confirming tdp2 mutation in spinocerebellar ataxia
TRANSCRIPT
ARTICLE OPEN ACCESS
Confirming TDP2 mutation in spinocerebellarataxia autosomal recessive 23 (SCAR23)Guido Zagnoli-Vieira BSc Francesco Bruni PhD Kyle Thompson PhD Langping He MD PhD
Sarah Walker PhD Arjan PM de Brouwer PhD Robert Taylor PhD FRCPath Dmitriy Niyazov MD
and Keith W Caldecott PhD
Neurol Genet 20184e262 doi101212NXG0000000000000262
Correspondence
Dr Caldecott
kwcaldecottsussexacuk
AbstractObjectiveTo address the relationship between mutations in the DNA strand break repair protein tyrosylDNA phosphodiesterase 2 (TDP2) and spinocerebellar ataxia autosomal recessive 23(SCAR23) and to characterize the cellular phenotype of primary fibroblasts from this disease
MethodsWe have used exome sequencing Sanger sequencing gene editing and cell biology bio-chemistry and subcellular mitochondrial analyses for this study
ResultsWe have identified a patient in the United States with SCAR23 harboring the same homozy-gous TDP2mutation as previously reported in 3 Irish siblings (c425+1GgtA) The current andIrish patients share the same disease haplotype but the current patient lacks a homozygousvariant present in the Irish siblings in the closely linked gene ZNF193 eliminating this asa contributor to the disease The current patient also displays symptoms consistent withmitochondrial dysfunction although levels of mitochondrial function in patient primary skinfibroblasts are normal However we demonstrate an inability in patient primary fibroblasts torapidly repair topoisomerase-induced DNA double-strand breaks (DSBs) in the nucleus andprofound hypersensitivity to this type of DNA damage
ConclusionsThese data confirm theTDP2mutation as causative for SCAR23 and highlight the link betweendefects in nuclear DNA DSB repair developmental delay epilepsy and ataxia
Current address Department of Biosciences Biotechnologies and Biopharmaceutics University of Bari Aldo Moro Bari Italy
From the Genome Damage and Stability Centre (GZ-V KWC) University of Sussex Falmer Brighton United Kingdom Wellcome Centre for Mitochondrial Research (FB KT LHRT) Institute of Neuroscience Newcastle University Tyne United Kingdom Sussex Drug Discovery Centre (SW) University of Sussex Falmer Brighton United KingdomDepartment of Human Genetics (APMdB) Donders Institute for Brain Cognition and Behaviour Radboud University Medical Center Nijmegen The Netherlands and MedicalGenetics (APMdB DN) Ochsner Health Center for Children New Orleans LA
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 authors
This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited
Copyright copy 2018 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1
DNA is under constant threat from attack by endogenous andexogenous electrophilic molecules1 and DNA topoisomeraseenzymes can introduce DNA breaks as abortive intermediatesof their activity2ndash4 Topoisomerase ldquopoisonsrdquo such as etopo-side inhibit the ligation activity of topoisomerase 2 (TOP2)thereby promoting the formation of abortive DNA double-strand break (DSB) intermediates that require DSB repairDSBs are repaired in cells by either homologousrecombinationndashmediated repair or by nonhomologous endjoining (NHEJ)2 The repair of TOP2-induced DSBs byNHEJ involves the enzyme tyrosyl DNA phosphodiesterase 2(TDP2) which removes trapped topoisomerase peptide fromthe 59-termini at the DSB and thereby allows the DNA ends tobe ligated3ndash5 The loss of TDP2 in mouse results in reducedexpression of gt100 genes in the brain6 and TDP2mutation inhumans has been associated with intellectual disability seiz-ures and ataxia6 a disease now denoted as spinocerebellarataxia autosomal recessive 23 (SCAR23) To date our un-derstanding of SCAR23 has been limited by the availability ofonly 3 Irish siblings with a mutation in TDP2 and by the lackof availability of fibroblast cell lines from these patients formolecular and cellular characterization Here we haveaddressed these limitations and identified an SCAR23 patientin the United States with the same homozygous TDP2 mu-tation as present in the Irish siblings confirming the associ-ation of this disease with mutated TDP2 In addition we havecharacterized at the molecular and cellular level primary pa-tient fibroblasts from the current SCAR23 patient
