early-onset parkinson disease caused by a mutation in ... · article open access early-onset...

ARTICLE OPEN ACCESS

Early-onset Parkinson disease caused bya mutation in CHCHD2 and mitochondrialdysfunctionRichard G Lee BSc (Hons) Maryam Sedghi MSc Mehri Salari MD Anne-Marie J Shearwood MSc

Maike Stentenbach BSc Ariana Kariminejad MD Hayley Goullee BSc (Hons) Oliver Rackham PhD

Nigel G Laing PhD Homa Tajsharghi PhDsect and Aleksandra Filipovska PhDsect

Neurol Genet 20184e276 doi101212NXG0000000000000276

Correspondence

Dr Tajsharghi

homatajsharghihisse

or Dr Filipovska

aleksandrafilipovskauwaeduau

AbstractObjectiveOur goal was to identify the gene(s) associated with an early-onset form of Parkinson disease(PD) and the molecular defects associated with this mutation

MethodsWe combined whole-exome sequencing and functional genomics to identify the genes asso-ciated with early-onset PD We used fluorescence microscopy cell and mitochondrial biologymeasurements to identify the molecular defects resulting from the identified mutation

ResultsHere we report an association of a homozygous variant in CHCHD2 encoding coiled-coil-helix-coiled-coil-helix domain containing protein 2 a mitochondrial protein of unknownfunction with an early-onset form of PD in a 26-year-old Caucasian woman The CHCHD2mutation in PD patient fibroblasts causes fragmentation of the mitochondrial reticular mor-phology and results in reduced oxidative phosphorylation at complex I and complex IVAlthough patient cells couldmaintain a protonmotive force reactive oxygen species productionwas increased which correlated with an increased metabolic rate

ConclusionsOur findings implicate CHCHD2 in the pathogenesis of recessive early-onset PD expandingthe repertoire of mitochondrial proteins that play a direct role in this disease

These authors contributed equally to the study and share first authorship

sectThese authors share senior authorship and are co-corresponding authors

From the Centre for Medical Research (RGL A-MJS M Stentenbach HG OR NGL HT AF) University of Western Australia and the Harry Perkins Institute for MedicalResearch Nedlands Western Australia Australia Department of Genetics (M Sedghi) University of Isfahan Isfahan Functional Neurosurgery Research Center (M Salari) ShohadaTajrish Neurosurgical Center of Excellence Shahid Beheshti University of Medical Sciences Tehran Iran Kariminejad-Najmabadi Pathology and Genetics Center (AK) Tehran IranSchool of Molecular Sciences (OR AF) The University of Western Australia Crawley Department of Diagnostic Genomics (NGL) PathWest QEII Medical Centre NedlandsWestern Australia Australia and Division Biomedicine and Public Health (HT) School of Health and Education University of Skovde Sweden

Funding information and disclosures are provided at the end of the article Full disclosure form information provided by the authors is available with the full text of this article atNeurologyorgNG

The Article Processing Charge was funded by the Australian Research Council

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2018 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Parkinson disease (PD MIM168600) is the most commonmovement disorder of aging and the second-most com-mon neurodegenerative disease after Alzheimer disease(MIM104300)12 PD has been associated with mutations inmultiple genes including PARK2 PINK1 PARK7 ATP13A2SCNA LRRK2 VPS35 EIF4G1 DNAJC13 and CHCHD23ndash6

Recent whole-exome sequencing (WES) of Japanese patientswith autosomal dominant or sporadic PD identified a hetero-zygous CHCHD2 missense change (pThr61Ile) in a familywith autosomal dominant late-onset PD5 Subsequently theidentical variant was identified in a Chinese family with auto-somal dominant PD6 Additional CHCHD2 variants includingpAla32Thr pPro34Leu and pIle80Val have been describedin 4 western European familial patients with PD7 The patho-mechanism of the CHCHD2 variants in these studies is how-ever unclear as is the physiologic role of CHCHD2

Here we identify a new CHCHD2 variant in a patient withautosomal recessive early-onset PD and establish the patho-genic mechanism of this variant We have shown that themutation results in a fragmented mitochondrial morphologyand reduced electron transport chain (ETC) activity

MethodsStandard protocol approvals registrationsand patient consentsThe study was approved by the ethical standards of the rel-evant institutional review board the Ethics Review Com-mittee in the Gothenburg Region (Dn1 842-14) and theHuman Research Ethics Committee of the University ofWestern Australia Informed consent was obtained frompatients included in this study after appropriate geneticcounseling Blood samples were obtained from patients theirparents and siblings

Clinical evaluationMedical history was obtained and physical examination wasperformed as part of routine clinical workup

Genetic analysisNext-generation sequencing (NGS) whole-exome andor tar-geted neuromuscular panel sequencing (WES or neuromus-cular sub-exomic sequencing [NSES]) was performed on thepatientrsquos DNA Confirmatory bidirectional Sanger sequencingwas performed in patients with PD and their family members(see e-Methods linkslwwcomNXGA85)

Cell culture and transfectionsDermal fibroblast cultures from the patient were establishedby standard protocols after written informed consent wasobtained Cultured fibroblasts from a healthy age-matchedindividual were used as a control Detailed methods are pro-vided in supplementary information

