Fatal Infantile Mitochondrial Myopathy and Renal Dysfunction Caused by Cytochrome c Oxidase Deficiency: Immunological Studies in a New Patient Massimo Zeviani, MD,” Ikuya Nonaka, MD, t Eduardo Bonilla, MD,” Eizo Okino, MD,S Maurizio Moggio, MD,*§ Sara Jones, BA,” and Salvatore DiMauro, MD”

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A 3-month-old female infant had profound generalized weakness, de Toni-Fanconi-Debre syndrome, and lactic acidosis. She required assisted ventilation and died at the age of 8 months. Muscle biopsy showed accumula- tion of mitochondria, glycogen, and lipid droplets. His- tochemical reaction and immunocytochemical stain for cytochrome c oxidase showed very weak results, but both reactions were normal in intrafusal fibers of the muscle spindle. In crude extracts of the patient’s muscle, cytochrome c oxidase activity was undetectable and en- zyme-linked immunosorbent assay showed decreased reaction at all dilutions of antiserum. These data indi- cate that the amount of immunoreactive enzyme protein is markedly decreased in muscle of patients with fatal infantile cytochrome c oxidase deficiency and renal dys- function.

Zeviani M, Nonaka I, Bonilla E, Okino E, Moggio M, Jones S, DiMauro S: Fatal infantile mitochondrial myopathy and renal dysfunction

caused by cytochrome c oxidase defiiency: immunological studies in a new patient.

Ann Neurol 17:414-417, 1985

Systematic biochemical studies of muscle biopsy speci- mens in patients with morphologically defined mito- chondrial myopathies have allowed identification of various defects of the electron transfer chain involving complexes I, 111, and IV [17, IS]. Defects of complex

From the “H. Houston Merritt Clinical Research Center for Muscu- lar Dystrophy and Related Diseases, Columbia University College of Physicians and Surgeons, New York, N Y 10032, the tNational Center for Nervous, Mental and Muscular Disorders, Tokyo, Japan, and SKanazawa University School of Medicine, Kanazawa, Japan.

Received Aug 16, 1984, and in revised form Oct 15. Accepted for publication Oct 15, 1984.

HDr. Moggio’s permanent address is: Dino Ferrari Clinical Research Center, 2nd Department of Neurology, Milano, Italy.

Address reprint requests to Dr DiMauro, 4-420 College of Physi- cians and Surgeons, 630 W 168th Street, New York. N Y 10032.

IV (cytochrome c oxidase) show marked clinical het- erogeneity [4]. Fatal infantile forms may be seen as myopathy alone or as myopathy associated with renal dysfunction (de Toni-Fanconi-Debre syndrome) or cardiopathy. A benign infantile form shows spontane- ous clinical improvement and a return of the muscle enzyme activity [G]. Isolated hepatopathy has been de- scribed in the second cousin of 2 siblings with fatal myopathy 131. Generalized defects may be seen as sub- acute necrotizing encephalomyelopathy (Leigh’s dis- ease) [l 1, 16, 291 or as progessive sclerosing poliodys- trophy (Alper’s disease) [23], and partial defects have been seen in cases of progressive external ophthalmo- plegia [13, 18, 20) and in 2 patients with en- cephalomyopathy and peripheral neuropathy 12 1 ]. Im- munological and immunocytochemical studies using antibodies against purified human cytochrome c oxi- dase may help clarify the different molecular defects underlying the various phenotypes. We present the results of immunological studies in a new patient with fatal infantile myopathy and de Toni-Fanconi-Debre syndrome.

Case Report A 3-month-old female infant was admitted to the Depart- ment of Pediatrics of Kanazawa University because of severe generalized weakness. She was born to healthy nonconsan- guineous parents after a normal full-term pregnancy and de- livery. There was no history of neuromuscular disease in the family. Birth weight was 3,200 gm; the patient was well for the first month of life, after which increasing “floppiness” was noted by her parents. At admission, there was severe hy- potonia with “frog-leg” posture and profound generalized weakness, with few spontaneous movements. There was no visceromegaly. Tendon reflexes were absent. She was lethar- gic but reacted to painful stimuli. The pupils reacted nor- mally and symmetrically to light, and facial and extraocular movements appeared normal.

The following laboratory tests yielded abnormal values (normal values in parentheses): glutamic oxaloacetic trans- aminase, 104 U/liter (less than 15); glutamic pyruvic trans- aminase, 54 U/liter (less then 22); lactate dehydrogenase, 864 U/liter (120 to 240); creatine kinase, 1 5 1 Uiliter (less than 50); serum lactate, 57.5 mg/dl (less than 20); and serum pyruvate, 3.58 mddl (less than 1.0); the concentration of lactic acid in the urine was 400 times greater than normal. There was also generalized aminoaciduria and glycosuria. Electroencephalography showed diffuse slow waves, and computed tomographic scan was normal.