MethodsPatient case reportThe patient is currently a 6-year-old caucasian boy fromthe United States presenting with developmental delaymicrocephaly and failure to thrive His mother and fatherare aged 29 years and 32 years respectively and there is noprevious family history of related disease but there ispossible consanguinity (3rd cousins) The patient startedwalking at the age of 14 months and talking at 3 years hishearing was reportedly normal but he did not have audi-tory brainstem response The patient is easily fatigued andhis parents reported that he eats excessively becomes ir-ritable and falls asleep sometimes for days or up to 2weeks He is now below the fifth percentile for weight and5 for height with a body mass index at lt5 He hasa G-tube and is followed by a dietitian and receives nutri-tional liquid supplements (PediaSure Abbott Lake ForestIL) He has a history of constipation and is followed up bya gastroenterologist He has a 12-year-old brother who is
also easily fatigued and a 14-year-old maternal half-sisterwith attention deficit hyperactivity disorder (ADHD)
The patient has difficulty keeping balance and has an ataxicgait He has been tested several times for abnormalities inblood urine and CSF and by MRI and EEG and only thelatter is abnormal Seizures began at the age of 5 months andhis EEGs show increased slowing and occasional spikes in theright posterior quadrant and during drowsiness he had 2generalized bursts of polyspike and slow-wave activity ata frequency of 4ndash5 Hz The patientrsquos 180K oligoarray verylong chain fatty acids carbohydrate-deficient transferrin CSFlactate and neurotransmitters and plasma amino acids werenormal
The patient exhibited several phenotypes consistent with mi-tochondrial dysfunction such as hypotonia low energy fati-gability hypersomnia failure to thrive short statureconstipation neutropenia hyponatremia cardiac arrhythmiaand gastrointestinal dysmotility Consistent with this electrontransport chain (ETC) studies on muscle biopsy at age 1 yearshowed a severe reduction of complex I + III and II + IIIactivity which satisfied the major Walker criteria after cor-rection for increased citrate synthase activity CoQ10 de-ficiency was suggested based on the complex I + III and II + IIIdeficiency and his metabolic tests were abnormal with thehigh lactate and high lactatepyruvate ratio The patient wasplaced on ubiquinol carnitine and leucovorin and anecdot-ally he responded well (particularly to liposomal ubiquinolthe active or reduced form of CoQ10) in terms of energy anddevelopmental progress He is reportedly no longer sleepy andlethargic and has a stable gait but is ldquostill behindrdquo in languageand cognitive skills and is in special education He still hasidiopathic fevers but has not been admitted to the hospital for15 months He does not have as many infections but still hasgastric dysmotility His seizures have reduced in frequency
Standard protocol approvals registrations andpatient consentsWe confirm that we have received approval from an in-stitutional ethics standards committee for this work and wehave written informed consent for research from the guardianof the patient for participation in this study
Exome sequencing and haplotype analysisWhole-exome sequencing (WES) (trio study) of the patient wasperformed by GeneDx using the Agilent Clinical ResearchExome kit to target the exonic regions and flanking splicejunctions of the genome These targeted regions were sequencedsimultaneously by massively parallel (NextGen Irvine CA)
