003 1-399 8/ 90/2 805-053 6$02.00/ 0 PEDIAT RIC R ESEARC H Copyright © 1990 Int ernational Pediatric Research Foundation, Inc.

Vol. 28, No. 5, 1990 Primed in U.S.A.

Cytochrome c Oxidase Deficiency SALVATO RE DIMAU RO, AN NE LOMBES, HIROFU MI NA KASE SHU JI MIT A G IAN MARI A FABRI Z I, HAN S-J U RGE N T RITSCHLE R, ED UAR'DO BONI LLA: ARMAN D F. MIRAN DA, DARR YL C. DEVIVO , AN D ER IC A. SCH O N H. Houston Merritt Clinical Research Center/or Mu scular Dystrophy and Related Diseases, Columbia

University College 0/ Physicians and Su rgeons, New York, New York 10032

ABSTRACT. Cytochrome c oxidase (COX) is a complex enzyme composed of 13 subunits, three of which are encoded by the mitochondrial DNA (mtDNA). The oth er 10 subunits are encoded by the nuclear DNA , synthesized in th e cytoplasm , and transported into the mitochondria. The complexit y of th e enzyme and its dual genetic control explain the heterogeneity of clinical phenotypes associated with COX deficiency. There are two major syndrom es, one characterized by muscle involvement (fatal infantile or benign infantile myopathy), the other dominated by brain disease (Leigh syndrome, myoclonic epilepsy with ragged red fibers, Menkes' disease). Partial defects of COX have been shown in muscle of patients with progressive external ophthalmoplegia, either alone (ocular myopathy) or as part of Kearns-Sayre syndrome. Biochemical studies have documented either muscle-specific or generalized defect s of CO X; CO X deficiency is reversible in the benign infantile myopathy. Immunologically detect abl e protein may be normal (benign myopath y) or variabl y decreased (fatal myopath y, Leigh syndrome). Th e subunit pattern of CO X is normal by immunoblot in patients with fatal myopathy and Leigh syndrome; a disproportionate decrea se of subunit II was seen in a patient with myoclonic epilepsy with ragged red fibers. Availability of the three mtDNA genes and of complementary DNA probes for eight of the 10 nuclear DNA-encoded subunits makes it possible to investigate the different diseases at the molecular level. Large deletions of mtDNA have been found in patients with ocular myopathy and Kearns -Sayre syndrome: the deleted mtDNA appear to be transcribed but not translated, thus explaining th e partial CO X deficiency. (Pediatr Res 28: 536-541, 1990) Abbreviat ions CO X, cytochrome c oxidase PEO, progressive external ophthalmoplegia mtDNA, mitochondrial DNA nDNA, nuclear DNA KSS, Kearns-Sayre syndrome RRF , ragged red fiber s M ERRF, myoclonic epilepsy with ragged red fibers CRM , cross-reacting material

