critical threshold during which the large cellular complement of mitochondrial of oxidati ve capacity, one might expect positive selective pres DNA becom es sequestered into man y smaller pools. During sure-preferential proliferation ofall mitochondria in those cells female gametogenesis, the ploidy of an individual mitochondrion with some defective organelles- because cells can respond to a becom es reduced to about one. After fertilization of the egg, the mismatch between oxidative capacity and demand by inducing first few cleavage divisions occur without growth of the zygote mitochondrial proliferation and increasing cellular ploidy of or apparent replication of mitochondria, with the total nu mber mitochondrial DNA (86, 87). We speculate that such selective of mitochondria in the zygote remaining fixed (Fig. 3). Thus, a mitochondrial proliferation may acco unt for the form ation of minor population of mutant mitochondria in the original ovum ragged red fibers. Furtherm ore, rare deletion events in mitochon may, by chance, become a major popul ation in certain cells of drial DN A of postm itotic tissues such as heart or brain may the blastocyst. Thus, it seems likely that stem cells ofvarious cell become selectively amplified in the course of norm al aging. lineages may, at rand om, receive different proportions of normal Evaluation of the relative roles of random drift and the effects and mutant mitochondria. The relative proportions of normal of such proposed selective pressures (80) in the production of the and mutant mitochondria in adult tissues, may, in part , reflect final phenotype associated with heteroplasmic mitochondrial the proportions contained in their progenitor cells. DNA mutations is an intriguing and difficult probl em . Is there In mammals, only a few cells in the blastocyst give rise to the something peculiar to skeletal mu scle and brain that invites embryo; most others are troph oblastic, and are thu s eventually selective proliferation of mutant mitochondria or is this simply discarded. It is by this mechani sm that hom oplasmy is usually ascertainment bias-patients with mitochondrial dysfunction maintained (8 1). In studies of cows with nonpathogenic poly predominating in other organs either not surviving or not being morphisms of mitochondrial DNA, Laipi s, Hauswirth , and col recognized? Excellent mathematical paradigms for analyzing leagues (82-85) docum ent ed occasional transitions to hetero such problems have already been developed by population biol plasm y, as well as rapid shifts between one apparently hom o ogists (88); biochem ical and molecular measures such as the ones plasmic state and another. Because these shifts occur in the discussed in this symp osium will be invaluable in testing such absen ce of any obvious selective pressure, they presumably result models. from rand om mitotic drift and embryonic sequestration. Similar processes may occur in patients with pathogenic mu "One cannot separate theoryfrom observation, or perhaps more tations of mitochondrial DNA , with added complexity super precisely, one must have a theory, however vague it may be." imposed by various selective pressures. In the specific case of 0. Kempthorne. partiall y deleted mitochondrial DNA, different selective pres sures may be exerted at different levels of organization. At the RE FER ENCES level ofan individual molecule with intact origins of replication, the deleted species, being smaller, may enjoy a replicative advan l. Kiloh LG . Nevi n S 195 1Progressive dystrophyof the ocular muscles (ocular myopathy). Brain 74:115- 143 tage over its intact, wild type counterpa rts. This effect would be 2. Petty RK H. Harding AE. Morgan-Hughes JA 1986 The clinical features of most evident in postmitotic tissues, allowing gradu al selective mitochondrial myopath y. Brain 109:915-938 accumulation th rough out life. At the level of the cell, selection 3. Egger J. Lake BD, Wilson J 1981 Mitochondrial cyto pathy. A multisystem may be a function of the proportion of normal mitochondria disorder with ragged red fibers on muscle biopsy. Arch Dis Child 56:741 752 and the oxidative dem ands on a particular cell. Early mammalian 4.Bastianse n LAK, Joosten EMG. De Rooij JAM . Hommes OR. Stadhouders embryogenesis in the fallopian tubes and shortly after implanta AM, Jaspar HHJ. Vccrkarnp JH. Bockelman H. van Hinsbergh VWM 1978 tion occurs at low oxygen concentrations. Thus, mutations af Ophthalmoplegia-plus:a real nosol ogicalentity.Acta NeurolScand 58:9-34 fecting oxidative phosphorylation may be selectively neutral 5. Berenberg RA, Pellock JM, DiMauroS, Schotland DL, Bonilla E, Eastwood A, Hays A, Vicale CT, BehrensM.ChutorianA, Rowland LP 1977 Lumping during the early sequestration and sorting of mitochond ria de orsplitting? "Ophthal moplegia-plus" or Kearns-Sayresyndrome?Ann Neu scribed above. However, at later stages, whenever a part icular rol 1:37-54 cell's capacity for oxidative metabolism drops below a critical 6. Leigh RJ. Zee DS 1983 The neurology of eye movemen ts. FA Davis Co, thresh old, one may expect negative selection, poor growth or Philadelphia. pp46-48 7. Roberst NK, Perloff JK. Kark RAP 1979 Cardiac conduction in the Kearns Sayre sy ndrome (a neuromuscular disorder associated wi th progressi ve ex ternalophthalmoplegia and pigmentary retinopathy). AmJ Cardiol44:1396 1400 8. CharlesR, HoltS, KayJM . EpsteinEJ, ReesJR1981 Myocardialultrastructure / \ and the deve lopmen t ofatrioventric ular block in Kearns-Sayre syndrome. Ci rc ulation63:214-219 ,/tI tt 9. Gallastegui J, Hariman RJ. Handler B. Lev M, Bharati S 1987 Cardiac I - ,-".- involvement inthe Kearns-Sayre syndrome. AmJ Cardiol60:385-388 10. Curless R. Flynn J, Bachynski B. Gregorious J. Benke P. 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Else vier Press, Amsterdam Only a few of thesecells give rise to the final embryo. Bychance, certain 15.Schneck L, Adachi M, Briet P, Wolintz A. Volk BW 1973 Ophthalmoplegia plus wi thmorphologicalandchemicalstudies ofcerebellarand muscletissue. cells acquire mostly mutant mitochondria (darkened ovals). Thus. a J Neurol Sci 79: 633-641 previously minor population of mutant species may become predomi 16. Olson W, Engel WK, Walsh GO, Ei naugler R 1972 Oculocra niosomatic nant in certain cell lineages. neuromusc ulardisease wi th"ragged-red" fi bers. Arch Neurol 26:193-211 HETEROPLASMY IN CPEO 547

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