GlossaryDSB = double-strand break ETC = electron transport chain FCS = fetal calf serum NHEJ = nonhomologous end joiningSCAR23 = spinocerebellar ataxia autosomal recessive 23 TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cellextract WES = Whole-exome sequencing
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sequencing on an Illumina HiSeq sequencing system with100bp paired-end reads Bidirectional sequence was assembledaligned to reference gene sequences based on human genomebuild GRCh37UCSC hg19 and analyzed for sequence var-iants in the selected genes or regions of interest using a custom-developed analysis tool (XomeAnalyzer) Capillary sequencingwas used to confirm all potentially pathogenic variants identi-fied in this individual Sequence alterations were reportedaccording to the nomenclature guidelines of the HumanGenome Variation Society The WES identified a homozygoussplice site mutation (c425+1GgtA) in the TDP2 gene Forcomparison of the current patient with the Irish pedigreepreviously reported6 we conducted haplotype analysis Var-iants were considered for homozygosity if they were (1) cov-ered by at least 4 reads or more (2) present in 80 of all readsor more (3) designated as a substitution (4) uniquely posi-tioned in the human genome and (5) present in the exomedata of both individuals Homozygous regions were de-termined using a sliding window accepting 2 or less homozy-gous variants per 10 variants assessed
Mitochondrial preparation and subcellularfractionationMitochondria were prepared as described previously7 with fewmodifications HeLa cells and fibroblasts (control and patient)were harvested resuspended in homogenization buffer (HB[06 M mannitol 10 mM Tris-HCl pH 74 1 mM (ethyleneglycol-bis(β-aminoethyl ether)-NNN9N9-tetraacetic acid)(EGTA) 01 bovine serum albumin (BSA) (wtvol]) andsubjected to differential centrifugation Mitochondria were pel-leted at 11000g for 10 minutes at 4degC and resuspended in HBthe postmitochondrial supernatant was retained after centrifu-gation (ldquopost-mito spinrdquo) For submitochondrial fraction prepa-ration HeLa cell mitochondria (300 μg) were treated with 16 μgof proteinase K on ice for 30minutes followed by the addition of5 mM phenylmethanesulfonyl fluoride (PMSF) This fractionwas pelleted at 11000g for 10minutes at 4degC and resuspended inHB Mitoplasts were obtained by resuspending PK-treated mi-tochondria in 9 volumes of 10 mM Tris-HCl (pH 74) andtreated with PK as described earlier Inner mitochondrialmembrane proteins were extracted in the presence of 100 mMNa2CO3 followed by centrifugation at 100000g for 15 minutesat 4degC Proteins (30 μg) from each fraction were loaded onto12 SDS-PAGE gel transferred to the polyvinylidene difluoride(PVDF) membrane and analyzed by immunoblotting usingprimary antibodies to apoptosis inducing factor (AIF) (NEB)eIF4E (Cell Signalling) EF-Tu (custom made) NDUFB8(Mitosciences) and TDP2 (see Western Blotting below)
Cell culture and vectorsHuman A549 cells were grown in Dulbecco Modified EagleMedium (Gibco ThermoFisher Waltham MA) containing10 fetal calf serum (FCS) 2 mM glutamine penicillin (100unitsmL) and streptomycin (100 μgmL) Human fibroblastswere grown in Minimum Essential Media (Gibco) containing15 FCS 2 mM glutamine penicillin (100 unitsmL) andstreptomycin (100 μgmL) All cells were grown at 5 CO2 at
37degC TDP2-mutated patient primary human fibroblasts wereestablished from a patientrsquos skin biopsy and were denoted 850-BR The control human fibroblast cell line 1-BR3 (denotedhere for simplicity as 1-BR) was previously derived from anunrelated normal individual and has been described pre-viously8 For complementation experiments we used 1-BRcells that were immortalized previously with hTERT (denotedas 1-BR hTERT) and a derivative of 850-BR immortalized inthe current study by retroviral-mediated hTERT expressionand selected in a medium containing 1 μgml puromycin(denoted as 850-BR hTERT) For complementation withhuman TDP2 850-BR hTERT cells were transfected with ei-ther empty eGFP-N1 vector or eGFP-N1 construct encodingGFP-tagged human TDP2 (denoted as TDP2-GFP-N1) andstable transfectants selected for 21 days by growth in a mediumcontaining 05mgmLG418 (Gibco ThermoFisherWalthamMA) TDP2-GFP-N1 was generated by PCR amplificationof the human TDP2 ORF using the primers TDP2_FW(59-AAAGAATTCATGGAGTTGGGGAGTTGCCTG-39)and TDP2_RV (59-AAAGGATCCAATATTATATCTAAGTTGCACAGAAGACC-39) and subcloning the PCRproduct into the EcoRIBamHI sites of eGFP-N1
