Cytochrome C Oxidase Biogenesis in a Patient with a Mutation in COX10

ampullopetal deflection and inhibition of the superior canal.1,9 Clockwise direction of the slow phase indi- Cytochrome c Oxidase cates predominant inhibition of the right superior ca- Biogenesis in a Patient with nal that might be more affected than the left one. The permanent oscillopsia in our patient might be a Mutation in COX10 Gene explained by abnormal visual inhibition of VOR, Marieke J. H. Coenen, MSc,1 which in association with gaze-evoked nystagmus and Lambert P. van den Heuvel, PhD,1 Cristina Ugalde, PhD,1 the abnormal clinical aspect of smooth pursuit might Marike ten Brinke,1 Leo G. J. Nijtmans, PhD,1 be linked to cerebellar lithium toxicity. Indeed, these Frans J. M. Trijbels, PhD,1 Skadi Beblo, MD,2 Esther M. Maier, MD,2 Ania C. Muntau, MD,2 different cerebellar oculomotor symptoms are known 1 to occur with lithium therapy even within the range of and Jan A. M. Smeitink, MD, PhD that drug’s therapeutic blood level.11 In conclusion, this article adds important data to the We report a cytochrome c oxidase (COX)–deficient pa- clinical description and pathophysiological understand- tient, clinically affected with Leigh-like disease, with a ing of superior canal dehiscence syndrome. Vertical os- homozygous mutation in the COX10 start codon. Two- cillopsia and pulse-synchronous nystagmus may be ob- dimensional gel electrophoresis showed a decrease of served in bilateral symptomatic forms as a result of an fully assembled COX without the accumulation of par- abnormal communication between the inner ear and tially assembled COX subcomplexes. Western blot analy- intracranial space. sis with antibodies directed to COX subunits I, II, and IV showed a decrease of these subunits in this patient compared with control. Overexpression of the COX10 We thank Dr G. Rambaud for referring the patient described in this protein in the patient’s fibroblasts proved that the de- article. tected mutation was indeed the disease cause.

References Ann Neurol 2004;56:560–564 1. Minor LB, Solomon D, Zinreich JS, Zee DS. Sound- and/or pressure-induced vertigo due to bone dehiscence of the superior semicircular canal. Arch Otolaryngol Head Neck Surg 1998; Human cytochrome c oxidase (COX) consists of 13 124:249–258. subunits; three of these are encoded by the mitochon- 2. Carey JP, Minor LB, Nager GT. Dehiscence or thinning of drial DNA. Because of the bigenomic origin of the bone overlying the superior semicircular canal in a temporal complex, isolated COX deficiencies can be caused by bone survey. Arch Otolaryngol Head Neck Surg 2000;126: 137–147. mutations in either the mitochondrial or the nuclear 3. Younge BR, Khabie N, Brey RH, Driscoll CL. Rotatory nys- genome. In contrast to complexes I, II, and III, no tagmus synchronous with heartbeat: a treatable form of nystag- mutations have yet been described in any nuclear- mus. Trans Am Ophthalmol Soc 2003;101:113–117; discus- encoded structural subunit of COX.1–3 However, six sion 117–118. genes involved in COX biogenesis have been linked to 4. Brantberg K, Bergenius J, Mendel L, et al. Symptoms, findings and treatment in patients with dehiscence of the superior semi- COX deficiency in humans (SURF1, SCO1 and SCO2, 4–13 circular canal. Acta Otolaryngol 2001;121:68–75. COX10, COX15, and LRPPRC). The COX10 and 5. Cremer PD, Minor LB, Carey JP, Della Santina CC. Eye COX15 proteins play a role in the mitochondrial heme movements in patients with superior canal dehiscence syndrome biosynthetic pathway. COX10 catalyzes the conversion align with the abnormal canal. Neurology 2000;55:1833–1841. of protoheme to heme O. COX15 exerts its role in the 6. Mong A, Loevner LA, Solomon D, Bigelow DC. Sound- and pressure-induced vertigo associated with dehiscence of the roof next step, in which heme O is converted to heme A, an 10 of the superior semicircular canal. AJNR Am J Neuroradiol essential group for the functioning of complex IV. 1999;20:1973–1975. To date, three patients harboring mutations in COX10 7. Deutschlander A, Strupp M, Jahn K, et al. Vertical oscillopsia in bilateral superior canal dehiscence syndrome. Neurology 2004;62:784–787. 8. Rambold H, Heide W, Sprenger A, et al. Perilymph fistula as- From the 1Department of Paediatrics, Nijmegen Centre for Mito- sociated with pulse-synchronous eye oscillations. Neurology chondrial Disorders, University Medical Centre Nijmegen, Nijme- 2001;56:1769–1771. gen, The Netherlands; and 2Metabolic Department, Dr. von 9. Hirvonen TP, Carey JP, Liang CJ, Minor LB. Superior canal Hauner Children’s Hospital, Ludwig-Maximilians-University, Mu- dehiscence: mechanisms of pressure sensitivity in a chinchilla nich, Germany. model. Arch Otolaryngol Head Neck Surg 2001;127: Received Apr 15, 2004, and in revised form Jun 14. Accepted for 1331–1336. publication Jun 15, 2004. 10. Leigh RJ, Zee DS. The neurology of eye movements. 3rd ed. Published online Sep 30, 2004 in Wiley InterScience Philadelphia: F. A. Davis, 1999. (www.interscience.wiley.com). DOI: 10.1002/ana.20229 11. Corbett JJ, Jacobson DM, Thompson HS, et al. Downbeating nystagmus and other ocular motor defects caused by lithium Address correspondence to Dr Van den Heuvel, Department of Pae- toxicity. Neurology 1989;39:481–487. diatrics, University Medical Centre Nijmegen, 6500 HB Nijmegen, The Netherlands. E-mail: [email protected]