THE ENZYME

COX , the last component of th e respiratory chain (com plex IV), catalyzes th e transfer of reducing equivalents from cytochrom e c to molecular oxygen. T he energy generated by th is reaction sustains a transm emb rane proto n-pum ping activity. Th e holoenzyme contains two (possibly th ree) copper atoms an d two unique hem e A iron porp hyrins bou nd to a multisubu nit protein frame embedded in the mitochondrial inner memb rane (I , 2). In mam ma ls, the apoprotei n is com posed of 13 sub un its. Th e three larger polypeptides (subunits I to III) are associated with th e prosthetic groups an d perform both catalytic and protonpu mping activities. Th ey are en coded by mtDNA and are synthesized in mitochond ria; both th eir coding sequences and primary structures have been establish ed in humans (3). The 10 sma ller subunits [IV, Va, Vb, VIa , v n, VIc, VIla , VIlb, VIle, and VIII, according to th e nom en clat ure of Kadenbach et at. (4)] are encoded by nDNA and synthesized in th e cyto plasm. All except subunit VIII are synthesized as precursors carrying N H zterm inal basic presequ ences th at allow them to be transport ed int o the mit ochondria (5, 6). T he function s of th e nD NAencoded subu nits (which are m issing in procaryotes) have not been fully elucida ted , but they may mod ulate the catalytic fun ction, optimizi ng it to th e metabolic requireme nts of different tissues. In agreem ent with this concept was the dem onstration (based on electrophoretic mobility, am ino acid sequences, and antibody specificity) that some of the COX subunits are tissuespecific and developm entally regulated (2). Character izatio n of the huma n nDNA-encoded subunits of COX at th e molecular level has progressed very rapidly, and fulllength eDNA are now available for all sub units except VIlb and VIlc (7- 14). When th ese cDNA were used as prob es in No rthern analyses of RNA isolated from different hum an tissues, it was fou nd th at only one subunit, VIa, is tissue specific. Altho ugh prelim inary evidence suggests that subunit VIla may also be tissue specific, a surprising result was that subunit VIII, for which at least two isoform s are known to exist in othe r species, is expr essed as a single form in primates. So far, the only unequivocal chro mosomal assignment has been obtained for the gene encod ing COX VIII, which is localized to chromosome II (14). THE D ISEASES

Repri nt req uests: Dr. Salva tore DiMau ro, Room 4-420, College of Physician s a nd Surgeons. 630 West 168th Street, New York , NY 10032. Sup ported by Center grant s from N INCDS (NS 11766) and th e Mu scular Dystrophy Association, and by a gen erous don ation fro m Libero and G raziella Danesi . A.L. was supported by postdoctoral fellowships from the Fon dati on po ur la Recherche Medicate. the Ministere Fran cais des Relati on s Extericures, a nd the Muscular Dystrophy Associati on . H.-J.T. was supported by a fellowship from the DAAD, Bonn , West German y.

COX deficien cy was first report ed in trichopoliod ystrophy (Menkes' disease), an X-link ed recessive disord er of copper meta~o li s m causing infan tile seizures, developme nta l regression , hair abnor malities, tortu ou s arter ies, fragile bones, hypopigmentat ion, and temperatu re insta bility (15). T hese diverse ma nifestations have been attributed to secon dary deficiencies of copperdepend ent enzymes, of which CO X is one. In 1977, the sam e group of investigators (15) reported COX deficiency in two unrelated and clinically different patient s. Van Biervlict et at. ( 16) described an infant with severe generalized myopath y and renal dysfunctio n. T here was no clinical involve-

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ment of other organs, and the child died of respiratory failure at 4 mo of age. There was co mplete lack of COX acti vity and cytochrome aa, and partial defect of cytochrome b in muscl e mit ochondria. Th e second patient was a girl who died of respiratory insufficiency at 6 y of age after suffering from a syndrome that included ataxia, opti c atrophy, and dementia (17). Neuropathologic examination showed the characteri stic lesion s of subacute necrotizing encepha lomyelopathy (Leigh synd rome). COX acti vity was und etectable in muscle mito chondria and decreased in heart, but normal in liver. During the years that followed, numerous other cases of COX deficien cy were reported, mo st of which fall into two groups exempl ified by the two pati ents described in 1977: in on e group m yopathy is the principal, if not the only , manifestation; in the other group, a multisystem disord er is dominated by brain disea se (Table I). Myopathies. Two forms of myopathy have been described, both presenting soon after birth with severe generalized weakness, respiratory distress, and lactic acidosi s, but each having a very d ifferent course and prognosis. Children with fatal infantile m yopath y have a relentlessly downhill course and die of respiratory failure before I y of age ( 16, 18- 25). Although heart, liver, and brain are clinicall y spared, man y patients have renal disease with glycosuria, phosphaturia, and gene ralized aminoaciduria (De Toni -Fanconi syndrome). Ped igree anal ysis in info rmative fam ilies suggests autosomal recessive transmission. Different clini cal phenotypes, probably not related genetically to th e fatal infantile myopathy, are characterized by th e association of myopathy and cardiomyopathy in the same patient (26, 27), or by the coexistence of myopathy and hep atopathy in the same fam ily [but not in the same patients (28)]. Children with beni gn in fantile myopath y also present with severe weakness and often need assisted ventilation and gavage feeding earl y in life, but improve spontaneousl y and are usuall y normal by 2 or 3 y of age (25, 29-3 1). Lactic acidosis, which is initially even more severe than in th e fatal form , also rem its sponta neously. Although potentially ben ign, this m yopathy is life-threatening in the first months of life; therefore, it would be important to identify pathologic or biochemical features that might help in the differential diagnosi s between fatal infantile and benign myopathies.

