Mutations in APOPT1, Encoding a Mitochondrial Protein, Cause Cavitating Leukoencephalopathy with Cytochrome c Oxidase Deficiency

Abstract

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation. Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation. Cytochrome c oxidase (COX, complex IV [cIV], E.C. 1.9.3.1) is the terminal component of the mitochondrial respiratory chain (MRC), operating the electron transfer from reduced cytochrome c to molecular oxygen. The redox reaction is coupled with proton translocation across the inner mitochondrial membrane, thus contributing to the formation of the mitochondrial membrane electrochemical potential (ΔΨ). ΔΨ is eventually utilized by the F1F0-ATP synthase (complex V) to produce ATP, the universal energy currency of the cell. Human COX is composed of several subunits:1Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1921) Google Scholar, 2Yoshikawa S. Shinzawa-Itoh K. Tsukihara T. Crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution.J. Bioenerg. Biomembr. 1998; 30: 7-14Crossref PubMed Scopus (79) Google Scholar the three largest are encoded by mitochondrial DNA (mtDNA) genes and form the catalytic core of the enzyme. The remaining 11 nuclear-encoded subunits, some of which have tissue-specific isoforms,3Arnold S. Kadenbach B. Cell respiration is controlled by ATP, an allosteric inhibitor of cytochrome-c oxidase.Eur. J. Biochem. 1997; 249: 350-354Crossref PubMed Scopus (183) Google Scholar are deemed to play an ill-defined regulatory role. COX deficiency (MIM 220110) is one of the most common biochemical abnormalities found in mitochondrial disorders, but about half of all cases remain genetically undefined.4DiMauro S. Tanji K. Schon E.A. The many clinical faces of cytochrome c oxidase deficiency.Adv. Exp. Med. Biol. 2012; 748: 341-357Crossref PubMed Scopus (46) Google Scholar Mutations in mtDNA or nuclear DNA genes encoding COX subunits are exceptionally rare, suggesting that direct damage of the structural components of cIV is likely to cause embryonic lethality in most cases. Conversely, early-onset COX deficiency is often due to mutations in assembly factors of the enzyme,5Ghezzi D. Zeviani M. Assembly factors of human mitochondrial respiratory chain complexes: physiology and pathophysiology.Adv. Exp. Med. Biol. 2012; 748: 65-106Crossref PubMed Scopus (107) Google Scholar SURF1 (MIM 185620) being the most commonly affected gene.6Tiranti V. Hoertnagel K. Carrozzo R. Galimberti C. Munaro M. Granatiero M. Zelante L. Gasparini P. Marzella R. Rocchi M. et al.Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.Am. J. Hum. Genet. 1998; 63: 1609-1621Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 7Wedatilake Y. Brown R.M. McFarland R. Yaplito-Lee J. Morris A.A. Champion M. Jardine P.E. Clarke A. Thorburn D.R. Taylor R.W. et al.SURF1 deficiency: a multi-centre natural history study.Orphanet J. Rare Dis. 2013; 8: 96Crossref PubMed Scopus (93) Google Scholar SURF1 mutant individuals typically present with Leigh syndrome (LS), an early-onset, rapidly progressive encephalopathy characterized by bilateral focal necrotizing lesions in the basal ganglia and brainstem nuclei. In addition, a number of mutations in genes involved in mtDNA expression or translation are consistently associated with isolated or predominant COX deficiency,8DiMauro S. Hirano M. Pathogenesis and treatment of mitochondrial disorders.Adv. Exp. Med. Biol. 2009; 652: 139-170Crossref PubMed Scopus (29) Google Scholar, 9Mootha V.K. Lepage P. Miller K. Bunkenborg J. Reich M. Hjerrild M. Delmonte T. Villeneuve A. Sladek R. Xu F. et al.Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics.Proc. Natl. Acad. Sci. USA. 2003; 100: 605-610Crossref PubMed Scopus (473) Google Scholar, 10Almalki A. Alston C.L. Parker A. Simonic I. Mehta S.G. He L. Reza M. Oliveira J.M. Lightowlers R.N. McFarland R. et al.Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.Biochim. Biophys. Acta. 2014; 1842: 56-64Crossref PubMed Scopus (56) Google Scholar, 11Dallabona C. Diodato D. Kevelam S.H. Haack T.B. Wong L.J. Salomons G.S. Baruffini E. Melchionda L. Mariotti C. Strom T.M. et al.Novel (ovario)leukodystrophy related to AARS2 mutations.Neurology. 2014; 82: 2063-2071Crossref PubMed Scopus (145) Google Scholar including some mutations in mitochondrial tRNA-encoding genes or in nuclear-encoded mtDNA translation proteins (e.g., LRPPRC [MIM 607544] or several mitochondrial aminoacyl tRNA synthetases). As part of a long-standing project aimed at identifying novel genes responsible for COX deficiency, we present here the identification of deleterious mutations in APOPT1 (Apoptogenic-1, previously APOP-1 or C14ORF153), encoding a mitochondrial protein. This gene was identified by whole-exome sequencing (WES) analysis in three individuals from independent cohorts of subjects with isolated COX deficiency and subsequently in three additional unrelated children on the basis of a distinctive brain MRI pattern present in all. Informed consent for participation in this study was obtained from the parents of all investigated subjects, in agreement with the Declaration of Helsinki and approved by the Ethical Committees of the Centers participating in this study, where biological samples were obtained. A