Mitochondrial isolation and cell lysisMitochondria were isolated from fibroblasts as previouslydescribed8 Cell lysates were prepared using a buffer con-taining the following 150 mMNaCl 01 (volvol) TritonX-100 and 50 mM Tris-HCl (pH 80) Protein concentra-tion was determined using a bicinchoninic acid (BCA)assay

Fluorescence microscopyDetailed methods are described in the supplementary mate-rial linkslwwcomNXGA85

Long-range PCR and mitochondrial DNA copynumber quantitative PCRDetailed methods are described in the supplementary mate-rial The sequences of all primers used for this study aredetailed in table e-1 linkslwwcomNXGA85

SDS-PAGE and immunoblottingMitochondria (25 μg) isolated from control and patientfibroblasts were separated on 4ndash12 Bis-Tris gels (Invi-trogen) and transferred onto a polyvinylidene difluoride(PVDF) membrane (Bio-Rad) All antibodies used aredetailed in the supplementary information

Mitochondrial protein synthesisDe novo mitochondrial protein synthesis was analyzed infibroblasts using 35S-radiolabeling of mitochondrially enco-ded proteins in the presence of emetine as previously de-scribed9 The cells were suspended in phosphate-bufferedsaline (PBS) and 20 μg of protein was resolved on a 125SDS-PAGE gel and radiolabelled proteins were visualizedon film

RespirationRespiration was measured in fibroblasts as previously de-scribed10 The full methods are detailed in supplementaryinformation

Cell function measurementsJC-1 mitochondrial mass dihydroethidium (DHE) and MTSassay full methods are detailed in supplementary information

GlossaryBCA = Bicinchoninic acidDHE = dihydroethidium ETC = electron transport chainMICOS =mitochondrial contact site andcristae organizing system NGS = next-generation sequencing OXPHOS = oxidative phosphorylation PBS = phosphate-buffered saline PD = Parkinson disease RCR = respiratory control ratio ROS = reactive oxygen speciesWES = whole-exomesequencing

2 Neurology Genetics | Volume 4 Number 5 | October 2018 NeurologyorgNG

Statistical analysisAll data are reported as mean plusmn SEM Statistical differenceswere determined using a two-tailed Student t test JC-1 DHEmitochondrial mass and MTS data are expressed as a percentof average control fibroblast values for the respective mediatreatment

Data availabilityStudy data for the primary analyses presented in this reportare available upon reasonable request from the correspondingand senior author

ResultsClinical characteristics of the patientA 30-year-old right-handed Caucasian woman (V2) was bornto healthy consanguineous parents aged 55 and 59 years(figure 1A) There was no significant family history and shedenied a history of other diseases or past drug use The clinicalpresentations were consistent with PD Symptoms developedrapidly at age 26 years including resting tremor mostly af-fecting her left arm and bradykinesia Neurologic examinationat age 28 years showed general bradykinesia rigidity restingtremor in both hands and legs but most prominent in the leftside with superimposed action-induced myoclonus She alsoshowed hypomimic face and mydriatic pupils with sluggishreaction to the light indicating autonomic dysfunction Therewere no pyramidal or cerebellar signs On fundoscopic ex-amination Kayser-Fleischer-like rings were negative Neithersphincter dysfunction nor orthostatic hypotension was pres-ent Laboratory evaluation including thyroid function liverfunction serum and urine copper and ceruloplasmin wasunremarkable Brain MRI was also unremarkable The patientshowed several clinical features of mitochondrial dysfunctionsuch as neuropsychological disturbance dysautonomia andmyoclonus Low-dose levodopa was started with dramaticresponse but after 1 month she had severe dyskinesia andafter 3 months motor fluctuation anxiety and insomnia be-came the main issues and she had dopamine dysregulationsyndrome After 6 months psychiatric problems includingaggression depression and impulsiveness were additionalsymptoms At follow-up at age 30 years PD symptoms hadnot progressed and became stable However she showedsigns of cognitive decline and orthostatic hypotention addedto her symptoms

Genetic findingsData fromNGSof DNA from 1 affected (V2) and 1 unaffectedfamily member (V1) were analyzed No likely pathogenicmutations were identified in the genes included in the targetedpanel of the 336 neuromuscular disease genes WES was per-formed on the patient and her unaffected sister No rare likelypathogenic heterozygous variants in the PD-associated geneswere identified The filtering strategy of initially concentratingon homozygous coding variants in known neurogenetic diseasegenes selected based on variant databases Human Genome