Partial defects of COX , so metimes manifested onl y by scattered histochemically negati ve fibers in muscle biopsies, are commo nly seen in patients with PEO and morphologic alterations of mitochondria [RRF (32- 34)]. The pathogeni c significance of thi s partial COX deficiency remained unclear until recentl y, when molecular genetic analysis provided a logical explanation for thi s findin g (see below). Encephalomyopathies. A number of multisystem disorders dominated clin ically by brain dysfun ction have been associated with generalized COX deficienc y. The most common appears to be subacute necrotizing enceph alom yelopath y (Leigh syndrome): 22 patients have been reported (17 , 35-42), and we have recently encountered 14 more (Van Cost er R, Lombes A, DeVivo DC , Chi TL, Dodson E, Rothman S, Orrecchio EJ, Grover W, Berry GT, Schwartz JF, Habib A, DiMauro S, unpublished observations) . Leigh syndrome is a devastating encephalopathy of infancy or childhood, characterized by psychomotor regression, that usually becomes manifest around I y of age and is accompan ied by a spectrum of neurologic signs including ataxia, optic atrophy, ophthalmoplegia, pt osis, nystagmus, dystonia, tremor, pyramidal signs, and respiratory abnormalities. The characteristic neuropathologic lesions are symmetrical areas of necrosis involvin g preferentially mid-br ain , pons , basal ganglia , thalamus, and optic nerves. Micr oscop ically, there is cystic cavitation, vascular proliferation, neu ron al loss, and dem yelination. Muscle histochemistry is normal, but electron microscopy ma y show an increased number of mitochondria. Two or more siblings were affected in six famili es and there was parental consanguinity in two , suggesting autosomal recessive transmi ssion . However, the predominance of affected males (2 1 of 32 patients) is un explained. Leigh syndro me may be du e to other bioch emical cau ses, such as pyru vate deh ydrogenase com plex deficiency, and th e biochem ical error rem ain s unknown in many pati ents. CO X deficienc y was also shown in muscl e biop sies from two unrelated pat ients with Alpers disease (progressive sclerosing poliodystroph y), a neurodegenerative disorder of infancy dominated by intractable epilepsy and associated with liver disease (43). CO X deficiency has been found in still another mitochondrial encephalomyopathy kno wn by the acronym MERRF (44-47). As the name implies, MERRF is characterized by mitochondrial myopathy and myoclonus. Ataxia, generalized seizures, hearing

Table 1. COX deficiency: phen otypic expression and residual COX (%)* Ph enot ype D iso rders affecti ng mu scle exclusively o r predom ina ntly Fatal infa ntile myopath y Myopa thy o nly Myopath y and neph ropath y Myopathy and card iopath y Myo path y a nd hepatop ath y Ben ign infan tile myop ath y Disorders atTecti ng predomi nan tly the bra in Sub acute necroti zing ence phalom yelo pat hy (Leigh syndrome)

Alpers synd rome MERR F T richo poliodystrophy (Me nkes' disease) Other PEO Oc ular myopathy

KSS Enceph alomyopathy in adults MN GI E syndromet

* Mod ified fro m Schon et al. (60). t Myo-n eur o-gastrointestin al ence phalopat hy.

