OBSERVATION Deficiency Due to a Novel SCO2 Mimics Werdnig-Hoffmann Disease

Leonardo Salviati, MD; Sabrina Sacconi, MD; Minerva M. Rasalan, MD; David F. Kronn, MD; Alex Braun, MD; Peter Canoll, MD, PhD; Mercy Davidson, PhD; Sara Shanske, PhD; Eduardo Bonilla, MD; Arthur P. Hays, MD; Eric A. Schon, PhD; Salvatore DiMauro, MD

Background: in the SCO2 have been Results: Findings from muscle histochemistry studies associated with fatal cardioencephalomyopathy. showed virtually undetectable cytochrome c oxidase ac- tivity, but normal reaction. Bio- Objective: To report a novel SCO2 mutation with promi- chemical analysis in muscle confirmed a severe isolated nent involvement mimicking spinal muscu- cytochrome c oxidase deficiency. Pathologic findings of lar atrophy (Werdnig-Hoffmann disease). the were unremarkable, but the ventral horns of the spinal cord showed moderate-to-severe loss of mo- Patient and Methods: An infant girl presented at tor neurons and astrocytosis. Sequencing of the SCO2 gene birth with generalized weakness, , and lactic showed the common E140K mutation, and a novel 10 acidosis. At 1 month of age she developed hypertrophic base–pair duplication of nucleotides 1302 to 1311, which cardiomyopathy and died of failure 1 month later. disrupts the reading frame of the messenger RNA and gives Neuroradiological studies were unremarkable. Muscle rise to a truncated . biopsy specimens showed groups of atrophic and hypertrophic fibers, but mutation screening of the SMN Conclusion: The SCO2 mutations should be consid- gene was negative. Histochemical and biochemical ered in the differential diagnosis of children with spinal studies of respiratory chain complexes were performed, muscular atrophy without mutations in the SMN gene. and the whole coding region of the SCO2 gene was sequenced. Arch Neurol. 2002;59:862-865

YTOCHROME c oxidase fected.2 We report a novel SCO2 muta- (COX) deficiency is argu- tion associated with severe involvement ably the most frequent of spinal cord motor neurons and clinical mitochondrial abnormal- and pathologic features resembling spinal ity in children, usually muscularatrophytypeI(Werdnig-Hoffmann presentingC as . In the past disease). few years, several molecular defects have been associated with COX deficiency, in- REPORT OF A CASE cluding mutations in 3 mitochondrial From the Departments of DNA–encoded COX subunits and in 4 A 3.2-kg girl was born from healthy, un- (Drs Salviati, nuclear COX–assembly : SURF1,1 related parents at 39 weeks’ gestation by Sacconi, Davidson, Shanske, SCO2,2 SCO1,3 and COX10.4 cesarean section because of a breech pre- Bonilla, Schon, and DiMauro), Pathology (Drs Canoll, Bonilla, The SCO2 gene encodes a 266 amino sentation. She required delivery room re- and Hays), and Genetics and acid protein that is imported into the mi- suscitation for respiratory distress. She had Development (Dr Schon), tochondrion and is probably required for generalized weakness and hypotonia, with Columbia University, New the correct assembly of in the ho- minimal spontaneous movements, weak York, NY; Department of loenzyme.5 Mutations in the SCO2 gene cry, and stridor. Grasp and suck reflexes Pediatrics, University of have been associated with a fatal infantile were absent and she had a high-arched pal- Padova, Padova, Italy form of cardioencephalomyopathy. At or ate. In the following days, tongue fascicu- (Dr Salviati); Department soon after birth, patients develop severe lations became apparent. of Neurology, University hypertrophic cardiomyopathy, encepha- Results of routine tests were nor- of Modena, Modena, Italy (Dr Sacconi); and the lopathy, and , and they die of car- mal. Serum creatinine, ammonia, amino ac- Departments of Pediatrics diac failure within the first year of life. Cy- ids, and urinary organic acid levels were (Drs Rasalan and Kronn) and tochrome c oxidase activity is markedly normal. The blood lactate level was el- Pathology (Dr Braun), New reduced in muscle, heart, and brain, while evated (48.6 mg/dL; reference range, Ͻ19.8 York Medical College, Valhalla. and fibroblasts are less severely af- mg/dL) while pyruvate level was normal.