Mutation Database and ClinVar and the most recent literatureallowed the identification of homozygous CHCHD2 (chro-mosome 7) and TOP1MT (chromosome 8) variants A novelhomozygous missense mutation in exon 2 of CHCHD2(c211GgtC) (pAla71Pro) was identified The size of the ho-mozygous region covering CHCHD2 variant on chromosome7 was 158Mb In addition a homozygous missense variant ofTOP1MT (c661GgtA ENST00000523676 transcript IDNM_0012584471) changing aspartic acid to asparagine(pAsp221Asn) was identified No rare likely pathogenicheterozygous or homozygous variants in the PD-associatedgenes including ATP13A2 CHCHD10 DNAJC13 EIF4G1LRRK2 PARK2 PARK7 PARK15 PINK1 SNCA and VPS35were identified in the exome sequencing data The appearanceof CHCHD2 and TOP1MT variants was examined in availablefamily members by sequencing analysis Sanger sequencingconfirmed segregation of both variants compatible with a re-cessive pattern of inheritance The unaffected parents (IV1 IV2) were both heterozygous for the CHCHD2 variant and thehealthy sister (V1) was not a carrier (figure 1B) Both parentsand the sister were heterozygous carriers of the TOP1MTvariant (figure 1C) The c211GgtC (pAla71Pro) (CHCHD2)variant was excluded in East and South Asian European Af-rican Latino and Ashkenazi Jewish Allele Frequency Com-munities (AFC) in the ExAC database the GenomeAggregation Database (gnomAD) and the 1000 Genomedatabase The TOP1MT variant (c661GgtA pAsp221Asnrs143769145) was a rare heterozygous variant reported inthe AFC (frequency 0025) the ExAC (frequency0026) and in the gnomAD (frequency 0023) data-bases the variant was identified in the homozygous state Insilico combined annotation dependent depletion (CADD)analysis of the missense variants revealed high deleteriousscores (CHCHD2 [pAla71Pro] 32000 and TOP1MT[pAsp221Asn] 26000) In silico analysis predicted bothCHCHD2 and TOP1MT substitutions to be potentiallydisease causing (MutationTaster mutationtasterorg) The2 amino acid residues affected are highly conserved acrossspecies (figure 1D) The CHCHD2-substituted residue islocated within the central conserved hydrophobic domainthe transmembrane element of the CHCHD2 protein(figure 1E) In addition the entire coding sequence ofCHCHD2 was analyzed in 2 affected individuals of 2 largefamilies with familial early-onset PD of Iranian origin TheCHCHD2 pAla71Pro substitution was not identified andwe did not detect any other variant in this gene

Clinical status of confirmed carriersCosegregation studies confirmed that the parents were car-riers of CHCHD2 and TOP1MT variants and the unaffectedsister was heterozygous for the TOP1MT variant Given thatthe previously reported CHCHD2 variants are mostly asso-ciated with late-onset dominant PD carrier parents were ex-amined by a neurologist They showed no evidence ofneurologic or movement disorder by history However theparents are still younger than the average age of PD onset(lt60)11

NeurologyorgNG Neurology Genetics | Volume 4 Number 5 | October 2018 3

Figure 1 Pedigree and genetic findings

(A) Pedigree of the family The affected individual (V2) is represented with a shaded symbol (B) Sanger sequence analysis demonstrates the presence ofhomozygous variants in CHCHD2 and (C) TOP1MT in the patient and the segregation of the variants (D) Multiple sequence alignment confirms that thepAla71Pro (CHCHD2) and pAsp221Asn (TOP1MT) substitutions affect evolutionarily conserved residues (shaded) (E) Mutated residues and CHCHD2 proteinstructure previously identified heterozygous missense variants associated with familial PD (black arrows) and the currently identified homozygous variant(red arrow) are all located in exon 2 The CHCHD2 pAla71Pro amino acid substitution is located within the central conserved hydrophobic domain at thetransmembrane element


Reduction of CHCHD2 but not TOP1MT causesthe PD pathologyTo investigate the molecular mechanisms behind this form ofearly-onset PD dermal fibroblasts were established from thepatient and compared with controls established from a healthyindividual We investigated the protein abundance by immu-noblotting in patient and control fibroblasts to determinewhich variant was pathogenic We observed a small reductionin TOP1MT levels whereas the CHCHD2 reduction wasmore pronounced (figure 2A) As TOP1MT is the top-oisomerase that catalyzes supercolied mtDNA relaxation12 weinvestigated mtDNA stability and abundance Long-range PCRshowed nomtDNA fragmentation and changes in mtDNA sizein patient cells (figure 2B) Furthermore qPCR analysis showedno significant difference in mtDNA copy number indicatingthat the TOP1MT variant has negligible effects on mtDNAstructure (figure 2 B and C)

CHCHD2 has been shown to regulate mitochondrial morphol-ogy in Drosophila melanogaster13 Therefore we investigated mi-tochondrial morphology in patient cells using MitoTrackerOrange In glucose medium we identified differences in mor-phology and fusion between patient and control cells character-ized by a reduced reticular network fragmentation of filamentousmitochondria and accumulation of granular bodies around thenucleus (figure 2D) We cultured both fibroblasts in galactose

medium that stimulates a dependence on aerobic metabolismcompared with cells grown in glucose media which can rely onanaerobic metabolism Growth in galactose exacerbated differ-ences in mitochondrial morphology seen in patient cells (figure2D) Our experiments excluded the mtDNA abnormalities thatwould be linked to TOP1MT dysfunction and identified changesin mitochondrial morphology that is related to the proposedfunction and membrane association identified for CHCHD2 assummarized in figure e-1 linkslwwcomNXGA85