537

Tissue (s) atTected (% COX)

Mus cle « 10%) Mus cle « 10%); kidn ey (40 %) Mus cle « 10%); heart ( 12%) Mu scle « 10%); liver « 10%) Mu scle «10% ret urn ing to no rm al) Mu scle ( 14%); brain (32%) Liver (6%); kid ney (35%); Heart (12 %); fibrob lasts (22%) Mus cle (10 %; 42 %) Mu scle (30 %); brai n (29 %) Heart (18 %); liver (4 1%) Mu scle (54%); brain (47 %) Heart (59 %); liver (23%)

Muscle (25 % norm al) All tissues (vari ab ly decreased) Muscle (28-43 %); brain (?) Muscle (18 %): liver (6 %)

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DIMAURO ET AL.

loss, and dementia are common additional features. This disorder is transmitted by nonmendelian, maternal inheritance, which implies a genetic defect of mtDNA (44-47). Neuropathologic alterations are most prominent in the dentate nucleus of the cerebellum and in the inferior olivary nuclei, with neuronal loss and gliosis (47). Findings similar to those described above for patients with PEO and RRF (partial COX deficiency in muscle and histochemical evidence of COX-negative fibers) are seen in patients with a mitochondrial encephalomyopathy called KSS. KSS is characterized by the onset before age 20 of PEO and pigmentary retinopathy, plus at least one of the following: complete heart block, cerebrospinal fluid protein level above 100 mg/dl., and cerebellar syndrome (48). Although some patients have features that are intermediate between pure PEO (ocular myopathy) and typical KSS, in our experience these patients with "probable KSS" are relatively few (48). Other multisystem disorders with partial COX deficiency are less well characterized. One such syndrome has been called myoneuro-gastrointestinal encephalopathy because the patient, a German woman who died at age 42, had had chronic malnutrition due to maladsorption, PEO, limb weakness and wasting, polyneuropathy, and computed tomography scan evidence of leukodystrophy (49). Muscle biopsy showed RRF, and COX activity was decreased in muscle and liver. We have recently studied a brother and sister of German descent (but unrelated to the first patient) who have a virtually identical clinical picture and partial COX deficiency in muscle (Lombes, unpublished observations). Their parents were first cousins, suggesting that myo-neuro-gastrointestinal encephalopathy is inherited as an autosomal recessive trait. As mentioned above, COX deficiency appears to be a secondary phenomenon in trichopoliodystrophy (Menkes' disease). HISTOCHEMICAL BIOCHEMICAL AND IMMUNOLOGIC STUDIES

Histochemical and biochemical studies of patients with different forms of COX deficiency provided the first independent support of Kadenbach's suggestion that there are tissue-specific COX isozymes. For instance, as suggested by clinical and pathologic observations, COX deficiency in patients with fatal infantile myopathy was confined to skeletal muscle, sparing heart, liver, and brain (21). Partial COX deficiency was documented in the kidney of patients with myopathy and nephropathy (20, 21) and in the heart of at least one patient with myopathy and cardiomyopathy (26). Muscle histochemistry confirmed the selective involvement of muscle: the enzyme stain was absent in extrafusal fibers but present in intrafusal fibers of muscle spindles and in smooth muscle of blood vessels (22, 50). Immunologic studies using antibodies against human heart COX holoenzyme showed a decreased amount of CRM in muscle of patients, both by ELISA and by immunocytochemistry (21). Electrophoretic study of the mutant enzyme after immunoprecipitation failed to show any alteration in the subunit pattern (21). However, availability of antibodies against the individual subunits now permits a more detailed analysis of the mutant enzyme. Immunohistochemistry of muscle from a patient showed lack of COX VIIb,c and decreased amounts of COX II and III (51), but in muscle biopsies from four other patients we found a selective defect of COX VIla (5Ia). A defect of COX VIla would be in agreement with the suspected tissue-specific nature of this subunit, but these data have to be confirmed by Western analysis. The spontaneous clinical recovery in children with the benign infantile myopathy correlates with a gradual return of COX activity in muscle, which can be demonstrated both histochemically and biochemically. In one patient, the enzyme activity increased from 6 to 33 to 174% of normal in biopsies taken at 2, 7, and 36 mo (29). Histochemistry showed that only scattered fibers stained for COX activity in the first biopsy, but the number of positive fibers increased with time. Immunocytochemistry,