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 A chest x-ray film was normal at birth, but at 4 weeks showed heart enlargement. Echocardiography revealed hy- PATIENTS AND METHODS pertrophic cardiomyopathy. Brain computed tomographic HISTOCHEMICAL AND and magnetic resonance imaging scans were normal. BIOCHEMICAL STUDIES An electromyogram showed spontaneous activity in several muscles. Motor unit amplitudes and durations Histochemical analyses of muscle biopsy specimens were slightly decreased. Nerve conduction studies were and postmortem tissue samples were performed as 6 unsuccessful due to electrical interference in the inten- described previously. The activities of respiratory sive care unit. Results of the mutation screening of the chain were measured in muscle extracts as described.7 SMN gene was negative. The child’s condition rapidly worsened. She was in- MOLECULAR GENETIC STUDIES tubated after an episode of aspiration and attempts to wean her from the mechanical ventilator were unsuccessful. Genomic DNA was isolated from the patient’s muscle She died of heart failure at 7 weeks of age. as described.8 The SCO2 gene region was amplified as described.2 Polymerase chain reaction products were sequenced with internal primers using the ABI RESULTS PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and the 310 Automatic Sequencer Analysis of respiratory chain activities in muscle (Applied Biosystem, Perkin Elmer, Foster City, extracts showed severe isolated COX deficiency (0.18 Calif). Polymerase chain reaction fragments were µmol/min per gram of tissue; mean [SD] control value, also subcloned in a pCRIITOPO Vector using a Topo TA Cloning kit (Invitrogen, Carlsbad, Calif). Clones 2.80 [0.52] µmol/min per gram of tissue). were sequenced using internal SCO2 primers. Routinehistologicexaminationofmusclerevealedneu- Restriction fragment length polymorphism analy- rogenic abnormalities, with large groups of atrophic fibers sis of the E140K mutation was performed as described.2 and separate groups of hypertrophic fibers (Figure 1A). Histochemistry showed normal checkerboard staining for succinate dehydrogenase, while COX activity was vir-

A B C

D

Figure 1. and spinal cord of patient with SCO2 mutations. A, Paraffin section of autopsy muscle shows large groups of atrophic fibers and 1 fascicle of hypertrophic fibers (hematoxylin-eosin, original magnificationϫ20). B, This transverse cryosection of the patient’s muscle biopsy specimen shows absent staining for cytochrome c oxidase in both type I and type II fibers and in intramuscular blood vessels. C, Normal control (histochemical stain for cytochrome c oxidase, original magnification, original magnification ϫ50). D, Paraffin section of lumbar ventral horns shows marked loss of motor neurons and subtle astrocytosis. Eosinophilic inclusions in 2 of these nerve cells are indistinct in this magnification (hematoxylin-eosin, original magnification ϫ50).

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 A other allele was normal. This second child, a boy, is 2 months old and appears healthy.

COMMENT

Mutations in the SCO2 gene cause fatal infantile cardio- encephalomyopathy characterized by severe COX defi- ciency in affected organs. All patients described to date harbored a common E140K mutation. While the first 6