At the age of 3 months the patient suddenly developed apnea and required assisted ventilation. She was kept on a respirator and died at 8 months of age. Permission for post- mortem examination was denied.

Materials and Methods A muscle biopsy was performed at 5 months of age; portions of muscle were processed for histochemical analysis and elec- tron microscopy by methods described previously [8} . Con- trol muscle was obtained by diagnostic biopsy from patients,

414

including infants, who were ultimately deemed to be free of neuromuscular diseases. Histochemical staining for cyto- chrome c oxidase was performed using the technique de- scribed by Seligman and co-workers 1251. For immunoscain- ing, unfixed sections from the patient’s muscle and normal human muscle were processed in sequence through the fol- lowing steps: (1) incubation for 3 hours at room temperature with preimmune serum and normal rabbit serum diluted (1: 100 to 1: 1000) in phosphate-buffered saline (PBS); ( 2 ) 30-minute wash in three changes of PBS; (3) incubation for 1 hour at room temperature with protein A-peroxidase di- luted 1 : 30 in PBS; and ( 4 ) 30-minute wash in three changes of PBS. The immunoperoxidase stain was developed for 10 minutes by incubating the tissue sections in a solution of 0.38% diaminobenzidine and 0.05% H202 in 0.5 M Tris buffer (pH, 7.4). The sections were finally washed in distilled water and mounted with glycerol gel 17, 241.

For biochemical analysis, the muscle was homogenized in nine volumes of 0.15 M KCI, 50 mM Tris HCI (pH, 7.4) in all-glass motor-driven homogenizers. Cytochrome c oxidase and citrate synthase were measured in the supernatant fluid after centrifugation at 1,000 g for 15 minutes by methods described previously 163.

Cytochrome c oxidase was purified from human heart by the method of Fowler and associates 191, and as modified by Tzagoloff and McLennan [27], as described in detail else- where [2]. Antibodies were raised in rabbits by repeated intradermal injections of enzyme protein (1 mg); the im- mune serum was stored frozen in small aliquots.

Enzyme-linked immunosorbent assay (ELISA) was per- formed as described previously 12) except that crude muscle extracts were used instead of mitochondria1 preparations. To ensure that normal and pathological extracts contained com- parable amounts of mitochondria, they were appropriately diluted with extraction medium so that they contained simi- lar activities of citrate synthase.

Results and Discussion The clinical and laboratory features of this child were similar to those reported in 6 other patients 15, 10, 15, 19, 28): severe generalized weakness not present at birth but manifested during the first weeks, lactic acidosis, and de Toni-Fanconi-Debre syndrome. All 6 patients died before 4 months of age; our patient lived to 8 months, with assisted ventilation for the last 5 months of life. Family history was noninformative in our patient, as it had been in 2 other cases 15, 101. Of the remaining 4 patients, 2 were siblings [20) and the other 2 each had a sibling who had died at 11 and 12 weeks of age, respectively, with similar symptoms 115, 28). Parents were asymptomatic and there was no con- sanguinity in any of the 6 previously reported cases.

In our patient, as in the others with the same syn- drome, morphological studies showed accumulation of mitochondria in many fibers, which appeared “ragged red” upon modified Gomori trichrome staining; they also showed increased staining with oxidative enzyme reaction, increased glycogen content, and increased number of lipid droplets. In normal muscle, the his-

Fig I . Frozen sections illustrating the cytochrome c oxidtse reac- tion of (a) normal human musck, in which the reaction is more intense in type IFbers. (b) The patient’s muscle, in which in- tense reaction is seen only in intrafusalfibers of a muscle spindle (arrow).

tochemical reaction for cytochrome c oxidase stained type I fibers more intensely than type I1 fibers (Fig la). In the patient’s muscle the stain was very faint in all fibers but, in sharp contrast to extrafusal fibers, in- trafusal fibers of muscle spindles stained normally (Fig lb). Immunostaining of frozen sections showed similar results: compared with the reaction of normal muscle (Fig 2a), the immunoperoxidase reaction in the patient was very weak in extrafusal fibers but normal in in- trafusal fibers and also in smooth muscle of blood ves- sels (Fig 2b). These results indicate that the decrease of cytochrome c oxidase activity in muscle is accom- panied by a decrease of immunologically reactive en- zyme protein and that intrafusal fibers of the muscle spindle contain a different isoenzyme of cytochrome c oxidase, which was not affected by the genetic defect of the muscle enzyme in our patient.

