0031-3998/03/5302-0224 PEDIATRIC RESEARCH Vol. 53, No. 2, 2003 Copyright © 2003 International Pediatric Research Foundation, Inc. Printed in U.S.A.

Mutation Screening in Patients With Isolated c Oxidase Deficiency

SABRINA SACCONI, LEONARDO SALVIATI, CAROLYN M. SUE, SARA SHANSKE, MERCY M. DAVIDSON, EDUARDO BONILLA, ALI B. NAINI, DARRYL C. DE VIVO, AND SALVATORE DIMAURO Department of Neurology [S.Sa., L.S., C.M.S., S.Sh., M.M.D., E.B., A.B.N., D.C.D.V., S.D.], Columbia University College of Physicians and Surgeons, New York, New York 10032, U.S.A.; Department of Neurology [S. Sa.], University of Modena, Via Del Pozzo 71, 44100, Modena, Italy; and Center for Rare Diseases, Department of Pediatrics [L.S.], University of Padova, Via Giustiniani 3, 35128, Padova, Italy

ABSTRACT

Cytochrome c oxidase (COX) deficiency has been associated in a patient with and one novel SCO2 with a variety of clinical conditions and can be due to mutations in in a patient with hypertrophic cardiomyopathy. These data nuclear or mitochondrial . Despite recent progress in our show that heterogeneous clinical are associated with understanding of the molecular bases of COX deficiency, the ge- COX deficiency, that mutations in mtDNA COX genes are rare, and netic defect remains elusive in many cases. We performed mutation that mutations in additional genes remain to be identified. (Pediatr screening in 30 patients with biochemical evidence of isolated COX Res 53: 224–230, 2003) deficiency and heterogeneous clinical phenotypes. Sixteen patients had various forms of encephalomyopathy, and six of these had the neuroradiological features of Leigh syndrome. Four patients had Abbreviations encephalohepatopathy, six had hypertrophic cardiomyopathy, and COX, (EC 1.9.3.1) four had other phenotypes. We studied the three mtDNA genes mtDNA, mitochondrial DNA encoding COX subunits, the 22 mtDNA tRNA genes, and seven nDNA, nuclear DNA COX assembly genes: SCO1, SCO2, SURF1, COX10, COX11, SSCP, single strand conformational polymorphism COX15, and COX17. We report two novel pathogenic SURF1 LS, Leigh syndrome

Cytochrome c oxidase (COX), complex IV of the mitochondrial different polypeptides, and the delivery and of the pros- respiratory chain (EC 1.9.3.1), catalyzes the transfer of reducing thetic groups into the holoenzyme. In theory, COX deficiency equivalents from cytochrome c to molecular and utilizes may result from mutations in structural subunits of the or the energy generated by this reaction to pump protons across the in ancillary required for its assembly (2). mitochondrial inner membrane. COX, active as a dimer, com- To date, pathogenic mutations have been described in the three prises 13 subunits, two heme groups (a and a3), three ions mtDNA genes and in four COX assembly genes, SCO1 (3), SCO2 (two in the CuA site and one in CuB site), a zinc ion, and a (4), COX10 (5), and SURF1 (6, 7). These result in a variety of magnesium ion (1). The biogenesis of COX requires the interplay clinical phenotypes. However, in some patients with isolated of two genomes. Mitochondrial DNA (mtDNA) encodes the three COX deficiency, the molecular defects remain elusive. larger subunits (COX I, COX II, and COX III) that compose the In a series of 30 patients with COX deficiency and unknown catalytic core of the enzyme and contain the prosthetic groups. molecular causes, we screened for mutations in the three mtDNA Nuclear DNA (nDNA) encodes the 10 smaller COX subunits, COX genes, the 22 mtDNA tRNA genes, the four nDNA COX- which have regulatory and structural functions, and several acces- assembly genes already associated with disease in humans (SURF1, sory proteins, which control the folding and maturation of the SCO1, SCO2, and COX10), and three candidate COX-assembly genes (COX11, COX15, and COX17). The yield was meager: one

Received April 2, 2002; accepted October 11, 2002. patient had two novel mutations in SURF1, and one had a novel Correspondence: Salvatore DiMauro, M.D., 4-420 College of Physicians and Surgeons, mutation in SCO2 associated with the common E140 K mutation. 630 West 168th St, New York, NY 10032, U.S.A.; e-mail [email protected] This work was supported by National Institutes of Health Grants PO1HD32062 and NS11766 and by a grant from the Muscular Dystrophy Association. L.S. is supported by METHODS grant 439b from Telethon Italia and by a scholarship from the University of Padova. S.Sa. and L.S. contributed equally to this work. We studied 30 patients with isolated COX deficiency [COX DOI: 10.1203/01.PDR.0000048100.91730.6A activity in muscle (referred to citrate synthase) below 33% of the