B patients were compound heterozygotes for this muta- tion and another ,2,9 3 patients homozy- gous for the E140K mutation have also been reported. These children had a milder clinical , with later onset and slower progression.10 Hypertrophic cardiomyopathy is the clinical hallmark of the disease and is usually the cause of death. However, as illustrated by our patient, it is not always the presenting symptom. is also present, but does not have the typical neuroradiological or neuropathological C features of Leigh syndrome. This is in contrast to mutations 1 in SURF-1, another COX-assembly gene, which are consis- MLLLTRSPTAWHRLSQLKPRVLPGTLGGQALHLRSWLLSRQGPAETGGQGQPQGPGLRT 11 RLLITGLFGAGLGGAWLALRAEKERLQQQKRTEALRQAAVGQGDFHLLDHRGRARCKAD tently associated with the Leigh syndrome phenotype. FRGQWVLMYFGFTHCPDICPDELEKLVQVVRQLEAEPGLPPVQPVFITVDPERDDVEAM Brain involvement varied in the 3 patients with SCO2 ARYVQDFHPRLLGLTGSTKQVAQASHSYRVYYNAGPKDEDQDYIVDHSIAIYLLNPDGL mutations previously described by us.2 The first patient FTDYYGRSRSAEQISDSVRRHMAAFRSVLS had early proliferation reminescent of Leigh syn- 2 MLLLTRSPTAWHRLSQLKPRVLPGTLGGQALHLRSWLLSRQGPAETGGQGQPQGPGLRT drome. The second patient had of the globus pal- RLLMAADHRPVRGWTRWGLAGPEG lidus and athrophy of the hippocampus, deep gray nu- clei, white matter, and . The third infant had Figure 2. A, Sequence of genomic DNA showing the heterozygous E140K mutation (G→A transition at nucleotide 1541). B, Sequence of the allele changes suggestive of a migrational disorder, with patches harboring the 10– duplication (underlined) cloned in a pCRIITOPO of cortical dysplasia in the left temporal lobe and focal Vector. C, sequence of wild type (1) and patient’s (2) SCO2 heterotopia in the cerebellum. The spinal cord was stud- . Underlined, the predicted the mitochondrial targeting sequence, in bold the 21 abnormal amino acids on the C-terminus. ied in 2 of these patients. One showed atrophy of the de- scending spinal tracts, diffuse , and moderate patchy loss of motor neurons. The other had mild gliosis and tually undetectable in both types I and II fibers and in in- white matter spongiosis. tramuscular blood vessels (Figure 1B). Conversely, the patient described herein had no brain Findings from a pathologic examination of the brain abnormalities, but the pathologic changes in the spinal were unremarkable. In the ventral horns of the spinal cord, cord were severe and closely resembled those of Werdnig- there was moderate to severe loss of motor neurons and Hoffmann disease. There were, however, subtle differences astrocytosis (Figure 1D). Sparse remaining motor neu- from the typical neuropathological features of Werdnig- rons showed central chromatolysis and rare large eo- Hoffmann disease, including abnormal shape of many mo- sinophilic inclusions. The ventral roots showed loss of tor neurons, absence of typical ballooned cells, more se- nerve fibers and glial bundles that expressed glial fibril- vere involvement of the sensory component of the periph- lary acidic protein. eral , and minor changes in the pyramidal Sequencing of the SCO2 gene revealed the E140K mu- tracts. tation (Figure 2A) and an insertion at nucleotide (nt) 1312. In the muscle biopsy specimen, neurogenic changes To better define this insertion, we amplified the patient’s were striking, suggesting that the severe weakness and SCO2 gene and subcloned the fragment in pCRIITOPO Vec- hypotonia of this patient were not simply due to a COX- tor. Clones were screened by restriction fragment length deficient myopathy, but were largely attributable to de- polymorphism for the presence of the E140K mutation. nervation. Neurogenic changes in muscle were also seen Negative clones were sequenced and revealed a 10–base pair in patients with the milder clinical phenotype, who were duplication of nt-1312 to nt-1321 (Figure2B) that dis- homozygous for the E140K mutation, but spinal cord pa- rupts the reading frame of the messenger RNA and gives thology was not described. Our data indicate that dener- rise to a truncated 83 amino acid polypeptide in which the vation in our patient was not due to involvement of the 21 amino acids at the C-terminus are abnormal (Figure 2C). peripheral nervous system, but rather to alterations of This is the first nucleotide insertion reported in the SCO2 the anterior horn cell of the spinal cord, as in Werdnig- gene. Analysis of DNA from the parents showed that the Hoffman disease. father carried the E140K mutation and the mother carried Mutations in the SMN gene are present in more than the 10–base pair insertion. 95% of the patients with Werdnig-Hoffmann disease,12 We have also performed prenatal diagnosis on am- but were excluded in our patient. Hence, the anterior horn niocytes from a second pregnancy of this couple. The fe- disease in this child seems to be a phenocopy of Werdnig- tus was heterozygous for the E140K mutation, but the Hoffmann disease caused by the SCO2 mutations.