however, showed presence of CRM even in fibers lacking COX activity: the presence of inactive enzyme protein was confirmed by ELISA. The reversibility of COX suggests at least two hypothetical explanations. If the genetic error affects mtONA, there could be a gradual selection of fibers containing a majority of wild-type mitochondrial genomes over those that contain mostly mutant mtONA. In favor of this hypothesis is the histochemical observation that fibers seem to be affected in an all-or-none fashion: it is the number of normal fibers that increases with time, not the intensity of the enzyme reaction in each fiber. Against the hypothesis, however, is the lack of evidence of maternal transmission for this disorder. If the genetic error affects nONA, the mutation may involve a subunit that is not only tissue-specific but also developmentally regulated. Mutations of a fetal or neonatal muscle isozyme would be corrected when the mature isozyme starts to be expressed. In patients with Leigh syndrome, there is a generalized but partial defect of COX. In a study of five patients, we found that the residual activity varied in different tissues but tended to be similar in the same tissue from different patients: it was approximately 35% of normal in brain, but only 15% in muscle, and less than 10% in liver (40). The expression of COX deficiency in cultured skin fibroblasts in most (but not all) patients suggests that prenatal diagnosis may be feasible in families with one affected child. Prenatal diagnosis was recently performed by determination of COX activity in a biopsy of chorionic villi (51). Abnormal susceptibility of the enzyme to inhibition by oxidized cytochrome c was reported in fibroblasts from one patient (52), but we found normal affinity of COX for reduced cytochrome c in muscle and brain from three other patients. The amount of CRM was variably decreased in different tissues (40), and the decrease appeared proportional to the loss of COX activity in fibroblasts (53). No alteration of the subunit pattern was found in mitochondria isolated from brain or cultured fibroblasts (40, 54). In our patient with MERRF syndrome, residual COX activity varied considerably from tissue to tissue and even in different specimens from the same tissue, in keeping with maternal inheritance (46). Biochemical errors due to mutations of mtONA ought to be generalized but variably expressed in different tissues and within each tissue depending on the relative proportion of normal and mutant mtONA [heteroplasmy (46)]. The Km for cytochrome c was abnormally low, suggesting a defect of the mtONA-encoded COX II (47). Immunologic studies showed that the holoenzyme was decreased, but subunit II was decreased more than the holocomplex or the nONA-encoded COX IV. Although the biochemical defect was confined to COX in our patient, a combined defect of complexes I and IV was reported in two members of another family with MERRF (46). MOLECULAR GENETIC STUDIES

The dual genetic control of the enzyme makes COX deficiencies particularly interesting disorders from the genetic point of view. In theory, at least, we could envision the following genetic causes of COX deficiency: I) mutations of nuclear genes encoding the 10 smaller COX subunits; 2) mutations of mtDNA affecting directly or indirectly the three COX genes; 3) mutations of genes controlling the many steps involved in the import of nONA-encoded COX subunits from the cytoplasm into the mitochondria; and 4) mutations of regulatory genes controlling the rate of synthesis and the assembly of mtONA and nONAencoded subunits. Human pathology offers examples of the first two categories; COX deficiencies due to the molecular errors postulated in categories 3 and 4 have not yet been documented, but are likely to occur. The muscle-specific COX deficiencies exemplified by the fatal and benign infantile myopathies are probably due to mutations in one of the two nONA-encoded subunits that are known or