224 MUTATION SCREENING IN COX DEFICIENCY 225 normal mean; other respiratory chain enzyme activities within The entire coding region of SCO1, SCO2, COX10, COX11, normal ranges]. Table 1 lists clinical and biochemical features. It COX15, and COX17 was amplified as summarized in Table 2. should be noted that four patients with typical Leigh syndrome SURF1 (7); mtDNA tRNA genes (12); and COX I, COX II, and (LS) had already been screened for SURF1 and SCO2 mutations COX III were amplified as described (13). and found negative by Sue et al. (8). They were included in this Mutation screening was performed by direct sequencing of study to be screened for other defects. Biopsies were ob- SURF1, SCO2, COX I, COX II, COX III, and mtDNA tRNA tained with the informed consent of parents or guardians, and all genes, using the ABI PRISM Dye Terminator Cycle Sequenc- studies were approved by the Institutional Review Board of ing Ready Reaction Kit and 310 Automatic Sequencer (Ap- Columbia University College of Physicians & Surgeons. plied Biosystem, Perkin Elmer, Foster City, CA, U.S.A.). The Biochemical analysis. Measurements of respiratory chain remaining genes were studied by single strand conformational activities were performed in as de- polymorphism (SSCP) analysis. scribed (9). COX activity was measured in in one patient SSCP analysis. A total of 100 ng of genomic DNA was and in in two patients. amplified as described in Table 2. Reactions were carried out in ␮ Histochemical analysis. . Muscle biopsies were stained for 25 L of 10 mM Tris-HCl (pH 8.9); 1.5 mM MgCl2; 0.4 mM COX and succinate dehydrogenase as described (10). each forward and reverse oligonucleotides; 0.2 mM each DNA analysis. DNA was extracted from tissues according to dATP, dGTP, and dTTP; 0.02 mM dCTP and 1 ␮Ci of ␣32P standard protocols (11). dCTP; and 1.25 units of TAQ DNA polymerase (Roche, Basel,

Table 1. Age of COX activity Patient onset/(death) Sex Clinical (% of controls) Family history Group 1: Leigh Syndrome 1 3 mo M Leigh syndrome 24 Negative 2 Birth F Leigh syndrome 13 Negative 3 2 mo M Leigh syndrome 9 Negative 4 2 mo F Leigh syndrome 4 Negative 5 6 mo F Leigh syndrome 23 Parental consanguinity 6 2 mo F Leigh syndrome 16 Negative Group 2: Encephalomyopathy 7 Birth M , lactic acidosis 28 Negative 8 Birth (9 days) F Encephalomyopathy 30 Negative 9 Birth M Encephalomyopathy, , lactic acidosis 14 Negative 10 Birth M Encephalomyopathy 3 Negative 11 Birth (18 days) F Encephalomyopathy 11 Parental consanguinity, one affected sister. 12 Birth M Encephalomyopathy, lactic acidosis 21 Negative 13 Birth (10 mo) M Encephalomyopathy, lactic acidosis 28 One affected brother 14 6 mo M Encephalomyopathy seizures 24 Negative 15 4 mo F Encephalomyopathy, lactic acidosis 22 Negative 16 3 mo M Encephalomyopathy 19 One affected sister Group 3 Encephalopathy, Myoclonus, and optic atrophy 17 Birth F Encephalopathy, seizures, myoclonus, optic atrophy 25 One affected sister 18 Birth F Encephalomyopathy, myoclonus, optic atrophy lactic 27 Negative acidosis Group 4 Hepatoencephalopathy 19 Birth (3 mo) M Hepatoencephalopathy, HCMP, 5* Negative 20 7 mo (12 mo) M Hepatoencephalopathy, seizures 16 Negative 21 Birth M Hepatoencephalopathy 1** Negative 22 Birth (9 mo) F Hepatoencephalopathy 12 Negative Group 5 Hypertrophic cardiomyopathy 23 2 years F HCMP 27 Negative 24 Birth (12 mo) F HCMP 6* Negative 25 Birth (16 days) M HCMP, lactic acidosis 21 Negative 26 Birth M HCMP, encephalomyopathy 5 Negative 27 Birth F HCMP, myopathy 29 Negative 28 Birth M HCMP, encephalomyopathy 7 One affected brother Group 6: Other 29 Birth F Isolated myopathy 27 Negative 30 3 years M Myoclonus, white matter disease, cerebellar atrophy, 26 One affected brother high CSF lactate * COX activity measured in heart. ** COX activity measured in liver. 226 SACCONI ET AL.