The role of other CHCHD family proteins in the mito-chondrial contact site and cristae organizing system(MICOS) complex1415 lead us to investigate how reducedCHCHD2 expression affects MICOS complex subunits Al-though levels of the intermembrane protein CHCHD3 werereduced in patient cells levels of the lipid binding proteinsAPOO and APOOL were unchanged (figure 2E) We con-clude that although CHCHD2 does not have a direct role inMICOS complex function or stability the CHCHD2 variantresults in reduction of the membrane-associated proteinCHCHD3

Reduced CHCHD2 expression results inOXPHOS dysfunctionImpaired oxidative phosphorylation (OXPHOS) complexformation and function has been identified in PD and

Figure 2 CHCHD2 but not TOP1MT contributes to PD pathogenesis

(A) SDS-PAGE and immunoblottingwere performed on 25 μg of iso-lated mitochondria from controland patient fibroblasts (B) Mito-chondrial DNA integrity was ex-amined using long-range PCR ofcontrol and patient fibroblast DNADNA was separated on a 1 aga-rose gel stained with ethidiumbromide (C) Mitochondrial DNAcopy number was examined usingquantitative PCR of control and fi-broblast DNA (D) Mitochondrialmorphology of control and patientfibroblasts grown in either highglucose or galactose media wasexamined using MitoTracker Or-ange and visualized by fluores-cence microscopy The scale barrepresents 10 μm Arrows indicaterefractile granules characteristic ofnetwork fragmentation (E)Twenty-five micrograms of iso-lated mitochondria from controland patient fibroblasts was ana-lyzed for mitochondrial contactsite and cristae organizing system(MICOS) protein levels by immu-noblotting SDHA was used asa loading control


drug-induced parkinsonism16 Therefore we investigatedOXPHOS complex subunit levels by immunoblotting Wefound reduced levels of complex I IV and V subunitswhereas complexes II and III showed negligible reductionsin patient cells (figure 3A) De novo mitochondrial trans-lation examined using 35S-methionine showed no differencein mitochondrial translation rates between control and pa-tient cells (figure 3B) This indicates that the CHCHD2mutation causes changes in OXPHOS subunit abundancethrough reduced protein stability and morphologic changesnot impaired protein synthesis

Next we investigated ETC activity by measuring mitochon-drial respiration at each complex (figure 3C) We show re-duced oxygen consumption at complexes I and IV but notcomplexes II and III suggesting that CHCHD2 may regulateelectron transfer between these complexes as previouslysuggested1317 We also examined the respiratory control ratio(RCR) by measuring oxygen consumption with succinate androtenone under ATP-generating conditions (phosphorylatingstate 3) and non-ATP generating conditions (non-phosphorylating state 4) We identified significant decreasesin the RCR in patient cells grown in glucose and galactosewhich is characteristic of an uncoupling of the ETC and ATP

production (figure 3D) This indicates that reductions inOXPHOS activity are likely due to impaired mitochondrialmorphology and electron transport between complexes

To test whether CHCHD2 dysfunction resulted in PD weperformed rescue experiments by expressing the wild-typeCHCHD2 or TOP1MT proteins in the control and patientcells (figure 4) The fragmented mitochondrial network wasrestored to filamentous reticular appearance (figure 4A)and respiration was restored to levels comparable to thecontrol fibroblast when rescued by expression of the wild-type CHCHD2 protein but not by expression of the wild-type TOP1MT protein (figure 4B) These experimentsvalidated the causative role of the CHCHD2mutation in PDpathology

Mutation in CHCHD2 causes increasedoxidative stressNext we investigated the mitochondrial membrane potential(Dψm) using JC-1 There were no significant changes in theDψm in patient cells grown in glucose or galactose indicatingthat the proton motive force maintains the Dψm (figure 5A)The decrease in membrane potential was greater in patientcells treated with FCCP indicating a reduction in maximal

Figure 3 Patient cells show impaired OXPHOS complex levels and function

(A) SDS-PAGE and immunoblottingwere performed on 25 μg of isolatedmitochondria from control and pa-tient fibroblasts (B) Mitochondrialprotein synthesis was examined incontrol and patient fibroblasts using35S radiolabeling of mitochondrialtranslation products Twenty micro-grams of cell lysate was separated ona 125 SDS-PAGE gel and visualizedby autoradiography (C) Oxygenconsumption of specific respiratorycomplexes was measured in controland patient fibroblasts using anOROBOROS respirometer (D) Phos-phorylated (state 3) and non-phosphorylated (state 4) respirationwas measured in the presence of10 mM succinate05 μM rotenone indigitonin-permeabilized control andpatient cells to determine the re-spiratory control ratio under bothglucose and galactose media con-ditions Data are presented as meanplusmn SEM and were analyzed usinga Studentrsquos t test p lt 005 p lt001


respiratory capacity This reduction was greater under galac-tose conditions (figure 5 A and B) The reduced stability ofOXPHOS subunits and decreased respiratory capacity wereindependent of mitochondrial mass (figure 5C)

Next we investigated reactive oxygen species (ROS) pro-duction18 Patient cells showed significantly increased su-peroxide levels when grown in glucose which was furtherexacerbated when cells were grown in galactose (figure5D) As ROS have been shown to affect metabolic rate andviability we investigated the metabolic consequences ofincreased ROS levels Patient cells showed a mild meta-bolic increase under glucose conditions and a further in-crease under galactose conditions indicating that theincrease in ROS is not inherently cytotoxic but is sufficientto modulate the metabolic rate in skin fibroblasts(figure 5E)