CRISPRCas9 gene editingA549 TDP2minusminus cells were created as previously reported9 Inbrief we used a 17-bp (minus the PAM) RNA sequencetargeting TDP2 exon 4 (59-GTAGAAATATCACATCT-39)which was selected using the tool E-CRISP (e-crisporgE-CRISP) and cloned into the guide RNA vector 41824(AddGene) The TDP2 guide construct was cotransfectedwith hCas9 expressed from plasmid 41815 (AddGene) usingAmaxa Nucleofector plataform (Lonza Basel Switzerland)Kit T program X-001 Transfected cells were enriched byselection in 1 mgmL G418 (Thermofisher) for 5 days beforeisolation of single clones and screening for loss of TDP2expression by Western blotting
Sanger sequencingDNA was extracted from 850-BR cells using the DNeasyBlood amp Tissue kit (Qiagen Manchester UK) PCRreactions used Phusion HF-DNA Polymerase (NEB) andthe following primers TDP2_FW GCCAGTGTT-GACCTAACCAATGAAGA TDP2_RV CTGTAGAAA-TATCACATCTGGGCTGTACC ZSCAN9_FWATGAAGTAACCAAGACTGAGGACAGAGAG and ZSCAN9_RVAGACCAGCTCAGCCACTGTGTGGATCT PCR productswere purified before sequencing using a QIAquick PCR purifi-cation kit (Qiagen)
Western blottingAnti-TDP2 antibody was used at 15000 in Western blottingand has been described previously10 Anti-Actin (SigmaA4700) was used at 12000 Western blot assessment ofOXPHOS components in patient primary fibroblasts wasconducted as described previously11 using antibodies againstNDUFB8 (Abcam cat ab110242) SDHA (Abcam catab14715) UQCRC2 (Abcam cat ab14745) COXI (Abcam
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 3
cat ab14705) ATP5A (Abcam cat ab14748) SDHB(Abcam cat ab14714) COX II (Abcam cat 110258) andVDAC1 (Abcam cat ab14734)
Clonogenic survival assays1-BR and 850-BR primary fibroblasts cells were plated ontofeeder layers (see below) and 3 hours later treated with indicatedconcentrations of etoposide for 21 days to allow the formation ofmacroscopic colonies which were rinsed in phosphate bufferedsaline (PBS) and fixedstained in 70 ethanol1 methyleneblue A549 and TDP2minusminus A549 were plated 4 hours beforetreatment with the indicated concentrations of etoposide for 12days and stained as described earlier The surviving fraction ateach dose was calculated by dividing the average number of
colonies (gt50 cells) in treated dishes by the average number inuntreated dishes For feeder layers 1-BR cells were irradiated (35Gy) and plated 24 hours before use at 5 times 104 cells10 cm dish
Tyrosyl DNA phosphodiesterase assaysWhole-cell extract (WCE) was prepared by resuspension of1-BR or 850-BR primary fibroblast cell pellets (1times106cells) in100μL lysis buffer (40 mM TrisHCl pH 75 100 mM NaCl01 Tween-20 1 mM DTT 1 mM PMSF 1x EDTA freeprotease cocktail inhibitor) followed by 30 minutes of in-cubation on ice and mild sonication The WCE was clarifiedby centrifugation for 10 minutes at 4degC at 16000g ina microfuge and the protein concentration quantified usingthe bicinchoninic acid (BCA) assay reagent (ThermoFisher)
Figure 1 TDP2 splice site mutation in an individual from the United States with SCAR23