Table 2. Primers PCR Conditions DMSO % COX10 EX1 agacaccacgctctcctttc 94° 3 min, (94° 45 s, 55° 45 s, 72° 30 s) 33 cycles, 5 ctcgcacgtggtaataagag 72° 7 min EX 2 gggaggtgtagtcatcatttg 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 ggcagaaagtaacagagtaag 72° 7 min EX 3a aaccatttgagagcatttggg 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 ccctacatctattgagtcttc 72° 7 min EX 3b gatagaactagagccagactc 94° 3 min (94° 30s, 60° 30s, 72° 30s) 35 cycles, 5 taagacaggacctgcagttc 72° 7 min EX 4 tacagttgggactcctgttg 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 acagccatctaggaaaaagtg 72° 7 min EX 5 gttcagtactaaagcggaag 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 gtgggaaatgattcagatgaac 72° 7 min EX 6* tgatcactccaggttctctg 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 gtgctcatatgagactccac 72° 7 min EX 7a tgatgactgcctttgtctcc 94° 3 min, (94° 45s, 55° 45s, 72° 30s) 33 cycles, 5 ggagatgtacgcattgatgg 72° 7 min EX 7b ctggacatcaccacatggac 94° 3 min, (94° 30s, 62° 30s, 72° 30s) 30 cycles, 5 gcaatttctcttcttgtgttcc 72° 7 min COX11 EX 1a gatttgacctctcgtccctg 94° 3 min, (94° 30s, 61° 30s, 72° 30s) 35 cycles, 5 gaaagggttcgagctcttagg 72° 7 min EX 1b gagagaggactgaggtggcttg 94° 3 min, (94° 45s, 60° 45s, 72° 60s) 33 cycles, 5 atttgtcttgcacccccacctc 72° 7 min EX 2 tagatcatcaacacctaaaagtattg 94° 3 min, (94° 30s, 53° 30s, 72° 45s) 35 cycles, 5 tagtcctctgacagtttaagtgatg 72° 7 min EX 3 aggtattatggtctgatgaataggc 94° 3 min, (94° 30s, 54° 30s, 72° 45s) 33 cycles, 5 cactaagtttcatactaaaccgtttc 72° 7 min EX 4* tcacgaagtgtactggaatgctaag 94° 3 min, (94° 30s, 53° 30s, 72° 45s) 35 cycles, gataaaagctgaacttgttctctcaag 72° 7 min COX17 EX 1 tgcctcttctgcgcacgcg 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 35 cycles, 5 tctccggctgcgcactgac 72° 7 min EX 2 gtgttttgtctcactccccatc 94° 3 min, (94° 30s, 57° 30s, 72° 30s) 36 cycles, 5 caggtgaactacctttcagag 72° 7 min COX15 EX 1 cgacccggaagtgcttctc 94° 3 min, (94° 30s, 63° 30s, 72° 30s) 32 cycles, 5 cctttcactccatccccgc 72° 7 min EX 2 gccattccctgtttctccag 94° 3 min, (94° 30s, 60° 30s, 72° 30s) 35 cycles, 5 tctcctcagtcaacctgtgc 72° 7 min EX 3 ccttttgtgagtaatccagcc 94° 3 min, (94° 30s, 62° 30s, 72° 30s) 34 cycles, 5 aagatcaaatgggcctactgg 72° 7 min EX 4 aggatgtttcctcctcctcc 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 37 cycles, 5 gaagctcctgggagcatttc 72° 7 min EX 5 gttgaacagatcattttaaccttg 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 35 cycles, 5 tatccaaaagaaccaaaagcgg 72° 7 min EX 6 cctctcattgttcctgcaac 94° 3 min, (94° 30s, 54° 30s, 72° 30s) 35 cycles, 5 cttagagtgcaatgcaaacag 72° 7 min EX 7 ctagtatttggtctcttcttcc 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 35 cycles, 5 caaagcctatgataggctcc 72° 7 min EX 8 acctggcacttctatctaatg 94° 3 min, (94° 30s, 54° 30s, 72° 30s) 35 cycles, 5 ctttgtattggggatactgtc 72° 7 min EX 9a gttactgcctttcctattgcc 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 35 cycles, 5 gcccaagttcttatgatctctg 72° 7 min EX 9b attgttgactgctgaattaatgtg 94° 3 min, (94° 30s, 55° 30s, 72° 30s) 35 cycles, 5 actgttttcagttgccactgtc 72° 7 min SCO1 EX 1a gacagagcgactccttccg 94° 3 min, (94° 45s, 58° 45s, 72° 30s) 32 cycles, 5 gttcccaggcaatagccag 72° 7 min EX 1b gagtcttgctgaggcagttctg 94° 3 min, (94° 45s, 59° 45s, 72° 30s) 34 cycles, 5 agtgtctgaggttacgaccaag 72° 7 min EX 2 catcatggcctgtgtgtat 94° 3 min, (94° 30s, 56° 30s, 72° 1m) 33 cycles, 5 ccaggaagacctttataca 72° 7 min EX 3 tgtcgatatgtttttgtctcctt 94° 3 min, (94° 45s, 60° 45s, 72° 30s) 34 cycles, 5 ttgtttagttagtgatggctttc 72° 7 min EX 4 ccagcacgtcctatactctc 94° 3 min, (94° 30s, 56° 30s, 72° 1m) 33 cycles, 5 ctatttcacaaggcactgtaagg 72° 7 min EX 5 tggcttctcatttttcactgtc 94° 3 min, (94° 45s, 60° 45s, 72° 30s) 34 cycles, 5 ctcttctcagaagctagtcag 72° 7 min EX 6 gcttattttggtaatctttgtcacac 94° 3 min, (94° 45s, 56° 45s, 72° 30s) 34 cycles, 5 tctcatggtatgaaggccattc 72° 7 min SCO2 EX2 A* ttcagatgggtggctggtct 94° 3 min, (94° 1m, 56° 1m, 72° 30s) 35 cycles, 10 ctgttcgcttttgctgctgc 72° 7 min EX2 B* gaacccggctgctgatcac 94° 3 min, (94° 1m, 56° 1m, 72° 30s) 35 cycles, 10 ggcctgcattgtagtacacg 72° 7 min EX2 C* gcctgtcttcatcactgtgg 94° 3 min, (94° 1m, 56° 1m, 72° 30s) 35 cycles, 10 tacacctgcgcagagaagag 72° 7 min * Exon studied by direct sequencing. MUTATION SCREENING IN COX DEFICIENCY 227 Switzerland). Samples were denatured and separated on a 6% MDE polyacrylamide (BME, Rockland, ME, U.S.A.), with 5% glycerol, according to the manufacturer’s protocol. Single- stranded conformers were visualized by autoradiography using BIOMAX film (Kodak, Rochester, NY, U.S.A.). Samples with abnormal patterns were sequenced as above. Analysis of insertions/deletions. harboring heterozy- gous insertions or deletions were amplified, and PCR products were subcloned in a pCRIITOPO Vector using a Topo TA Cloning kit (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. Plasmid DNA was extracted using a Plasmid Mini Kit (Qiagen Inc., Valencia, CA, U.S.A.) and se- quenced using M13R and M13–40 primers. Hydropathy plots were calculated with the Kyte-Doolittle algorithm using DNA Strider 1.2 software (CEA, Gif-Sur-Yvette, Cedax, France).