DiscussionHere we have identified homozygousCHCHD2 andTOP1MTvariants in a Caucasian woman presenting with characteristicfeatures of PD at 26 years Both healthy parents were hetero-zygous CHCHD2 and TOP1MT variant carriers but not theunaffected sister The likely pathogenicity of the variants wassupported by the results of the prediction tools PolyPhen-2SIFT and CADD where the TOP1MT variant (c661GgtApAsp221Asn rs143769145) was identified at a low frequencyof 0023ndash0025 in the Genome Aggregation Databases withno homozygotes The mutated residues of both CHCHD2 andTOP1MTare highly conserved during evolution Taken togetherour finding suggests that bothCHCHD2 andTOP1MTmight becausative genes of recessive early-onset PD Mitochondrial dys-function has been shown to be a key factor in PD pathogenesis inboth animal models and patients with disease-associated genes

Figure 4 CHCHD2 but not TOP1MT expression rescues molecular defects

(A) Mitochondrial morphology of controland patient fibroblasts in the presenceor absence of either wild-type CHCHD2 orwild-type TOP1MT was examined usingMitoTracker Orange and visualized by fluo-rescence microscopy The scale bar repre-sents 10 μm Arrows indicate refractilegranules characteristic of network fragmen-tation (B) Oxygen consumption of respiratorycomplexes was measured in control patientfibroblasts and patient fibroblasts express-ing either wild-type CHCHD2 or wild-typeTOP1MT using an OROBOROS respirometerData are presented as mean plusmn SEM andwere analyzed using a Studentrsquos t test p lt005 p lt 001


PINK1 and CHCHD2 being involved in mitochondrialfunction1319ndash21 Recently however the association ofCHCHD2 with PD has displayed mixed results in differentpopulations671922ndash25 suggesting that it is infrequent andethnic specific Here we have used cultured patient fibroblaststo establish the pathogenic mechanisms of the identifiedCHCHD2 and TOP1MT variants and expand the repertoireof genes implicated in the pathology of PD

Homozygous variants of both CHCHD2 and TOP1MT affectedprotein levels albeit more pronounced for CHCHD2 In patientfibroblasts the reduced CHCHD2 expression caused mito-chondrial network fragmentation when cells were forced to relyon oxidative phosphorylation This is consistent with previousstudies that implicated CHCHD2 in mitochondrial morphologyand its importance for energy production1326 The morphologyand respiration defects in the patient fibroblasts were rescued byexpressing wild-type CHCHD2 providing evidence that theCHCHD2 mutation contributes to the PD pathology ReducedTOP1MT expression causes increased negative supercoiling ofmtDNA resulting in reduced mtDNA replication26 Howeverthe lack of changes in mtDNA integrity or copy number inpatient fibroblasts indicated that the TOP1MT variant hada negligible effect on TOP1MT function Furthermore thecontribution of the TOP1MT variant to the PD pathogenesis

was excluded because expression of this protein did not rescuemitochondrial morphology and function

Changes in mitochondrial morphology have been previouslylinked with PD-characteristic OXPHOS deficiencies1620212728

Reductions in complexes I and IV subunits were consistent withreduced activities of these complexes in patient cells Althoughreduced complex IV activity has been previously reported in PDthere is much less evidence for impaired complex IV activitycontributing to PDdevelopment27 The reduction in complex IVformation is consistent with studies finding an interactionbetween CHCHD2 and the cytochrome cndashbinding proteinMICS11329 The reduction in complex activity and function wasconsistent with changes in the mitochondrial membrane andindicated an uncoupling of the ETC and impaired electrontransfer through the ETC3031 as seen previously17 It is possiblethat reduced CHCHD2 expression causes impaired MICS1function therefore impairing movement of electrons fromcomplex III to IV by cytochrome c as previously suggested17

The Dψm was not significantly altered in patient fibroblastsdespite inefficient electron transfer and reduced respiratorycapacity The greater difference in uncoupled state shownunder galactose conditions indicates that when patient cellsare made to rely on aerobic energy sources they can

Figure 5 Patient cells demonstrate increased oxidative stress

(A) JC-1 assay was used to examine the strength of the proton motive force in both control and patient fibroblasts under high glucose (A) and galactose (B)media conditions both in the absence of FCCP (+Dψm) and the presence of FCCP (minusDψm) (n = 6) (C) Mitochondrial mass was measured in control and patientfibroblasts using nonyl acridine orange (NAO) under high glucose conditions (n = 6) (D) Reactive oxygen species (ROS) levels were measured in control andpatient fibroblasts using dihydroethidium (DHE) under both high glucose and galactose media conditions (n = 6) (E) Metabolic activity was measured incontrol and patient fibroblasts using anMTS assay (n = 6) All data are presented as mean plusmn SEM and were analyzed using a Studentrsquos t test p lt 005 p lt0001


compensate for abnormalities in ETC coupling and theelectron leak through increasing the electrons flowingthrough the OXPHOS complexes Although increasing elec-tron flow may allow cells to maintain the proton motive forceit has negative consequences through ROS formation