(A) Pedigree analysis White symbolswild-type TDP2 Black symbols ho-mozygosity for the TDP2 splice sitemutation c425+1GgtA (IVS3+1GgtA) inthe proband (ldquo1rdquo) Dotted symbolsheterozygosity for c425+1GgtA (IVS3+1GgtA) (B) Sanger sequencing of theproband demonstrating (left) thehomozygous TDP2 mutation c425+1GgtA (IVS3+1GgtA) and (right) the ab-sence of the ZSCAN9 variantc914AgtG Patient sequences areshown at the bottom and referencesequences are shown at the top The2 nucleotides relevant to the muta-tions are underlined (C) Pathologicfeatures of the Irish and US patients(D) Haplotypes of Irish patients(51670 Irish pedigree) and the cur-rent patient from the United States(1583786) carrying the homozygousc425+1GgtA variant in TDP2 Only thevariants bordering and directly withinthe homozygous regions and witha minor allele frequency of 5 areshown Variants are indicated by theiraccession number in the dbSNP da-tabase and the position of c425+1GgtA is in bold The minimal over-lapping region is delimited byrs9396886 and rs749338 and is 151Mb in size Black bars represent thehomozygous haplotype as inferredfrom exome sequencing data TDP2 =tyrosyl DNA phosphodiesterase 2
4 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
Clarified WCE (15μg total protein) was incubated with 40nM TDP2 substrate (Cy5-59Tyrosine-ssDNA19-BHQ) orTDP1 substrate (BHQ-ssDNA13-39Tyrosine-Cy5) in re-action buffer (50 mM TrisHCl pH80 10 mM MgCl280 mM KCl and 1 mM DTT 005 Tween-20) in a totalvolume of 6μL at room temperature and Cy5 fluorescencewas measured at 640 nm at the indicated time intervals ona BMG PHERAstar plate reader
DSB repair assaysCells were grown on coverslips until confluent and thentreated for 30 minutes with 25μM etoposide or irradiatedwith x-rays (2 Gy) After treatment cells were rinsed andfixed for 10 minutes in PBS containing 4 para-formaldehyde at the indicated time points Cells werepermeabilized (20 minutes in PBS-02 Triton X-100)
blocked (1 hour in PBS-5 BSA) and incubated with anti-γH2AX (Millipore 05-636 12500) and anti-CENP-F(Abcam ab5 12500) antibodies for 3 hours in PBS con-taining 5 BSA Cells were then washed (3 times 5 minutes inPBS containing 01Tween-20) incubated for 1h with thecorresponding Alexa Fluor conjugated secondary antibody(11000 5 BSA) and washed again as described earlierFinally cells were counterstained with DAPI (SigmaGillingham UK) and mounted in VECTASHIELD (VectorLabs Peterborough UK) Images were acquired on anautomated wide-field microscopy Olympus ScanR system(motorized IX83 microscope) with ScanR Image Acquisi-tion and Analysis Software 20times045 (LUCPLFLN20timesPH) dry objectives and Hamamatsu ORCA-R2 digitalCCD camera C10600 For the analysis of G0G1 cells inpatient primary fibroblasts only CENP-F-negative cells
Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
(A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts as measured by immunoblotting (B)TDP1 and TDP2 biochemical assay Hydrolysis of a 39-phosphotyrosyl (TDP1 assay) or 59-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus resulting in elevated fluorescence at 640 nm (C) Real-time (0ndash30minutes)measurements of 59-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) orWCE (15μg protein)from 1-BR normal or 850-BR patient primary fibroblasts using the single-stranded oligonucleotide (40 nM) with a 59-phosphotyrosyl-linked Cy5 fluorophoreand 39-BHQ (D) Real-time measurements (0ndash30 minutes) of 39-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 39-phospho-tyrosyl-linked Cy5 fluorophore and 59-BHQ Data are mean of 3 independent experiments plusmnSEM Statistical significance was determined by two way analysisof variance (ANOVA) p lt 0001 BHQ = black hole quencher TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cell extract
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were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
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850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
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is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
