BACKGROUND: Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. METHODS: Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. RESULTS: We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. CONCLUSIONS: We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested.Supported by Telethon-Italy grant GGP15041 (to DG); Telethon-Italy Network of Genetic Biobank grant GTB12001J; ERC advanced grant ERC FP7-322424 (to MZ). MRC QQR grant MC_UP_1002/1

Ardissone, Anna

Fernandez-Vizarra, Erika

Ghezzi, Daniele

Martinez Lyons, Anabel

Moroni, Isabella

Reyes, Aurelio

Robinson, Alan J

Zeviani, Massimo

English

PubMed

Background Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aalong mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. Methods Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. Results We found compound heterozygous mutations in COA7: a paternal c.410A > G, p.Y137C, and a maternal c.287+1G > T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. Conclusions We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested

Lyons A. M.

Ardissone A.

Reyes A.

Robinson A. J.

Moroni I.

Ghezzi D.

Fernandez-Vizarra E.

Zeviani M.

Archivio istituzionale della ricerca - Università di Padova

COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency

Background Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins.Methods Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts.Results We found compound heterozygous mutations in COA7: a paternal c. 410A>G, p.Y137C, and a maternal c. 287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase.Conclusions We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested

A.M. Lyons

A. Ardissone

A. Reyes

A.J. Robinson

I. Moroni

D. Ghezzi

E. Fernandez-Vizarra

M. Zeviani

AIR Universita degli studi di Milano

Apollo (Cambridge)