The increased ROS levels were not cytotoxic and in contrastpatient fibroblasts demonstrated increased metabolic rateAlthough mild increases in ROS levels have been shown toincrease metabolic rate and proliferation in certain cells suchas fibroblasts neuronal cells show reduced viability32ndash35 Themitochondrial toxicity of oxidized dopamine derivatives incombination with the inherently low mitochondrial mass ofDA neurons may potentiate mitochondrial dysfunctionresulting from reduced CHCHD2 expression and causeneuronal cell death33 As dermal fibroblasts may not fullyemulate the characteristics of DA neurons further in-vestigation is needed to understand how increased ROS levelsalter metabolic function in DA neurons and how they po-tentiate mitochondrial dysfunction resulting from reducedCHCHD2 expression

Recent findings demonstrate that mutations in CHCHD2 andits paralogue CHCHD10 are associated with multiple neuro-degenerative disorders2536ndash38 The existence of a complexcontaining CHCHD2 and CHCHD10 may explain sharedfeatures between disorders3940 In addition CHCHD10knockdown and knockout models show altered respiratoryactivity and OXPHOS subunit levels3940 Further in-vestigation is required to fully understand how this complexregulates mitochondrial and neurologic functions

Author contributionsA Filipovska and H Tajsharghi study concept and design RGLee M Sedghi M Salari A-MJ Shearwood M StentenbachA Kariminejad H Goullee and O Rackham acquisition ofdata RG Lee M Sedghi M Salari A-MJ ShearwoodM Stentenbach A Kariminejad H Goullee O RackhamH Tajsharghi and A Filipovska analysis and interpretationRG Lee A-MJ Shearwood H Goullee and H Tajsharghistatistical analysis RG Lee O Rackham NG LaingH Tajsharghi and A Filipovska critical revision of themanuscript for important intellectual content O RackhamNG Laing H Tajsharghi and A Filipovska study supervision

AcknowledgmentThe authors thank the family members who provided samplesand clinical information for this study The authors thank DrMaryam Gholami for obtaining skin biopsy

Study fundingThe study was supported by grants from the EuropeanUnionrsquos Seventh Framework Programme for research tech-nological development and demonstration under grantagreement no 608473 (H Tajsharghi) and the Swedish Re-search Council (H Tajsharghi) NG Laing is supported byNHMRC Principal Research Fellowship (APP1117510)

H Goullee by NHMRC EU Collaborative GrantAPP1055295 O Rackham by a Cancer Council WA ResearchFellowship and A Filipovska is supported by NHMRCSenior Research Fellowship (APP1005030) and NHMRC andAustralian Research Council Discovery projects (toA Filipovska and O Rackham) The funders had no role in thedesign of the study and collection analysis decision to publishinterpretation of data or preparation of the manuscript

DisclosureRG Lee M Sedghi M Salari A-MJ ShearwoodM Stentenbach A Kariminejad H Goullee and O Rack-ham report no disclosures NG Laing has received a speakerhonorarium from the World Muscle Society has receivedtravel funding from the Asian Oceanian Myology CentreSanofi and the Ottawa Neuromuscular Meeting serves onthe editorial board of Neuromuscular Disorders receivespublishing royalties for The Sarcomere and Skeletal MuscleDisease Springer Science and Business Media LandesBioscience 2008 and has received research support fromthe Australian National Health and Medical ResearchCouncil the US Muscular Dystrophy Association Associa-tion Francaise contre les Myopathies Foundation BuildingStrength for Nemaline Myopathy Motor Neuron DiseaseResearch Institute of Australia Western Australian Healthand Medical Research Infrastructure Fund and the Perpet-ual Foundation H Tajsharghi and A Filipovska report nodisclosures Full disclosure form information provided bythe authors is available with the full text of this article atNeurologyorgNG

Received February 28 2018 Accepted in final form June 18 2018

References1 Liu G Bao X Jiang Y et al Identifying the association between Alzheimerrsquos disease

and Parkinsonrsquos disease using genome-wide association studies and protein-proteininteraction network Mol Neurobiol 2015521629ndash1636

2 Mhyre TR Boyd JT Hamill RW Maguire-Zeiss KA Parkinsonrsquos disease SubcellBiochem 201265389ndash455

3 Thomas B Beal MF Parkinsonrsquos disease Hum Mol Genet 200716R183ndashR194

4 Lill CM Genetics of Parkinsonrsquos disease Mol Cell Probes 201630386ndash3965 FunayamaM Ohe K Amo T et al CHCHD2mutations in autosomal dominant late-

onset Parkinsonrsquos disease a genome-wide linkage and sequencing study LancetNeurol 201514274ndash282

6 Shi CH Mao CY Zhang SY et al CHCHD2 gene mutations in familial and sporadicParkinsonrsquos disease Neurobiol Aging 201638217e9ndash217e13

7 Jansen IE Bras JM Lesage S et al CHCHD2 and Parkinsonrsquos disease Lancet Neurol201514678ndash679

8 Rackham O Davies SMK Shearwood AMJ Hamilton KL Whelan J Filipovska APentatricopeptide repeat domain protein 1 lowers the levels of mitochondrial leucinetRNAs in cells Nucleic Acids Res 2009375859ndash5867