560 © 2004 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services Fig 1. Molecular analysis of COX10 genomic DNA. (A) COX10 DNA sequence of control and patient blood; the arrow indicates the mutation in the start codon of COX10. (B) Restriction endonuclease analysis pattern with BstZI. The fragment harboring the mutation could be digested, whereas the control was undigested. The length of the fragments (in base pairs) are indicated on the right site of the figure. p, patient; f, father; m, mother; w, wild type. have been described.8,9 Here we report a new patient, tivities were measured in skin fibroblasts and muscle (slightly phenotypically classified as suffering from a Leigh-like modified from the method described previously14). disease, with a mutation in the start codon of the COX10 gene. The effect of this mutation on the as- Analysis of COX10 DNA sembly or stability of mitochondrial COX has been an- A group of 11 patients with an isolated COX deficiency at alyzed by two-dimensional blue-native electrophoresis. least expressed in cultured skin fibroblasts were included in this study. DNA was isolated from patients’ fibroblasts and parents’ blood.15 The oligonucleotide primers used for the Case Report amplification of COX10 genomic DNA were described pre- The male patient was born at term as the first child of viously.8 The DNA sequence was analyzed on an ABI 377 consanguineous parents. At 5 months’ age, he devel- sequencer (Perkin-Elmer, Oak Brook, IL). To confirm the oped progressive failure to thrive, and pronounced mo- presence of the mutation, we performed restriction fragment tor agitation was noted. Gross motor development was length polymorphism analysis with BstZI (Promega, Madi- severely delayed at 7 months. At this age, the patient sion, WI). showed generalized muscular hypotonia with persistent head lag at traction, ataxia, hypermetria, exaggerated COX10 Complementary DNA Construct, Virus tendon reflexes with enlarged reflex zones, low- Production, Infection, and Measurement amplitude nystagmus, and saccadic eye movements. of Enzyme Activity 9 Ocular fixation was weak. He was not able to grasp. The retroviral vector was created as described previously. Laboratory evaluation showed metabolic acidosis with COX activities were measured before and after overexpression of COX10 protein (as described by Capaldi and col- elevated serum and cerebrospinal fluid lactate concentra- leagues16 and Srere17). tions. Magnetic resonance imaging of the brain showed slight atrophy and hyperintense lesions in the thalamus, Protein Electrophoresis olives, and the nucleus ruber, a pattern comparable to One- and two-dimensional blue-native electrophoresis were Leigh-like disease. Biochemical COX activity was signif- performed with digitonin-isolated mitochondria.18 Sodium icantly reduced in muscle and fibroblasts (0.15 COX/ dodecyl sulfate polyacrylamide gel electrophoresis (SDS- citrate synthase [CS]; control range, 0.52–2.08; and PAGE) was performed according to the method of Schagger 0.22 COX/CS; control range, 0.68–1.19 COX/CS, re- and von Jagow.19 Proteins were transferred to a PROTAN spectively). The boy died at 9 months of age of acute nitrocellulose membrane (Schleicher & Schnell, Keene, NH). pneumonia and cardiorespiratory failure. Prenatal diag- Western blotting was performed using anti-COXI, anti- nosis was performed in a later pregnancy. Normal COX COXII, anti-COXIV (all from Molecular Probes, Eugene, activity was found in chorionic villi, and the mother OR), anti–mitochondrial HSP70 (Alexis, Molecular Probe, Eugene, OR), and peroxidase-conjugated anti–mouse immu- gave birth to a healthy girl. noglobulin G (Molecular Probes). The signal was detected by enhanced chemiluminescence with ECL Plus (Amersham Materials and Methods Biosciences, Arlington Heights, IL). Cell Culture and Biochemical Measurements Human skin fibroblasts were cultured in M199 (Life Tech- Results nologies, Bethesda, MD) supplemented with 10% fetal calf Eleven patients with decreased COX activity estab- serum and antibiotics. Mitochondrial OXPHOS complex ac- lished in cultured fibroblasts (data not shown) were an-