SHORT REPORTCOA7 (C1orf163/RESA1) mutations associated withmitochondrial leukoencephalopathy and cytochromec oxidase deficiencyAnabel Martinez Lyons,1 Anna Ardissone,2 Aurelio Reyes,1 Alan J Robinson,1Isabella Moroni,2 Daniele Ghezzi,3 Erika Fernandez-Vizarra,1 Massimo Zeviani11MRC-Mitochondrial BiologyUnit, Cambridge,Cambridgeshire, UK2Department of ChildNeurology, Milan, Italy3Unit of MolecularNeurogenetics of theFondazione IstitutoNeurologico “Carlo Besta”,Milan, ItalyCorrespondence toProfessor Massimo Zeviani andDr. Erika Fernandez Vizarra,MRC Mitochondrial BiologyUnit, Wellcome Trust/MRCBuilding, Hills Road,Cambridge CB2 0XY, UK;mdz21@mrc-mbu.cam.ac.uk;emfvb2@mrc-mbu.cam.ac.ukReceived 1 August 2016Accepted 30 August 2016To cite: Martinez Lyons A,Ardissone A, Reyes A, et al.J Med Genet 2016;53:846–849.ABSTRACTBackground Assembly of cytochrome c oxidase (COX,complex IV, cIV), the terminal component of themitochondrial respiratory chain, is assisted by severalfactors, most of which are conserved from yeast tohumans. However, some of them, including COA7, arefound in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containingfive Sel1-like tetratricopeptide repeat sequences, whichare likely to interact with partner proteins.Methods Whole exome sequencing was carried out ona 19 year old woman, affected by early onset,progressive severe ataxia and peripheral neuropathy, mildcognitive impairment and a cavitating leukodystrophy ofthe brain with spinal cord hypotrophy. Biochemicalanalysis of the mitochondrial respiratory chain revealedthe presence of isolated deficiency of cytochrome coxidase (COX) activity in skin fibroblasts and skeletalmuscle. Mitochondrial localization studies were carriedout in isolated mitochondria and mitoplasts fromimmortalized control human fibroblasts.Results We found compound heterozygous mutationsin COA7: a paternal c.410A>G, p.Y137C, and amaternal c.287+1G>T variants. Lentiviral-mediatedexpression of recombinant wild-type COA7 cDNA in thepatient fibroblasts led to the recovery of the defect inCOX activity and restoration of normal COX amount. Inmitochondrial localization experiments, COA7 behavedas the soluble matrix protein Citrate Synthase.Conclusions We report here the first patient carryingpathogenic mutations of COA7, causative of isolatedCOX deficiency and progressive neurological impairment.We also show that COA7 is a soluble protein localizedto the matrix, rather than in the intermembrane space aspreviously suggested.INTRODUCTIONCytochrome c oxidase (COX) is the terminal com-ponent of the mitochondrial respiratory chain(MRC). COX transfers four electrons from cyto-chrome c to oxygen. The energy liberated by thisredox reaction sustains the translocation of fourprotons from the matrix to the intermembranespace (IMS) and contributes to the formation ofthe mitochondrial electrochemical gradient. Inhumans, COX deficiency can be either isolated orcombined to defects in other MRC complexes.Phenotypes may vary1 2 from multisystem disordersto isolated myopathies or encephalopathies.3 4Isolated COX deficiency may be caused bymutations in mitochondrial DNA (mtDNA) ornuclear DNA genes encoding structural COX subu-nits, but in most cases is due to recessive mutationsin nuclear genes encoding factors involved in COXbiogenesis.5 COA7 is a putative COX assemblyfactor containing five Sel1-like repeat domains(Interpro IPR006597), is conserved in animals, butneither in plants nor in fungi, and has beenreported to localise in the mitochondrial IMS.6Here, we describe the first biallelic pathogenicCOA7 mutations in a patient with isolated COXdeficiency and leukoencephalopathy, and show thatthe protein is localised in the inner mitochondrialcompartment, predominantly in the matrix.CASE REPORTThe proband is a 19-year-old woman, first child ofhealthy unrelated parents. Her family history wasunremarkable. She was born at term after a normalpregnancy. The perinatal period was uneventfuland her early development was referred to asnormal, but after 1 year of age, psychomotor delaybecame evident. She started walking autonomouslyat 22 months, with poor balance and frequent falls.At 3 years of age, she developed a demyelinatingsensorimotor neuropathy and a brain MRI dis-closed supratentorial leukodystrophy. During herchildhood, the clinical signs remained stable. At10 years, her walking difficulties worsened, andlimb weakness and tremor ensued. The neuro-logical evaluation showed dysarthria, dysmetria,ataxic gait and hyporeflexia in the four limbs withmuscle wasting. She was able to walk alone onlyfor a few steps with an ataxic gait. Mild cognitiveimpairment was documented (IQ 75, WISC-Rscale). Histological analysis of a muscle biopsyshowed hypo/atrophy of fibres. The clinical evolu-tion was slowly progressive. At her last follow-upexamination, at 19 years of age, she was able towalk alone only with ankle-foot orthotic aids andhad developed a marked dorsal-lumbar scoliosis.Other clinical signs were stable. Neurophysiologicalstudies confirmed worsening of her mixed axonaldemyelinating peripheral neuropathy. Brain andspinal cord MRI showed mild extension of signalabnormalities and extensive cavitations in the cere-bral white matter; the cerebellum and brainstemwere spared but the spinal cord was thin with noobvious focal lesions (figure 1A). Plasma lactatewas 2.9 mM (n.v. <2.1).846 Martinez Lyons A, et al. J Med Genet 2016;53:846–849. doi:10.1136/jmedgenet-2016-104194Mitochondrial