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presumed to be tissue specific, CO X VIa or COX VIla. This is in keeping with the apparent autosomal recessive mode of transmission of th ese disorde rs and with preliminary immunohistochemical evidence that COX VIla is defective in muscle of patients with fatal infantile myopathy. Because a mtDNA defect has been co nsidered in the etiology of th e benign form of COX deficient m yopathy, we performed Southern analysis of total muscle DN A from four patients, and found no dete ctable deletion ofmtD NA (48). Th ese studies, however, do not exclude the possibility th at a point mutat ion or sma ll deletion may affect one of the mitochondrial COX genes. Leigh synd rome associat ed with COX deficiency is clearl y transmitted as an autosom al recessive trait. The generalized natu re of the enzyme defect suggests in volvem ent of one of the eight nontissue-spe cific nuclear genes. Evidence for a nDNAencoded mutat ion comes also from experiments in which COXdeficient fibroblasts from a patient with Leigh syndrome were fused with a variant strain of HeLa cells (55). Prolonged cultivation of the hybrids in appropriate medi a led to preferential loss of HeL a cell mtD NA . COX acti vity was normal in cell clones that had lost almost all the HeLa cell mtDNA, suggesting that the enzyme defect was corrected by a nDNA-en cod ed factor from the HeLa parental cell. Ho wever, as mentioned above, Western analysis in different laboratori es has failed to show a specific defect of a single subunit. We are performing syste ma tic Nort hern analysis of mRNA extracted from different tissue s of Leigh pat ient s usin g cDNA probes for all available COX subunits. So far , we ha ve found no evidence to suggest altered size or abundanc e of messa ge for any subunit. Thus, Leigh syndrome might be due to the mutation of a nuclear regulatory gene controlling the assembly or stability of th e enzyme, but this remains to be pro ven. The matern al inhe ritance in MERRF syndro me suggests a mutat ion of mtDNA, but Southern anal ysis has ruled out major deletion s (46, 48). Ind irect biochemical and immunologic evidence in a patient studied by us (see above) suggested that the defect in volved COX II. H owever, Northern and Southern blots showed th at the gene for subunit II (as well as the gen es for subunits I, III, IV, and VIII) was of norm al size and normally transcrib ed (47). To document a point mutat ion of the gene for COX II will require dir ect sequencing, a task that will be complicated by th e heteroplasmy, that is, the coe xistence in the same tissue of normal and mutant mtDNA. Paradoxically, direct eviden ce of mtDNA mutations has been obtained in the two disorders that are usually not hered itary, PEO (ocular myopath y) and KSS. We found deletions ofmtDNA in muscl e biopsies from 32 pati ents, I S with typical or probable KSS and 17 with ocular myopath y (48) . No deletions were found in three pati ents with KSS and in 27 with ocular m yopathy. The deletions ran ged in size from 1.3 to 7.6 kb and were mapped to different sites in the mtDNA, but an identical 4.9-kb delet ion was found in 11 patients. One or more of the CO X genes were involved in some but not all deletions; for example, only the gene for subunit III (C03) was included in the more com mon 4.9-kb deleti on. The relative number of mutant mtDNA in muscle varied widely among patients, ran ging fro m 27 to 85 %. We found th at, in KSS, th e same mtDNA deletion was found in all tissues, but the relati ve proportions of normal and deleted m itochondrial genomes vari ed from tissue to tissu e. Becau se none of th e cases studied by us was famil ial, and no deletion was found in mu scle biopsie s from th e mothers of three KSS pat ients, it is likely that the deletion s are due to spontaneous mutations of mtDNA in the oocyte or in the zygote . We have shown that the partial CO X deficien cy observed in mu scle from patients with ocul ar m yopathy or KSS is not a specific defect. Biochemical analysis of six mitochondrial enzymes showed that th e mean activities of four enzymes of the respiratory chain- CO X, succinate-cytochrome c reductase, rotenone-sensitive NADH-cytochro me c reductase, and NADH deh ydrogenase-were significantly lower in muscle extracts from 22 pat ients with mtDNA