RESULTS

We studied 30 unrelated patients with isolated COX defi- ciency. COX activity in affected tissues ranged from 3% to 30% of control samples. Other respiratory chain enzyme ac- Figure 1. (A) Reverse-complemented sequence of the allele harboring the 608 tivities were normal. The biochemical finding of COX defi- T3C mutation (arrow). The black triangle indicates the 610 A3T polymor- B ciency was confirmed by histochemical analysis. None of the phism. ( ) Alignments of Surf1 polypeptides in different species. Note in our patient the L203P mutation (arrow) and the adjacent I204F polymorphism patients had evidence of ragged-red fibers on muscle biopsy. (underlined). (C) Hydropathy plots of the Surf1 region with the L203P All of our patients had onset of disease in early childhood mutation (aa 150–250). The arrow indicates the altered peak in the patient’s and clusters of clinical symptoms suggestive of mitochondrial . Note that the I204F substitution does not alter the plot. disorder (Table 1). They can be divided into six groups on the basis of the clinical phenotype. Group 1 patients (1–6) had clinical and neuroradiological features of LS; group 2 patients (7–16) had encephalomyopathy but lacked the neuroradiologi- cal features of LS. Group 3 patients (17, 18) had encephalop- athy, myoclonus, and optic atrophy. Group 4 patients (19–22) had hepatoencephalopathy as the main clinical feature. Group 5 patients (23–28) had hypertrophic cardiomyopathy. Group 6 patients (29 and 30) had clinical pictures not classifiable within any of the previous groups. We were able to detect pathogenic mutations in only two of these patients. Patient 1, a child with LS, was a compound heterozygote for SURF1 mutations. The first mutation was a T3C transversion at nucleotide 608 (Fig. 1A) that changes a highly conserved Figure 2. (A) Reverse-complemented sequence of the allele harboring the 675_692del. (B) Alignments of Surf1 polypeptides in different species. Un- leucine to a proline at position 203 (L203P). The second derlined the six amino acids deleted in the patient. mutation was an 18-bp 675_692del in exon 7 (Fig. 2A); the resulting protein lacked six highly conserved amino mtDNA-encoded COX subunits; in the 22 tRNA genes; or in acids (aa 226–231), and leucine at position 232 was changed to SCO1, COX10, COX11, COX15, and COX17. isoleucine. This is the first in-frame deletion reported in SURF1. Subcloning and sequencing the PCR fragment con- DISCUSSION taining exons 6 and 7 demonstrated that the two mutations were located in different alleles. COX deficiency is a relatively common biochemical finding Patient 28 with hypertrophic cardiomyopathy and encepha- underlying a variety of clinical conditions. It can occur as an lomyopathy had two mutations in SCO2. He harbored the isolated defect, or it can accompany defects of other respiratory common E140 K mutation and a novel T3C transition at nt chain enzymes containing mtDNA-encoded subunits (14). The 1575 (Fig. 3A) that changed a conserved leucine at position latter situation usually reflects mtDNA mutations, either large- 151 to a proline (L151P). We analyzed the DNA of his parents scale rearrangements of mtDNA or mutations in tRNA genes. and older brother, who had died some years earlier of a similar The genetic causes for isolated COX deficiency can be divided disease. The brother harbored both mutations, whereas the into two main groups. The first includes mutations in structural father carried the E140 K and the mother carried the L151P components of the enzyme, which have been described only in mutation. These three novel mutations were absent in 100 the three mtDNA-encoded COX subunits but not yet in any of controls. No pathogenic mutations were found in the three the 10 nuclear encoded polypeptides. These mutations are 228 SACCONI ET AL. Clinical data for SCO1 and COX10 mutations are scarce. SCO1 defects have been described in two brothers with neo- natal-onset hepatic failure and encephalopathy (3), whereas COX10 mutations have been reported in a family with enceph- alopathy and renal tubulopathy (4). These findings suggest that COX assembly pathways are, to a certain extent, tissue-specific (2). In addition, four patients with myoclonic epilepsy, , hearing loss, and isolated COX deficiency in muscle were found to harbor mutations in mtDNA tRNASer(UCN) (29). The Figure 