total of six individuals from five families were found to harbor mutations in APOPT1 (see below). The clinical features varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs presenting in adolescence. Encephalopathic episodes were characterized by acute loss of developmental milestones including ability to walk or sit, loss of speech, episodes with somnolence and seizure, and pyramidal signs rapidly evolving into spastic tetraparesis. In all cases, the clinical course subsequently tended to stabilize and in several subjects marked recovery of neurological milestones was observed over time. Brain MRI was characterized by a cavitating leukodystrophy, predominantly involving the posterior cerebral white matter and the corpus callosum in the acute stage, after which the abnormalities partially improved and then stabilized (Figure 1). A summary of the clinical features is presented in Table 1 (for further details contact the corresponding authors). The MRI features are summarized in Table S1 available online and a detailed description is provided in the legend of Figure 1.Table 1Clinical FeaturesAffected SubjectS1S2S3S4S5S06GenderfemalefemalemalemalemalefemaleYear of birth198719981997200020072009Siblings (affected / unaffected / otherwise affected)1 / 0 / 01 / 0 / 00 / 1 / 00 / 1 / 00 / 8 / 00 / 1 / 0Consanguinity parents−−+++−Pregnancy, delivery, neonatal periodnormalnormalnormalnormalnormalnormalInitial motor & cognitive developmentnormalnormalnormalnormalmildly delayednormalSingle Episode of RegressionAge at presentation (years)2.5never developed neurological signs3552Signs at presentationL-hemiparesis, somnolence, irritability, loss of ambulationNAdelayed speech, gait difficultiesgait difficultiesdysarthria and gait difficultiesfrequent falls and leg weaknessPreceding eventrecurrent vomiting and poor of spastic and with loss of and with loss of and with loss of due to on and and of at controlled with further somnolence with at of 11 further further further further spastic signs of and signs of and spastic and marked in a M. E. and of mitochondria in 1996; PubMed Google Scholar of from individual at and from individual at of profound of COX reaction and to a control (Figure on the from individual S1 showed the of mitochondria with and of the and analysis of individual complex M. F. S. E. E. F. I. L. M. et and molecular in children with complex deficiency.Biochim. Biophys. Acta. PubMed Scopus Google Scholar of individual S1 showed that cIV to synthase was of the in and in A decrease in complex was in and fibroblasts was in both and the reaction in was and The reaction in the individual was and of individual marked increase of in in reduced values of all the respiratory chain to showed the most severe in of the a decrease of and was in and biochemical of a from individual at showed profound COX deficiency, with a of of the and Table fibroblasts were available for further In and fibroblasts obtained from individual at a severe decrease in and was at showed of COX and and analysis of respiratory chain showed isolated of the control the reaction to COX was in fibroblasts (Figure to a control cell (Figure A summary of the is provided in Table for all cases with the of individual or of control of synthase of control of synthase of control of synthase of control of synthase of control were in and the values are and of the control The values of the control for and for are with an of control of synthase of control in a The were in and the values are and of the control The values of the control for and for are with an Mutations in SURF1 and mtDNA were in individuals and analysis showed or of individual S1 the in individual was by a increase in mtDNA L. T.M. Taylor R.W. and of mitochondrial DNA in individual by 30: PubMed Scopus Google Scholar to control was subsequently on DNA from individuals S1 and T.B. B. T. A. M. V. E. et in mitochondrial complex deficiency Med. Genet. 2012; PubMed Scopus Google Scholar after to common we for or by the two to a predicted of the of genes by this we then selected to associated with and novel genes that or predicted mitochondrial T.B. B. T. A. M. V. E. et in mitochondrial complex deficiency Med. Genet. 2012; PubMed Scopus Google Scholar As a a was identified in a gene on The number is predicted to a causing the synthesis of a protein This was by sequencing in both individuals S1 and and the parents were to of to in and and are and are are to in and S1 and are in a are was on individual identifying a in APOPT1 by the This is within the of individual to study APOPT1 we showed that is in the of the of the for the synthesis of a of the wild-type protein appeared to of 1 (Figure of APOPT1 was by this We then APOPT1 in five subjects characterized by cavitating with posterior and found mutations in three individuals and and presented with severe COX deficiency individual was investigated subjects with isolated cIV deficiency with or were but further mutations were of of APOPT1 was DNA from individual (Figure suggesting a of the corresponding and was identified in we of and we that the corresponding to the and analysis of the from the mutant showed the of the encoded by and The of causes a in the of APOPT1 and is predicted to in the of a In individual we found a a is