Coenen et al: Novel COX10 Gene and COX Assembly 561 alyzed for mutations in the COX10 gene. A homozy- tection of COX subcomplexes. The patient’s fibroblasts gous mutation in the COX10 start codon was detected display a general decrease of all subcomplexes as well as in one patient. The T3C transition of the second a lesser amount of holo COX (Fig 2B). This general base of the start codon (ATG) results in the abolition decrease of the subcomplexes was also detected with an of the start site for protein translation (Fig 1A). Besides antibody directed to COXII (data not shown). this mutation, two polymorphisms were detected in SDS-PAGE showed hardly detectable mitochondrial- control and COX-deficient patient material (comple- encoded COXI and COXII levels in the patient’s fi- mentary DNA G476A [dbSNP rs8077302] and broblasts (Fig 2C). The nuclear-encoded subunit A699G [dbSNP rs2230354]). The mutation present in COXIV steady state level was also reduced compared the COX10 start codon was confirmed by restriction with control levels. The effect of COX10 overexpres- enzyme analysis (Fig 1B). sion resulted in elevated levels of COXI and COXII in To investigate the functional significance of the de- patient and control material. COXIV level was in- scribed mutation, we infected patient and control fibro- creased after overexpression of COX10 but to a lesser blasts with a retrovirus containing human COX10 com- extent than the COXI and COXII levels (Fig 2C). plementary DNA. COX activity was established before Taken together, these results demonstrate that the mu- and after infection of the cells. Before infection, COX tation in the COX10 gene is responsible for the COX activity in the patient’s fibroblasts was 0.09 COX/CS. In defect observed in the patient. the patient cell lines, the COX activity rose to 0.51 COX/CS. Control fibroblasts infected with COX10 Discussion showed a COX-to-CS ratio of 0.64. This experiment This article describes the results of a mutational anal- suggests that the deficiency in the patient’s fibroblasts ysis study in the COX assembly gene COX10.In1of has been mainly rescued by overexpression of wild-type 11 patients who suffered from Leigh-like disease, a mu- COX10. tation in the start codon of COX10 was found. Both One-dimensional blue-native gel electrophoresis was parents were heterozygous for this mutation. Two ad- performed to investigate the effect of the mutation on the ditional in-frame ATG codons are present, 72bp formation of COX. A reduction in the amount of COX downstream and 174bp upstream of the predicted start could be detected in patient material compared with that codon. These may serve as alternative translational start in control (Fig 2A). Blue-native electrophoresis showed sites. However, these start sites will probably not lead that overexpression of COX10 results in an increase in the to a fully functional protein, as the COX enzyme ac- amount of COX holoenzyme complex (Fig 2A). tivity was substantially increased after overexpression of A two-dimensional gel (blue native/SDS) was also the COX10 protein. performed, as this method is more sensitive for the de- The effect of the mutation on the formation of

Fig 2. (A) Mitochondria were extracted from fibroblasts, and the protein complexes were separated on a 5–12% blue-native acryl- Ϫ amide gel. The gel was blotted. An antibody directed to COXI was used to detect COX in control (C) and patient (COX10 ) fibro- ϩ blasts and patient’s fibroblasts overexpressing COX10 (COX10 ). (B) For the separation of the individual subunits of control (upper panel) and patient (lower panel) fibroblasts, a 10% tricine–sodium dodecyl sulfate (SDS) gel was run in the second dimension, fol- lowed by western blotting. The directions of the first (blue-native [BN]) and second (SDS) dimension are shown in the upper-right corner. For the positioning of COX, a primary antibody against the COX subunit COXI was used. Lanes 1–3 indicate the previously described COX assembly intermediates; lane 4 indicates holo-COX. (C) For SDS polyacrylamide gel electrophoresis, 30␮g of whole-cell Ϫ ϩ lysates isolated from control (C) and patient (COX10 ) fibroblasts and patients’ fibroblasts overexpressing COX10 (COX10 ) was used. The proteins were separated on a 10% tricine–SDS gel. The gel was blotted and incubated with antibodies raised against COX subunits COXI, COXII, COXIV, and against mitochondrial HSP70 as a loading control.