9 Davies SMK Rackham O Shearwood AMJ et al Pentatricopeptide repeat domainprotein 3 associates with the mitochondrial small ribosomal subunit and regulatestranslation FEBS Lett 20095831853ndash1858

10 Sanchez MIGL Mercer TR Davies SMK et al RNA processing in human mito-chondria Cell Cycle Georget Tex 2011102904ndash2916

11 Lesage S Brice A Parkinsonrsquos disease frommonogenic forms to genetic susceptibilityfactors Hum Mol Genet 200918R48ndashR59

12 Zhang H Zhang YW Yasukawa T Dalla Rosa I Khiati S Pommier Y Increasednegative supercoiling of mtDNA in TOP1mt knockout mice and presence of top-oisomerases IIα and IIβ in vertebrate mitochondria Nucleic Acids Res 2014427259ndash7267

13 Meng H Yamashita C Shiba-Fukushima K et al Loss of Parkinsonrsquos disease-associated protein CHCHD2 affects mitochondrial crista structure and destabilizescytochrome c Nat Commun 2017815500

14 Ding CWu Z Huang L et al Mitofilin and CHCHD6 physically interact with Sam50to sustain cristae structure Sci Rep 2015516064


15 Li H Ruan Y Zhang K et al Mic60Mitofilin determines MICOS assembly essentialfor mitochondrial dynamics and mtDNA nucleoid organization Cell Death Differ201623380ndash392

16 Richardson JR Caudle WM Guillot TS et al Obligatory role for complex I inhibitionin the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) Toxicol Sci 200795196ndash204

17 Aras S Bai M Lee I Springett R Huttemann M Grossman LI MNRR1 (formerlyCHCHD2) is a bi-organellar regulator of mitochondrial metabolism Mitochondrion20152043ndash51

18 Murphy MP How mitochondria produce reactive oxygen species Biochem J 20094171

19 Liu Z Guo J Li K et al Mutation analysis of CHCHD2 gene in Chinese familialParkinsonrsquos disease Neurobiol Aging 2015363117e7ndash3117e8

20 Morais VA Haddad D Craessaerts K et al PINK1 loss-of-function mutations affectmitochondrial complex I activity via NdufA10 ubiquinone uncoupling Science 2014344203ndash207

21 Morais VA Verstreken P Roethig A et al Parkinsonrsquos disease mutations in PINK1result in decreased Complex I activity and deficient synaptic function EMBO MolMed 2009199ndash111

22 Liu G Li K CHCHD2 and Parkinsonrsquos disease Lancet Neurol 201514679ndash68023 Foo JN Liu J Tan E-K CHCHD2 and Parkinsonrsquos disease Lancet Neurol 201514

681ndash68224 Iqbal Z Toft M CHCHD2 and Parkinsonrsquos disease Lancet Neurol 201514

680ndash68125 Rubino E Brusa L Zhang M et al Genetic analysis of CHCHD2 and

CHCHD10 in Italian patients with Parkinsonrsquos disease Neurobiol Aging 201753193e7ndash193e8

26 Rambold AS Kostelecky B Elia N Lippincott-Schwartz J Tubular network formationprotects mitochondria from autophagosomal degradation during nutrient starvationProc Natl Acad Sci USA 201110810190ndash10195

27 Benecke R Strumper P Weiss H Electron transfer complexes I and IV of platelets areabnormal in Parkinsonrsquos disease but normal in Parkinson-plus syndromes Brain JNeurol 1993116(pt 6)1451ndash1463

28 JasonCannon R Tapias VM Na HMHonick AS Drolet RE Greenamyre JT A highlyreproducible rotenone model of Parkinsonrsquos disease Neurobiol Dis 200934279ndash290

29 Oka T Sayano T Tamai S et al Identification of a novel protein MICS1 that isinvolved in maintenance of mitochondrial morphology and apoptotic release of cy-tochrome c Mol Biol Cell 2008192597ndash2608

30 Brand MD Nicholls DG Assessing mitochondrial dysfunction in cells Biochem J2011435297ndash312

31 Jastroch M Divakaruni AS Mookerjee S Treberg JR Brand MD Mitochondrialproton and electron leaks Essays Biochem 20104753ndash67

32 Hwang O Role of oxidative stress in Parkinsonrsquos disease Exp Neurobiol 20132211ndash17

33 Valencia A Moran J Reactive oxygen species induce different cell death mechanismsin cultured neurons Free Radic Biol Med 2004361112ndash1125

34 Emdadul Haque M Asanuma M Higashi Y Miyazaki I Tanaka K Ogawa NApoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive qui-none generation in neuroblastoma cells Biochim Biophys Acta 2003161939ndash52

35 Liang CL Wang TT Luby-Phelps K German DC Mitochondria mass is low inmouse substantia nigra dopamine neurons implications for Parkinsonrsquos disease ExpNeurol 2007203370ndash380

36 Bannwarth S Ait-El-Mkadem S Chaussenot A et al A mitochondrial origin forfrontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 in-volvement Brain J Neurol 20141372329ndash2345

37 Johnson JOGlynn SMGibbs JR et alMutations in theCHCHD10 gene are a commoncause of familial amyotrophic lateral sclerosis Brain J Neurol 2014137e311