with to a being predicted deleterious by several (Figure In individual we identified two the present in individuals S1 and and a causing the of a were to of one and a was for the of the APOPT1 mutations and corresponding in the protein are summarized in Figure and Table APOPT1 is predicted to a mitochondrial protein an N-terminal mitochondrial targeting (Figure are present in the encoding at 1 and the predicted mature of APOPT1 from or are the is predicted to and (Figure APOPT1 was previously to have mitochondrial in cultured K. M. T. M. T. H. T. a novel protein but Biol. PubMed Scopus Google Scholar into we showed that both the human and proteins with a mitochondrial in and we the sequence from the most likely human APOPT1 for two the APOPT1 sequence is in is absent in all (Figure the human APOPT1 one in the of the and is that that are of the structure in the we APOPT1 for all further We human APOPT1 but showed by or We a encoding a human APOPT1 protein with the at the an we analysis on of D. Haack T.B. A. M. S. et al.Mutations in encoding a mitochondrial cause early-onset mitochondrial J. Hum. Genet. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar We two with the of the in D. Haack T.B. A. M. S. et al.Mutations in encoding a mitochondrial cause early-onset mitochondrial J. Hum. Genet. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar predicted and mature predicted (Figure that human APOPT1 an N-terminal mitochondrial targeting sequence of which is cleaved from the mature protein into the inner mitochondrial In to study the of the protein in a we to in and cell by a expression that a D. Haack T.B. A. M. S. et al.Mutations in encoding a mitochondrial cause early-onset mitochondrial J. Hum. Genet. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar we levels of after (Figure protein was by or in cell this was due to cell we a expressing the to of we were to the protein in expressing levels of the (Figure and to cell in to control these that the is but the corresponding protein is rapidly by a in standard further this fibroblasts from individual or a control with the were with for a proteasome The of a proteasome inhibitor on in to cell reactive oxygen and 2009; Google Scholar corresponding to the and mature were present in both cell in with the of in the cell and that APOPT1 protein is by the proteasome in standard we the levels of the APOPT1 protein to A. R. M. F. and of oxidative by on cultured human Cell PubMed Scopus Google Scholar or D. A. P. E. Gasparini P. V. Zeviani M. in an infantile mitochondrial associated with cytochrome c oxidase deficiency.Am. J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar We the cell to of or to a standard of an of of oxidative protein to with a at (Figure protein was treatment with In to the of to the predominant of the (Figure the of oxidative we the of reactive oxygen a in basal levels were mutant fibroblasts and control after or 1 for levels in mutant were higher in control fibroblasts (Figure in fibroblasts with the amount of was with being to that found in control with the higher suggesting a for APOPT1 in mitochondrial to (Figure Conversely, we obtained of a for APOPT1 in available a was on from individuals S1 and were observed by in individual S1 and in was found after treatment in mutant control fibroblasts we a in for in brain white We then investigated the amount and of the COX holocomplex by D. Haack T.B. A. M. S. et al.Mutations in encoding a mitochondrial cause early-onset mitochondrial J. Hum. Genet. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar of S1 and We found that the amount of both COX holocomplex and was reduced in both mutant in (Figure The of the corresponding to individual including were to The cIV was in fibroblasts from individual In of the levels of APOPT1 in individual we found a but increase in the of cIV and in these to individual and suggesting a for APOPT1 in cIV assembly were by of targeting the APOPT1 with the were a and marked knockdown of APOPT1 expression in cell of the control levels and myoblasts and control fibroblasts expressing and showed a dramatic decrease in cell after the (Figure the with the control were in myoblasts after the with that to in COX or in the amount of the holocomplex and and levels were in fibroblasts to (Figure or the of cell in the clinical of mitochondrial and in the of mitochondrial In six subjects to five unrelated we identified five mutant in encoding a mitochondrial protein of All mutations are predicted to in the synthesis of severely damaged protein species, due to or of an In the five subjects for the and biochemical of the mitochondrial respiratory chain was we a profound of COX in available cultured with marked of cIV holocomplex and In two a of was which was present in available samples from the subjects, suggesting a APOPT1 mutations were consistently associated with a brain MRI pattern characterized by and of cavitating white matter predominantly in the posterior of the cerebral and corpus This pattern was to the direct of APOPT1 in a of five subjects with cavitating leukodystrophy, three of to harbor mutations in this the two subjects APOPT1 mutations COX deficiency in or these that mutations in APOPT1 are responsible for a mitochondrial characterized