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 The reasons for the variability in the clinical expres- Corresponding author and reprints: Salvatore DiMauro, sion of SCO2 mutations are not totally clear. Patients ho- MD, 4-420 College of Physicians and Surgeons, 630 W 168th mozygous for the E140K mutation show a milder phe- St, New York, NY 10032 (e-mail sd12 @columbia.edu). notype, perhaps because the protein is still partially 10 active. However, there is no clear correlation between REFERENCES type of mutation and type of neurological involvement. This case illustrates the importance of screening for 1. Zhu Z, Yao J, Johns T, Fu K, et al. SURF1, encoding a factor involved in the bio- SCO2 mutations even in patients without the typical phe- genesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat Genet. 1998; notype, and especially in children with features of SMA 20:337-343. but without mutations in the SMN gene. Knowledge of 2. Papadopoulou LC, Sue CM, Davidson MM, et al. Fatal infantile cardioencepha- the molecular defect will, as in this case, make prenatal lomyopathy with COX deficiency and mutations in SCO2, a human COX assem- diagnosis and accurate genetic counseling possible for bly gene. Nat Genet. 1999;23:333-337. 3. Valnot I, Osmond S, Gigarel N, et al. Mutations of the SCO1 gene in mitochon- young couples who have already lost a child. drial cytochrome c oxidase deficiency with neonatal-onset hepatic failure and encephalopathy. Am J Hum Genet. 2000;67:1104-1109. Accepted for publication January 14, 2002. 4. Valnot I, von Kleist-Retzow JC, Barrientos A, et al. A mutation in the human heme Author contributions: Study concept and design (Drs A: gene (COX10) causes cytochrome c oxidase deficiency. Hum Mol Genet. 2000;9:1245-1249. Sacconi, Kronn, Shanske, and DiMauro); acquisition of 5. Glerum DM, Shtanko A, Tzagoloff A. SCO1 and SCO2 act as high copy suppres- data (Drs Salviati, Sacconi, Rasalan, Kronn, Braun, Canoll, sors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae. Shanske, Bonilla, and Hays); analysis and interpretation J Biol Chem. 1996;271:20531-20535. of data (Drs Salviati, Sacconi, Davidson, Shanske, Hays, 6. Sciacco M, Bonilla E. Cytochemistry and immunocytochemistry of mitochon- and DiMauro); drafting of the manuscript (Drs Salviati, Sac- dria in tissue sections. Methods Enzymol. 1996;264:509-521. 7. DiMauro S, Servidei S, Zeviani M, et al. Cytochrome c oxidase deficiency in Leigh coni, Rasalan, and Shanske); critical revision of the manu- syndrome. Ann Neurol. 1987;22:498-506. script for important intellectual content (Drs Sacconi, Kronn, 8. Sambrook J, Russell DW. In: Molecular Cloning: A Laboratory Manual. Cold Spring Braun, Canoll, Davidson, Bonilla, Hays, and DiMauro); Harbor, NY: Cold Spring Harbor Laboratory Press; 2001:6.4-6.11. obtained funding (Drs Shanske and DiMauro); adminis- 9. Jaksch M, Ogilvie I, Yao J, et al. Mutations in SCO2 are associated with a dis- tinct form of hypertrophic cardiomyopathy and cytochrome c oxidase defi- trative, technical, and material support (Drs Sacconi, Braun, ciency. Hum Mol Genet. 2000;9:795-801. Davidson, Shanske, and DiMauro); study supervision (Drs 10. Jaksch M, Horvath R, Horn N, et al. Homozygosity (E140K) in SCO2 causes de- Sacconi, Kronn, Davidson, Shanske, Bonilla, and Hays). layed infantile onset of cardiomyopathy and neuropathy. Neurology. 2001;57: This study was supported by grants PO1HD32062 and 1440-1446. NS11766 from the National Institutes of Health, Bethesda, 11. Sue CM, Karadimas C, Checcarelli N, et al. Differential features of patients with mutations in two COX assembly genes, SURF-1 and SCO2. Ann Neurol. 2000; Md, and by a grant from the Muscular Dystrophy Associa- 47:589-595. tion, Tucson, Ariz. Dr Salviati is supported by grant 439b 12. Schmalbruch H, Haase G. : present state [review]. Brain from Telethon Italia, Rome, Italy. Pathol. 2001;11:231-247.

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