geneticsMETHODSWhole exome sequencing (WES) was performed as described.7Fibroblasts from skin biopsies were immortalised by lentiviraltransduction using the pLOX-Ttag-iresTK vector (Tronolab,Addgene #12246). Primary and immortalised fibroblasts andHEK293T cells were grown in standard conditions. For mitochon-drial localisation studies, mitochondria and mitoplasts were preparedand trypsin-digested as previously described.8 9 For membrane asso-ciation studies, sonicated mitochondrial supernatants containing thesoluble fractions were separated from the membrane-enrichedpellets by ultracentrifugation. Membrane-containing pellets weretreated with increasing ionic strength buffers, and for the finalprotein dissociation, they were suspended in equal volumes of iso-tonic buffer containing 0.2% SDS. These fractions were analysed bywestern-blot (WB) immunodetection, using several proteins asmarkers of distinct mitochondrial compartments.The COA7 cDNA sequence was obtained from the IMAGEclone (ID: 4430419/IRATp970-0D0921D). PCR products werecloned into the lentiviral vector pWPXLd-ires-PuroR (derived frompWPXLd, Tronolab, Addgene #12258). Lentiviral particle produc-tion and transduction of target cells were performed as recom-mended (https://www.addgene.org/tools/protocols/plko/#E).Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) and blue native gel electrophoresis (BNGE) wereperformed as described.10 Antibodies were purchased fromProteintech, Abcam and Sigma.MRC and other enzymatic activities were measured asdescribed.11 12RESULTSBiallelic mutations in COA7 are present in the probandThis study was approved by the Ethical Committee of the ‘CarloBesta’ Neurological Institute, Milan, Italy, in agreement with theDeclaration of Helsinki. Informed consent was signed by theparents of the patient. We ruled out the presence of pathogenicmutations in mtDNA by Sanger sequencing. WES7 was thencarried out. After standard filtering steps and assuming a recessivetrait, we prioritised 13 genes with either one homozygous variantor two heterozygous variants. Of these 13 genes, the only oneencoding a mitochondria-targeted protein was COA7,6 whichcarried a (paternal) heterozygous nucleotide transition c.410A>G(NM_023077), p.Y137C and a (maternal) heterozygous G>Ttransversion affecting the first intronic nucleotide of the exon 2/intron 2 junction (c.287+1G>T). The two variants were notreported in the ExAc database (March 2016). The presence of bothmutations was validated by Sanger sequencing.COA7 mutations are associated with absence of the COA7proteinAmplification of the patient COA7 cDNA yielded two bands.The upper band, which had the same size as that amplified fromcontrol samples, showed the c.410A>G paternal missense muta-tion. The lower band was originated from the maternal allelecontaining the c.287+1G>T-splicing mutation, which producesthe deletion of the whole 141bp-long exon 2 of COA7 (figure1B). The missense mutation affects the highly conserved Tyr137,which is replaced by a non-conservative Cys residue, whereasthe splice site mutation produces the in-frame deletion of the 47amino acids encoded by exon 2. This deletion spans almost allof the first conserved Sel1-like domain and half of the second(figure 1C).Next, total protein lysates extracted from patient-derived cellswere analysed by WB using a specific anti-COA7 antibody. NoCOA7 cross-reacting material was immunodetectable, and theamount of two COX structural subunits (MT-CO2 andFigure 1 Clinical and genetic findings. (A) Transverse supratentorial (i), coronal (ii) and sagittal brain/spinal cord (iii) T2- fluid attenuated inversionrecovery (FLAIR) MRI sequences. (B) Sanger sequence of the mutated regions in cDNA from mutant fibroblasts. (C) Clustal W diagram. Boxed areascorrespond to mutations. Sel1-like conserved domains are underlined.Martinez Lyons A, et al. J Med Genet 2016;53:846–849. doi:10.1136/jmedgenet-2016-104194 847Mitochondrial geneticsMT-CO3) was reduced in the mutant sample, relative to controlcells, whereas that of subunits belonging to complex I andcomplex III was normal (figure 2A).COX activity and assembly are recovered after expression ofwild-type COA7In patient-derived immortalised fibroblasts, the levels of fullyassembled cIV and of the cIII2+cIV supercomplex were clearlylower relative to controls (figure 2B).In order to test complementation, wild-type (wt) COA7(COA7wt) was transduced in immortalised mutant fibroblasts.Robust overexpression of COA7wt was detected in both mutantand wt transduced cells, and, in mutant cells, this was associatedwith the recovery of normal amount of MT-CO2 subunit(figure 2C) and of both COX assembly (figure 2D) and activity(figure 2E).COA7 is localised in the mitochondrial matrixTo further characterise COA7, we isolated mitochondria fromHEK293T cells and performed WB analysis in organellar andsuborganellar fractions. Trypsin digestion was carried out in bothintact mitochondria or after stripping off the outer mitochondrialmembrane (mitoplasts), by either hypotonic shock or digitonintreatment. The outer membrane protein TOM70 was completelydigested by trypsin in mitochondria and mitoplasts; the IMSprotein cytochrome c was protected in mitochondria but progres-sively digested in mitoplasts, whereas the inner membraneprotein succinate dehydrogenase subunit A (SDHA) (part of cII)and the matrix protein citrate synthase were protected fromdigestion