DNA is under constant threat from attack by endogenous andexogenous electrophilic molecules1 and DNA topoisomeraseenzymes can introduce DNA breaks as abortive intermediatesof their activity2ndash4 Topoisomerase ldquopoisonsrdquo such as etopo-side inhibit the ligation activity of topoisomerase 2 (TOP2)thereby promoting the formation of abortive DNA double-strand break (DSB) intermediates that require DSB repairDSBs are repaired in cells by either homologousrecombinationndashmediated repair or by nonhomologous endjoining (NHEJ)2 The repair of TOP2-induced DSBs byNHEJ involves the enzyme tyrosyl DNA phosphodiesterase 2(TDP2) which removes trapped topoisomerase peptide fromthe 59-termini at the DSB and thereby allows the DNA ends tobe ligated3ndash5 The loss of TDP2 in mouse results in reducedexpression of gt100 genes in the brain6 and TDP2mutation inhumans has been associated with intellectual disability seiz-ures and ataxia6 a disease now denoted as spinocerebellarataxia autosomal recessive 23 (SCAR23) To date our un-derstanding of SCAR23 has been limited by the availability ofonly 3 Irish siblings with a mutation in TDP2 and by the lackof availability of fibroblast cell lines from these patients formolecular and cellular characterization Here we haveaddressed these limitations and identified an SCAR23 patientin the United States with the same homozygous TDP2 mu-tation as present in the Irish siblings confirming the associ-ation of this disease with mutated TDP2 In addition we havecharacterized at the molecular and cellular level primary pa-tient fibroblasts from the current SCAR23 patient
MethodsPatient case reportThe patient is currently a 6-year-old caucasian boy fromthe United States presenting with developmental delaymicrocephaly and failure to thrive His mother and fatherare aged 29 years and 32 years respectively and there is noprevious family history of related disease but there ispossible consanguinity (3rd cousins) The patient startedwalking at the age of 14 months and talking at 3 years hishearing was reportedly normal but he did not have audi-tory brainstem response The patient is easily fatigued andhis parents reported that he eats excessively becomes ir-ritable and falls asleep sometimes for days or up to 2weeks He is now below the fifth percentile for weight and5 for height with a body mass index at lt5 He hasa G-tube and is followed by a dietitian and receives nutri-tional liquid supplements (PediaSure Abbott Lake ForestIL) He has a history of constipation and is followed up bya gastroenterologist He has a 12-year-old brother who is
also easily fatigued and a 14-year-old maternal half-sisterwith attention deficit hyperactivity disorder (ADHD)
The patient has difficulty keeping balance and has an ataxicgait He has been tested several times for abnormalities inblood urine and CSF and by MRI and EEG and only thelatter is abnormal Seizures began at the age of 5 months andhis EEGs show increased slowing and occasional spikes in theright posterior quadrant and during drowsiness he had 2generalized bursts of polyspike and slow-wave activity ata frequency of 4ndash5 Hz The patientrsquos 180K oligoarray verylong chain fatty acids carbohydrate-deficient transferrin CSFlactate and neurotransmitters and plasma amino acids werenormal
The patient exhibited several phenotypes consistent with mi-tochondrial dysfunction such as hypotonia low energy fati-gability hypersomnia failure to thrive short statureconstipation neutropenia hyponatremia cardiac arrhythmiaand gastrointestinal dysmotility Consistent with this electrontransport chain (ETC) studies on muscle biopsy at age 1 yearshowed a severe reduction of complex I + III and II + IIIactivity which satisfied the major Walker criteria after cor-rection for increased citrate synthase activity CoQ10 de-ficiency was suggested based on the complex I + III and II + IIIdeficiency and his metabolic tests were abnormal with thehigh lactate and high lactatepyruvate ratio The patient wasplaced on ubiquinol carnitine and leucovorin and anecdot-ally he responded well (particularly to liposomal ubiquinolthe active or reduced form of CoQ10) in terms of energy anddevelopmental progress He is reportedly no longer sleepy andlethargic and has a stable gait but is ldquostill behindrdquo in languageand cognitive skills and is in special education He still hasidiopathic fevers but has not been admitted to the hospital for15 months He does not have as many infections but still hasgastric dysmotility His seizures have reduced in frequency