38 Muller K Andersen PM Hubers A et al Two novel mutations in conserved codonsindicate that CHCHD10 is a gene associated with motor neuron disease Brain JNeurol 2014137e309

39 Straub IR Janer A Weraarpachai W et al Loss of CHCHD10ndashCHCHD2 complexesrequired for respiration underlies the pathogenicity of a CHCHD10 mutation in ALSHum Mol Genet 201827178ndash189

40 Burstein SR Valsecchi F Kawamata H et al In vitro and in vivo studies of the ALS-FTLD protein CHCHD10 reveal novel mitochondrial topology and protein inter-actions Hum Mol Genet 201827160ndash177


DOI 101212NXG000000000000027620184 Neurol Genet

Richard G Lee Maryam Sedghi Mehri Salari et al dysfunction

Early-onset Parkinson disease caused by a mutation in CHCHD2 and mitochondrial

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Parkinson disease (PD MIM168600) is the most commonmovement disorder of aging and the second-most com-mon neurodegenerative disease after Alzheimer disease(MIM104300)12 PD has been associated with mutations inmultiple genes including PARK2 PINK1 PARK7 ATP13A2SCNA LRRK2 VPS35 EIF4G1 DNAJC13 and CHCHD23ndash6

Recent whole-exome sequencing (WES) of Japanese patientswith autosomal dominant or sporadic PD identified a hetero-zygous CHCHD2 missense change (pThr61Ile) in a familywith autosomal dominant late-onset PD5 Subsequently theidentical variant was identified in a Chinese family with auto-somal dominant PD6 Additional CHCHD2 variants includingpAla32Thr pPro34Leu and pIle80Val have been describedin 4 western European familial patients with PD7 The patho-mechanism of the CHCHD2 variants in these studies is how-ever unclear as is the physiologic role of CHCHD2

Here we identify a new CHCHD2 variant in a patient withautosomal recessive early-onset PD and establish the patho-genic mechanism of this variant We have shown that themutation results in a fragmented mitochondrial morphologyand reduced electron transport chain (ETC) activity

MethodsStandard protocol approvals registrationsand patient consentsThe study was approved by the ethical standards of the rel-evant institutional review board the Ethics Review Com-mittee in the Gothenburg Region (Dn1 842-14) and theHuman Research Ethics Committee of the University ofWestern Australia Informed consent was obtained frompatients included in this study after appropriate geneticcounseling Blood samples were obtained from patients theirparents and siblings

Clinical evaluationMedical history was obtained and physical examination wasperformed as part of routine clinical workup

Genetic analysisNext-generation sequencing (NGS) whole-exome andor tar-geted neuromuscular panel sequencing (WES or neuromus-cular sub-exomic sequencing [NSES]) was performed on thepatientrsquos DNA Confirmatory bidirectional Sanger sequencingwas performed in patients with PD and their family members(see e-Methods linkslwwcomNXGA85)

Cell culture and transfectionsDermal fibroblast cultures from the patient were establishedby standard protocols after written informed consent wasobtained Cultured fibroblasts from a healthy age-matchedindividual were used as a control Detailed methods are pro-vided in supplementary information

Mitochondrial isolation and cell lysisMitochondria were isolated from fibroblasts as previouslydescribed8 Cell lysates were prepared using a buffer con-taining the following 150 mMNaCl 01 (volvol) TritonX-100 and 50 mM Tris-HCl (pH 80) Protein concentra-tion was determined using a bicinchoninic acid (BCA)assay

Fluorescence microscopyDetailed methods are described in the supplementary mate-rial linkslwwcomNXGA85

Long-range PCR and mitochondrial DNA copynumber quantitative PCRDetailed methods are described in the supplementary mate-rial The sequences of all primers used for this study aredetailed in table e-1 linkslwwcomNXGA85

SDS-PAGE and immunoblottingMitochondria (25 μg) isolated from control and patientfibroblasts were separated on 4ndash12 Bis-Tris gels (Invi-trogen) and transferred onto a polyvinylidene difluoride(PVDF) membrane (Bio-Rad) All antibodies used aredetailed in the supplementary information

Mitochondrial protein synthesisDe novo mitochondrial protein synthesis was analyzed infibroblasts using 35S-radiolabeling of mitochondrially enco-ded proteins in the presence of emetine as previously de-scribed9 The cells were suspended in phosphate-bufferedsaline (PBS) and 20 μg of protein was resolved on a 125SDS-PAGE gel and radiolabelled proteins were visualizedon film

RespirationRespiration was measured in fibroblasts as previously de-scribed10 The full methods are detailed in supplementaryinformation

Cell function measurementsJC-1 mitochondrial mass dihydroethidium (DHE) and MTSassay full methods are detailed in supplementary information

GlossaryBCA = Bicinchoninic acidDHE = dihydroethidium ETC = electron transport chainMICOS =mitochondrial contact site andcristae organizing system NGS = next-generation sequencing OXPHOS = oxidative phosphorylation PBS = phosphate-buffered saline PD = Parkinson disease RCR = respiratory control ratio ROS = reactive oxygen speciesWES = whole-exomesequencing
















































































































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