562 Annals of Neurology Vol 56 No 4 October 2004 Table. Clinical Presentation and Disease Course of COX-Deficient Patients due to a Mutation in COX10

Characteristic Patient 1a Patient 2b Patient 3b Patient 4c

Sex Male Male Female Male Age of presentation (mo) 18 1⁄4 11⁄2 5 Age of death (mo) 24 5 4 9 Consanguinous parents ϩ NM Ϫϩ Convulsions ϩϪϪϪ Ataxia ϩϪϪϩ Hypotonia ϩϩϩϩ Pyramidal syndrome ϩϪϪϩ Eye fixation 2 ϪϪ2 Hypertrophic cardiomyopathy ϪϩϪϪ Anemia (blood) NM ϩϩϪ Blood lactate (mmol/L)d 3.8 114.6 CSF lactate (mmol/L)e 3.1 NM 1 6.6 CMRI NM NM Leigh Leigh-like Residual COX activityf NM 5% 16% 29% aPatient 1 is previously described in Valnot and colleagues.8 bPatients 2 and 3 are, respectively, Patient D and S from Antonicka and colleagues.9 cPatient 4 is described in this report. dControl range 0.6–2.1 mmol/L. eControl range 1.4–1.9 mmol/L. fThe residual COX activity is presented as a percentage of the lowest control value as determined in muscle. NM ϭ not mentioned; CSF ϭ cerebral spine fluid; CMRI ϭ cerebral magnetic resonance imaging.

COX was studied using blue-native gel electrophoresis, All COX10 patients described so far showed differ- which demonstrated that the mutation leads to a lesser ent clinical features (Table). However, the disease amount of holo COX when compared with control progression is very fast; after the first clinical symp- material. This finding was also confirmed by two- toms, all patients died within a few months. Other dimensional gel electrophoresis, in which a general de- general features are hypotonia and elevated blood lac- crease in COX assembly could be detected in patient tate levels, which could be detected in all patients. All material. Similar observations were obtained in other patients showed a residual activity of COX, suggest- patients harboring a mutation in COX10.9,10 It is clear ing that COX10 is not essential for the formation of from both approaches that a general decrease in COX complex IV but is essential for the maintenance of assembly occurs when COX10 is mutated. wild-type levels. In the previously described patients, The effect of the mutation in COX10 on COX pro- COX10 could have some residual COX activity, as all tein levels resulted in a severe reduction in the steady patients have missense mutations; in the patient de- state levels of COXI, COXII, and COXIV subunits in scribed here, the residual activity could be explained the patient’s fibroblasts. This result is in line with west- by the use of alternative start codons. However, it ern blot experiments performed on samples from an- may also be possible that another mechanism converts other patient with a COX10 mutation.8 some heme O to heme A. These observations show The increase in COX activity after COX10 overex- that it is difficult to establish a general clinical picture pression was also reflected in COX protein levels. SDS- for COX10-deficient patients.8,9 PAGE shows that COXI and COXII protein levels are increased by overexpression of COX10 in the patient’s fibroblasts. The levels were higher than those in the This work was supported by grants from the Prinses Beatrix Fonds (98-0108 L.v.d.H., J.S.) and Marie Curie Fellowship (MCFI-2000- control cell line. One explanation is that overexpression 02003, C.U.). of COX10 might lead to higher than normal levels of heme. The heme groups can stabilize COXI, which We thank our colleagues from the Nijmegen Centre for Mitrochon- drial Disorders for measurement of the enzyme activities and E. might lead to higher COXI steady state levels. In con- Shoubridge for his invaluable help. trast to COXI and COXII, the COXIV levels do not reach control levels after COX10 overexpression. These results together suggest that COX10 overexpression References might lead to an accumulation of the subcomplexes, 1. Yano T. The energy-transducing NADH: quinone oxidoreduc- which might result in a slightly disturbed assembly of tase, complex I. Mol Aspects Med 2002;23:345–368. complex IV. This is also reflected in the enzyme activ- 2. Ackrell BA. Cytopathies involving mitochondrial complex II. ity of complex IV. Mol Aspects Med 2002;23:369–384.

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From the 1Childhood Cancer Research Unit, Department of Methods 2002;26:327–334. 2 19. Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate- Woman and Child Health, Karolinska Hospital; Department of Laboratory Medicine, Division of Pathology, Huddinge University polyacrylamide gel electrophoresis for the separation of proteins in Hospital; and 3Institute of Environmental Medicine, Division of the range from 1 to 100 kDa. Anal Biochem 1987;166:368–379. Molecular Toxicology, Karolinska Institutet, Stockholm, Sweden. Received Apr 9, 2004, and in revised form Jun 15. Accepted for publication Jun 15, 2004. Published online Aug 31, 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.20223 Address correspondence to Dr Henter, Childhood Cancer Research Unit, Department of Woman and Child Health, Karolinska Hospi- tal, 171 76 Stockholm, Sweden. E-mail: [email protected]