delet ion s than in 20 pati ent s witho ut deletions , but the activi ties of succinate deh ydrogen ase and citrate syntha se were not different in th e two groups (48). All fou r affected enz ym es contain one or more subunits encoded by mtDNA, wh ereas the two unaffected enzymes ar e entirely controlled by nDNA. However, the observation that all four enzymes were affected irrespective of the site of the delet ion suggested th at the entire mitochondrial geno me may function as a single genetic unit rather th an as a series of gene s en cod ing individu al en zyme s. As th e transcription of mtDNA is pol ycistroni c, a deletion anyw here in th e genome co uld affect transcription and tr ansl ation, even of gene s not directl y enco m passed by the deletion . This concept is supported by in situ hybridizat ion stud ies of CO X in muscle from a pat ient with KSS and the "com mon" 4.9-kb deletion (56) . Fibers lacking histochem ical CO X react ion had a predominance of deleted mtDNA and delet ed mitochondrial mRNA (showing that the deleted genomes are tr anscribed). Immunocytochemistry of these same fibers showed th at the nuclear-encoded subunit IV was pres ent, whereas COX II was markedly decreased or absent, desp ite the fact that the gene for COX II was not included in the delet ion . DISCUSSION

T he co m plexity of COX and th e related varie ty of clin ical ph enotypes du e to COX deficien cy offer man y opportunities to gain insight int o th e structure, function, and co ntrol of th is multisubunit enz ym e. Because of the dual gen etic control of COX, th e study of patients may also help clarify th e coo rdination of nucl ear and mitochondrial gene expression. The availability of monospecific antibod ies against th e different subunits and cDNA probes for most of them should facilitate our understanding of th e different COX de ficiencies at the molecular level. Most studies have bee n co nd ucted in skeletal muscle, because th is tissue is both highly vulnerable to COX defici ency and easily accessible. The relati ve uniformity of cell population s in muscle furth er facilitates both histochemical and biochemical inv estigation s. Other tissue s th at are equally or more vulnerable to COX deficiency, such as the brain , remain to be stud ied . H istochemical studies of COX in th e brain of normal primates ha s revealed a fascin ating heterogene ity in the distribution of th e enzyme among different neu rons and within different areas of the same neuron (57). The possible existence of multiple COX isozymes in the brain could be studied by immunocytochemistry and in situ hybr idizati on, and th e presence and distributi on of residual COX activity ought to be in vestigated by the same techniques in bra ins of patients with Leigh syndro me , MERRF, or othe r forms of CO X deficiency affecting the nervou s system. System atic an alysis of cultured skin fibroblasts in pat ients with lactic acidos is will detect most cases of generalized CO X deficien cy, suc h as Leigh synd rom e (39 , 58), and similar studies have been suggested in liver biopsi es of patients with un expla ined gen eral ized metabolic diseases (59). As interest in mitochondrial diseases in general, and in COX deficiency in particul ar , extends outside the area of neurology, we can expect new clinical phenotypes to be added to the already varied spectrum of the COXdeficien t encephalomyopathies.

Ackn owledgment. The authors dedi cate thi s rep ort to the memory of Aldo Danesi. REFE RE NCES I. Capaldi RA, Malatesta F, Darle y-Us mar V 1983 Stru cture of cytoc hro me c ox idase. Bioch im Biophys Acta 726: 135- 148 2. Kadenbach B, Ku hn-Nent wig L, Buge U 1987 Evolut ion of a regulatory enzyme: cytochrome c oxidase (compl ex IV). Curr Top Bioenerg 15:114161 3. Anderson S, Banki er AT, Bar rell BG, DeBruijn MHL. Coul son AR, Drouin J. Eperson IC, Nierlich D P, Roe BA, San ger F, Schreier PH , Smi th AJ H, Smith H, Staden R, Yo ung IG 198 1 Seq uence a nd organization of the hum a n mit och ond rial genom e. Nature 290:457- 475

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