3. (A) Sequence of genomic DNA showing the heterozygous 1575 precise mechanism by which a mutation in a tRNA gene T3C (L151P) mutation (arrow). (B) Alignments of Sco2 polypeptides in produces isolated COX deficiency is still unclear. different species. Arrow indicates the L151P mutation. We surveyed all genes thus far associated with COX defi- ciency in a series of 30 patients with various clinical manifes- tations and biochemical evidence of isolated COX deficiency. usually sporadic and are associated with variable clinical phe- We also studied three COX-assembly genes, COX11, COX15, notypes, ranging from exercise intolerance and myoglobinuria and COX17, which are known to cause COX deficiency in to severe multisystemic disease. Ages at onset also vary from when mutated, but have not yet been associated with neonatal period to adulthood (2, 15). human disease. We decided not to screen the 10 nDNA- The second group includes mutations in COX-assembly genes. encoded COX genes because previous studies (29, 30), includ- The proteins encoded by these genes are not structural compo- ing one by our group (M. Hirano unpublished results), failed to nents of COX but are required for the correct folding and matu- show any mutation. ration of the various polypeptides and for the synthesis, delivery, We identified pathogenic mutations in only two patients. One and insertion of the prosthetic groups into the holoenzyme. They patient had hypertrophic cardiomyopathy and mutations in SCO2. have been studied extensively in yeast, and some of their human He had the classical phenotype seen in SCO2 defects: hypertro- homologues have been identified (Table 3) (16–22). Mutations in phic cardiomyopathy in the first days of life, encephalomyopathy, COX-assembly genes usually present in the neonatal period or in and rapid progression, with death in the second month of life. He early childhood, have an autosomal recessive pattern of inheri- harbored the common E140 K mutation and a novel L151P tance, and are associated with severe phenotypes (2). mutation. This leucine is conserved throughout evolution (Fig. SURF1 mutations have been reported in Ͼ40 patients (23). 3B). We aligned Ͼ80 SCO proteins from different eukaryotic and They are usually associated with classical LS, although they prokaryotic species. In some species, leucine is substituted either have also been reported in one patient with by valine or by isoleucine, both hydrophobic amino acids; no (24) and in another with normal neuroimaging, which was other was found in the reported sequences. We believe limited, however, to computerized tomography (25). that proline disrupts the secondary structure of the SCO2 protein SCO2 defects have been described in 10 patients (4, 26–28). in a region close to the putative copper binding site. Only one of All of them harbored one common mutation, E140 K. Patients the six patients with hypertrophic cardiomyopathy had mutations with the severe form of the disease are compound heterozy- in SCO2, confirming the notion that this clinical phenotype is gotes, harboring the E140 K and one other mutation. They genotypically heterogeneous (31). usually present soon after birth with hypertrophic cardiomy- The second patient, with typical features of LS, had muta- opathy, encephalopathy, and myopathy. One patient, however, tions in SURF1. He had an 18-bp deletion in exon 7 and a presented at birth with a Werdnig-Hoffmann–like syndrome 608T3C changing leucine 203 to proline and developed cardiomyopathy later (28). The severe form is (L203P). Interestingly, this mutation is associated with another fatal within the first year of life. In contrast, patients homozy- nucleotide change, a 610 A3T transversion that changes a gous for the E140 K mutation exhibit a milder phenotype, with nonconserved isoleucine to a phenylalanine (I204F), probably later onset and slower progression of disease (27). a neutral polymorphism. Leucine at position 203 is conserved throughout evolution (Fig. 1B), and the substitution with a Table 3. proline clearly alters the hydropathy plot of the protein, Yeast Human Function whereas the I204P change affecting the adjacent amino acid residue does not alter it (Fig. 1C). The resulting protein is COX10 COX10 Heme synthesis COX11 COX11 Copper metabolism: probably inserts a copper probably incorrectly folded. The 675_692del ablates six con-