The results of biochemical and immunotitration studies confirmed the cytochemical data: cytochrome c oxidase activity was undetectable in the patient’s mus-

Case Report: Zeviani et al: Cytochrome c Oxidase Deficiency 415

0.751

* UJ C Q,

5 050- m 0

c .-

.- c op

0 25-

I 0 2 103 I 0 4 105 106

Fig 2. Binding of antibodies against cytochrome c oxidase in fro- zen sections of (a) normal human mscle, in which the im- munoperoxi~se is intense in type Ifibers; of F ig 3. lmmunoreactivity b enzyme-linked immunosorbent assay

of crude muscle extracts from patient (solid circles) and control these fibws show discrete subsarcolemmal accumulations of stain (open circles), with progressive dilutions of purified antihuman (arrow). (6) The patient's muscle, in which extrafusal fibers cytochrome c oxidzsr IgG. (Ab = antibody.) show on(r background reaction, but there is clear binding of

Ab Dilutions

antibodies t o intrafusalfibers of the spindle (arrow) and t o smooth muscle of an artery (arrowhead).

obtained in a patient with fatal infantile cytochrome c oxidase deficiency but without de Toni-Fanconi-Debre syndrome 11, 2). In contrast, immunostaining of mus- Mitochondria1 Enzymes in Crude Muscle Extractsa

~~

Cvtochrome c cle biopsies in 1 child with the benign reversible form Subject Oxidase Citrate Synthase of cytochrome c oxidase deficiency 16) showed that the

Patient 0 5.59 Controls 2.50 t 0.50 8.81 5 3.08

(n = 35) (n = 19) Control range 1.76-3.74 4.12-16.50

"Activities expressed as pmol substrate utilized per minute per gram of tissue. Control values -C SD.

cle extract, whereas the activity of citrate synthase, an enzyme of the mitochondrial matrix, was only slightly decreased (Table). Immunotitration by the ELISA technique was performed on crude muscle extracts be- cause the patient's biopsy specimen was too small to allow isolation of mitochondria. At all dilutions of anti- serum the immune-linked alkaline phosphatase reac- tion was much less in the patient than in the control (Fig 3).

This is the first demonstration that immunologically reactive enzyme protein is lacking or defective in mus- cle of patients with fatal infantile cytochrome c oxidase deficiency and renal dysfunction. Similar results were

enzyme protein was present in early biopsy specimens that had exhibited a lack of activity in biochemical tests and present in fibers that showed no histochemical reaction (E. Bonilla and co-workers, unpublished re- sults, 1984). The enzyme protein was also present in all tissues studied by ELISA in a child who died at age 4 of Leigh's disease I 1 1): cytochrome c oxidase activity varied between 3 and 25% of normal in various organs (G. E. Hoganson and associates, in preparation).

These immunological studies demonstrate additional biochemical heterogeneity among the various pheno- types of cytochrome c oxidase deficiency.

The selective involvement of muscle in some of these disorders was clearly illustrated in our patient by the normal histochemical and immunocytochemical reactivity of the intrafusal fibers of the spindle. Our observations also highlight the difference in biochem- ical makeup between intrafusal and extrafusal muscle fibers. This is in agreement with immunocytochemical studies showing that in mammals only the small nu- clear bag fibers (bag 1 fibers) of the muscle spindle and

416 Annals of Neurology Vol 17 No 4 April 1985

some fibers in extraocular muscles stain brightly with antibodies against slow avian muscle myosin r223. Cytochrome c oxidase is composed of at least seven subunits, three of which (I to 111) are synthesized under the control of mitochondrial DNA, while the others are encoded by nuclear DNA 1261. The tissue specificity of cytochrome c oxidase has been attributed to one of the nuclear-coded subunits, possibly subunit VIa [12, 141. It is conceivable that a genetic error affecting this subunit may impair the assembly of the whole complex in muscle, but the molecular defects underlying the various phenotypes of cytochrome c oxidase deficiency remain to be defined.

Supported by Center Grants NS-11766 from the National Institute of Neurological and Communicative Disorders and Stroke and from the Muscular Dystrophy Association, and by Grant AM-25500 from the National Institute of Arthritis, Diabetes and Kidney Diseases. Dr Zeviani is the recipient of a postdoctoral research fellowship and Ms Jones of a predoctoral summer research fellowship, both from the Muscular Dystrophy Association.

We are grateful to Ms Mary Tortorelis for typing the manuscript.

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Case Report: Zeviani et al: Cytochrome c Oxidase Deficiency 417