Standard protocol approvals registrations andpatient consentsWe confirm that we have received approval from an in-stitutional ethics standards committee for this work and wehave written informed consent for research from the guardianof the patient for participation in this study
Exome sequencing and haplotype analysisWhole-exome sequencing (WES) (trio study) of the patient wasperformed by GeneDx using the Agilent Clinical ResearchExome kit to target the exonic regions and flanking splicejunctions of the genome These targeted regions were sequencedsimultaneously by massively parallel (NextGen Irvine CA)
GlossaryDSB = double-strand break ETC = electron transport chain FCS = fetal calf serum NHEJ = nonhomologous end joiningSCAR23 = spinocerebellar ataxia autosomal recessive 23 TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cellextract WES = Whole-exome sequencing
2 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
sequencing on an Illumina HiSeq sequencing system with100bp paired-end reads Bidirectional sequence was assembledaligned to reference gene sequences based on human genomebuild GRCh37UCSC hg19 and analyzed for sequence var-iants in the selected genes or regions of interest using a custom-developed analysis tool (XomeAnalyzer) Capillary sequencingwas used to confirm all potentially pathogenic variants identi-fied in this individual Sequence alterations were reportedaccording to the nomenclature guidelines of the HumanGenome Variation Society The WES identified a homozygoussplice site mutation (c425+1GgtA) in the TDP2 gene Forcomparison of the current patient with the Irish pedigreepreviously reported6 we conducted haplotype analysis Var-iants were considered for homozygosity if they were (1) cov-ered by at least 4 reads or more (2) present in 80 of all readsor more (3) designated as a substitution (4) uniquely posi-tioned in the human genome and (5) present in the exomedata of both individuals Homozygous regions were de-termined using a sliding window accepting 2 or less homozy-gous variants per 10 variants assessed
Mitochondrial preparation and subcellularfractionationMitochondria were prepared as described previously7 with fewmodifications HeLa cells and fibroblasts (control and patient)were harvested resuspended in homogenization buffer (HB[06 M mannitol 10 mM Tris-HCl pH 74 1 mM (ethyleneglycol-bis(β-aminoethyl ether)-NNN9N9-tetraacetic acid)(EGTA) 01 bovine serum albumin (BSA) (wtvol]) andsubjected to differential centrifugation Mitochondria were pel-leted at 11000g for 10 minutes at 4degC and resuspended in HBthe postmitochondrial supernatant was retained after centrifu-gation (ldquopost-mito spinrdquo) For submitochondrial fraction prepa-ration HeLa cell mitochondria (300 μg) were treated with 16 μgof proteinase K on ice for 30minutes followed by the addition of5 mM phenylmethanesulfonyl fluoride (PMSF) This fractionwas pelleted at 11000g for 10minutes at 4degC and resuspended inHB Mitoplasts were obtained by resuspending PK-treated mi-tochondria in 9 volumes of 10 mM Tris-HCl (pH 74) andtreated with PK as described earlier Inner mitochondrialmembrane proteins were extracted in the presence of 100 mMNa2CO3 followed by centrifugation at 100000g for 15 minutesat 4degC Proteins (30 μg) from each fraction were loaded onto12 SDS-PAGE gel transferred to the polyvinylidene difluoride(PVDF) membrane and analyzed by immunoblotting usingprimary antibodies to apoptosis inducing factor (AIF) (NEB)eIF4E (Cell Signalling) EF-Tu (custom made) NDUFB8(Mitosciences) and TDP2 (see Western Blotting below)