ion in CuB site of COX I served amino acids, including tryptophan at position 227 and COX15 COX15 Heme synthesis aspartate at position 231, which are conserved even in prokary- COX17 COX17 Copper transport to intermembrane space otic homologues of SURF1 (Fig. 2B). SCO1 SCO1 Copper metabolism, probably inserts a copper This patient was the only one of six with the classical ion in CuA site of COX II SCO2 SCO2 Copper metabolism, probably inserts a copper features of LS in which we could find SURF1 mutations.

ion in CuA site of COX II However, four of the six patients with typical LS had been part SHY1 SURF1 Unclear. Probably stabilizes one of the of a cohort previously screened by Sue et al. (8) for SURF1 intermediate complexes during COX mutations (and found negative). Thus, our LS group was biased assembly against SURF1 . Of the two unbiased LS patients, one MUTATION SCREENING IN COX DEFICIENCY 229 (50%) harbored SURF1 mutations, a frequency close to the reduced in patients (39, 40), including those harboring mis- 41% found by Sue et al. (8), although lower than that found in sense mutation (17). A similar approach could be used for a large series of European patients (10, 32, 33). Complemen- other COX-assembly genes. tation studies in European patients also showed that the ma- Further research will be designed to identify new genes jority of COX-deficient LS patients belong to a single comple- responsible for COX deficiency. We believe that functional mentation group (34, 35). Ethnic factors could account for the complementation studies are the most promising approach to lower frequency of SURF1 mutations in our American popu- the problem (2). We are currently performing such experiments lation, which includes patients of Hispanic, Arabic, Asian, and in fibroblasts from some of the patients in this series. African origin. Mutations in the region or in intronic regions not amplified by our primer set might have been overlooked, REFERENCES although our protocols (7) allow us to sequence all SURF1 1. Michel H, Behr J, Harrenga A, Kannt A 1998 Cytochrome c oxidase: structure and exons and all those intronic regions where mutations have been spectroscopy. Ann Rev Biophys Biomol Struct 27:329–356 reported thus far (16, 36, 37). It is noteworthy, however, that in 2. Shoubridge EA 2001 Cytochrome c oxidase deficiency. Am J Med Genet 106:46–52 3. 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