in both mitochondria and mitoplasts, as was COA7(figure 2F). Next, mitochondria were disrupted by sonication,and the ionic strength was increased by adding NaCl to solubleversus membrane fractions. As shown in figure 2G, COA7 waspredominantly immunodetected in the supernatant, similar tocitrate synthase, although a small percentage was also detected inthe pellet, before SDS treatment. Taken together, these resultsindicate that COA7 is predominantly localised in the mitochon-drial matrix, although a small aliquot appears to be associatedwith the inner mitochondrial membrane.DISCUSSIONCOA7 is involved in MRC biogenesis, as depletion by RNAi incultured cells clearly affected COX assembly.6 Here, we presentthe first pathological mutations in COA7 in humans, associatedwith a severe, slowly progressive mitochondrial neurological dis-order. We found biallelic mutations in one patient, according tothe autosomal recessive mode of inheritance suggested by thepatient’s family tree. The COA7 mRNA skipping of exon 2 inthe maternal allele and the drastic amino acid change in thepaternal allele suggested pathogenicity. Indeed, WBFigure 2 Western blot (WB) immunodetection and enzymatic activities. (A) WB immunodetection of SDS-PAGE in primary fibroblasts. P, patient,C1 and C2, control cell lines. (B) WB immunodetection of BNGE in immortalised patient (Pi) and control (C3i) fibroblasts. Immunovisualised subunitsare between brackets. (C) WB immunodetection of SDS-PAGE in immortalised patient fibroblasts (Pi) and a control (C4i). n.t., not transduced; mock,transduced with empty vector; COA7, transduced with COA7. (D) WB immunodetection of BNGE in COA7-transduced immortalised fibroblasts frompatient (Pi) and a control (C4i). (E) Cytochrome c oxidase (COX)/citrate synthase (CS) activity in immortalised fibroblasts of two controls (C3i, C4i),and the patient (Pi). Vertical bars indicate SEM. Statistical analysis was by ANOVA (*p<0.05; **p<0.01; ***p<0.001). (F) WB immunodetection ofSDS-PAGE of mitochondria and mitoplasts from HEK293T cells. (G) WB immunodetection of SDS-PAGE of soluble and membrane fractions fromintact mitochondria. ANOVA, analysis of variance.848 Martinez Lyons A, et al. J Med Genet 2016;53:846–849. doi:10.1136/jmedgenet-2016-104194Mitochondrial geneticsimmunodetection showed the virtual absence of the protein incultured cells. Furthermore, the expression of COA7wt restoredCOX assembly and activity in mutant cells.Our case was diagnosed as a mitochondrial encephalopathyand peripheral neuropathy. Although the onset was in infancy,the clinical course was slowly progressive, as the patient hasreached adulthood in relatively stable conditions. This could berelated to a partially dispensable role of COA7 in COXbiogenesis.Finally, our suborganellar fractionation studies, supported byin vitro import results, indicate that COA7 localises in the mito-chondrial inner compartment, mostly in the matrix, rather thanin the IMS, as previously proposed.6 We suggest that the pro-posed localisation to the IMS was in fact the consequence of aincorrect interpretation of results similar to ours, that is, protec-tion of COA7 from proteinase K-treated mitoplasts, thusindicating its localisation within the inner mitochondrialcompartment.6Future work is warranted to establish the interactions andpartners of COA7, its mechanisms of action and the steps of itsintervention during the formation of COX.Contributors AM-L carried out most of the molecular biology and cell biologyexperiments as part of her PhD programme; AA and IM carried out the clinicalfollow up; AR carried out WES and part of the work on COA7 mitochondriallocalisation; AJR provided bioinformatics management of the WES data; DG carriedout the biochemical diagnosis and part of the molecular biology; EF-V and MZorganised the experimental set-up, collected the data and wrote the manuscript.Funding Supported by Telethon-Italy grant GGP15041 (to DG); Telethon-ItalyNetwork of Genetic Biobank grant GTB12001J; ERC advanced grant ERCFP7-322424 (to MZ). MRC QQR grant MC_UP_1002/1.Competing interests None declared.Ethics approval Ethical Committee of the Fondazione Istituto Neurologico‘Carlo Besta’, Milan, Italy.Provenance and peer review Not commissioned; externally peer reviewed.Open Access This is an Open Access article distributed in accordance with theCreative Commons Attribution Non Commercial (CC BY-NC 4.0) license, whichpermits others to distribute, remix, adapt, build upon this work non-commercially,and license their derivative works on different terms, provided the original work isproperly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/REFERENCES1 Shoubridge EA. Cytochrome c oxidase deficiency. Am J Med Genet2001;106:46–52.2 DiMauro S, Tanji K, Schon EA. The many clinical faces of cytochrome c oxidasedeficiency. Adv Exp Med Biol 2012;748:341–57.3 Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol 2007;20:564–71.4 Zeviani M, Di Donato S. Mitochondrial disorders. 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COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency.

http://dx.doi.org/10.1136/jmedgenet-2016-104194

COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency.

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Archivio istituzionale della ricerca - Università di Padova

Archivio istituzionale della ricerca - Università di Padova

AIR Universita degli studi di Milano

Apollo (Cambridge)