by marked COX deficiency and a form of cavitating MRI in the acute are white matter abnormalities predominantly in the posterior of the cerebral involving the corpus In severe cases, the white matter abnormalities into the and the part of the corpus In the acute stage, on and are and within the white matter levels of are by proton are typically of the affected of and of and with a decrease in In to neurological by profound cIV rapidly progressive S. Tanji K. Schon E.A. The many clinical faces of cytochrome c oxidase deficiency.Adv. Exp. Med. Biol. 2012; 748: 341-357Crossref PubMed Scopus (46) Google Scholar the clinical course of encephalopathy to in some individuals developed severe neurological signs at an with motor evolving into spastic and severe cognitive (e.g., individual showed an with severe motor but or absent cognitive and in one the neurological was virtually at of the of some posterior white matter in all affected individuals and of a was in of clinical severely affected individual S1 and affected individual are the which the synthesis of a the clinical of this of the of the and in all cases both the MRI lesions and the clinical a chronic, long-surviving clinical in with the rapidly often of early-onset All APOPT1 mutant subjects are some in with the one being in third In some cases and S1 and with the of and in individual the of a the of have to the of clinical the of these remains subjects and showed with treatment 1). white matter an associated of or a fraction of infantile mitochondrial from to of the cases in I. M. A. E. white matter in children with mitochondrial PubMed Scopus Google Scholar, D. Zeviani M. mitochondrial Med. Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. D. P. A. C. P. D. et common pattern of brain MRI in mitochondrial with complex Med. Genet. PubMed Scopus Google Scholar In some cases, MRI are consistently associated with mutations in genes related to mitochondrial a number of mitochondrial aminoacyl tRNA including T. M. J. M. et synthetase deficiency causes with brain and and Genet. PubMed Scopus Google Scholar (MIM D. Haack T. D. A. L. E. J. et with and brainstem and caused by 2012; PubMed Scopus Google Scholar (MIM and C. Diodato D. Kevelam S.H. Haack T.B. Wong L.J. Salomons G.S. Baruffini E. Melchionda L. Mariotti C. Strom T.M. et al.Novel (ovario)leukodystrophy related to AARS2 mutations.Neurology. 2014; 82: 2063-2071Crossref PubMed Scopus (145) Google Scholar (MIM APOPT1 was identified in a in a study on cultured from The gene a mitochondrial protein deemed a its cell K. M. T. M. T. H. T. a novel protein but Biol. PubMed Scopus Google Scholar which by the Y. H. M. Y. T. K. T. H. of Biophys. PubMed Scopus Google Scholar further on this protein are that APOPT1 is targeted to and localized within of a predicted and of a N-terminal which for translocation across the inner mitochondrial membrane, that APOPT1 is localized in the mitochondrial In with we obtained of a for this protein. We observed mutant subjects and in both in and with in and cell of in or The levels of obtained in in standard conditions, levels of by of the protein the This was by the of the protein in to a proteasome with of fibroblasts led to a increase in the amount of the mature protein species, suggesting a for APOPT1 in mitochondrial In to further this we showed that fibroblasts from individual produce of wild-type APOPT1 to a of We that APOPT1 is by and a oxidative conditions, but otherwise in standard conditions, at in cultured A for proteins at in mitochondrial for which is in but is stabilized by the of the mitochondrial membrane mitochondrial for and in mitochondrial PubMed Scopus Google Scholar In addition, an to the by R. Salomons Y. L. T. of of 1 of Biol. PubMed Scopus Google Scholar COX deficiency is the biochemical of APOPT1 mutant subjects, is APOPT1 and cIV assembly or a and to play an in the of mitochondrial associated with COX S. M. M. F. S. and with cytochrome c oxidase deficiency in mitochondrial PubMed Scopus Google Scholar, B. S. I. M. The of cytochrome c oxidase in and Biophys. Acta. PubMed Scopus Google Scholar We showed that the expression of wild-type APOPT1 in mutant led to an increase in the amount of COX and a of to shRNA-mediated of APOPT1 expression in human myoblasts or fibroblasts to COX or increase but was associated with cell to of and cell This dramatic cell is likely to an acute to the of APOPT1 was observed in mutant in a of APOPT1 is that that COX deficiency are rapidly the are selected for COX In APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease by the combination of deficiency in COX and a neuroimaging presentation of cavitating with posterior cerebral and a APOPT1 is an mitochondrial which to by proteasome in of cell but stabilized and a related to remain including the clinical found in mutant is to the the of to its in mitochondrial and to the its and a structural and of COX We and for We the and DNA of and and the of DNA and cell of the of and the This was by and the Italian of the and of the Italian of and the the and of the in the of the for in an and the of and for and of the of the Italian of the for and the project and and were by the for is by a the for the and the of and with and Table S1 The for presented are in