Cell culture and vectorsHuman A549 cells were grown in Dulbecco Modified EagleMedium (Gibco ThermoFisher Waltham MA) containing10 fetal calf serum (FCS) 2 mM glutamine penicillin (100unitsmL) and streptomycin (100 μgmL) Human fibroblastswere grown in Minimum Essential Media (Gibco) containing15 FCS 2 mM glutamine penicillin (100 unitsmL) andstreptomycin (100 μgmL) All cells were grown at 5 CO2 at
37degC TDP2-mutated patient primary human fibroblasts wereestablished from a patientrsquos skin biopsy and were denoted 850-BR The control human fibroblast cell line 1-BR3 (denotedhere for simplicity as 1-BR) was previously derived from anunrelated normal individual and has been described pre-viously8 For complementation experiments we used 1-BRcells that were immortalized previously with hTERT (denotedas 1-BR hTERT) and a derivative of 850-BR immortalized inthe current study by retroviral-mediated hTERT expressionand selected in a medium containing 1 μgml puromycin(denoted as 850-BR hTERT) For complementation withhuman TDP2 850-BR hTERT cells were transfected with ei-ther empty eGFP-N1 vector or eGFP-N1 construct encodingGFP-tagged human TDP2 (denoted as TDP2-GFP-N1) andstable transfectants selected for 21 days by growth in a mediumcontaining 05mgmLG418 (Gibco ThermoFisherWalthamMA) TDP2-GFP-N1 was generated by PCR amplificationof the human TDP2 ORF using the primers TDP2_FW(59-AAAGAATTCATGGAGTTGGGGAGTTGCCTG-39)and TDP2_RV (59-AAAGGATCCAATATTATATCTAAGTTGCACAGAAGACC-39) and subcloning the PCRproduct into the EcoRIBamHI sites of eGFP-N1
CRISPRCas9 gene editingA549 TDP2minusminus cells were created as previously reported9 Inbrief we used a 17-bp (minus the PAM) RNA sequencetargeting TDP2 exon 4 (59-GTAGAAATATCACATCT-39)which was selected using the tool E-CRISP (e-crisporgE-CRISP) and cloned into the guide RNA vector 41824(AddGene) The TDP2 guide construct was cotransfectedwith hCas9 expressed from plasmid 41815 (AddGene) usingAmaxa Nucleofector plataform (Lonza Basel Switzerland)Kit T program X-001 Transfected cells were enriched byselection in 1 mgmL G418 (Thermofisher) for 5 days beforeisolation of single clones and screening for loss of TDP2expression by Western blotting
Sanger sequencingDNA was extracted from 850-BR cells using the DNeasyBlood amp Tissue kit (Qiagen Manchester UK) PCRreactions used Phusion HF-DNA Polymerase (NEB) andthe following primers TDP2_FW GCCAGTGTT-GACCTAACCAATGAAGA TDP2_RV CTGTAGAAA-TATCACATCTGGGCTGTACC ZSCAN9_FWATGAAGTAACCAAGACTGAGGACAGAGAG and ZSCAN9_RVAGACCAGCTCAGCCACTGTGTGGATCT PCR productswere purified before sequencing using a QIAquick PCR purifi-cation kit (Qiagen)
Western blottingAnti-TDP2 antibody was used at 15000 in Western blottingand has been described previously10 Anti-Actin (SigmaA4700) was used at 12000 Western blot assessment ofOXPHOS components in patient primary fibroblasts wasconducted as described previously11 using antibodies againstNDUFB8 (Abcam cat ab110242) SDHA (Abcam catab14715) UQCRC2 (Abcam cat ab14745) COXI (Abcam
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 3
cat ab14705) ATP5A (Abcam cat ab14748) SDHB(Abcam cat ab14714) COX II (Abcam cat 110258) andVDAC1 (Abcam cat ab14734)
Clonogenic survival assays1-BR and 850-BR primary fibroblasts cells were plated ontofeeder layers (see below) and 3 hours later treated with indicatedconcentrations of etoposide for 21 days to allow the formation ofmacroscopic colonies which were rinsed in phosphate bufferedsaline (PBS) and fixedstained in 70 ethanol1 methyleneblue A549 and TDP2minusminus A549 were plated 4 hours beforetreatment with the indicated concentrations of etoposide for 12days and stained as described earlier The surviving fraction ateach dose was calculated by dividing the average number of
colonies (gt50 cells) in treated dishes by the average number inuntreated dishes For feeder layers 1-BR cells were irradiated (35Gy) and plated 24 hours before use at 5 times 104 cells10 cm dish
Tyrosyl DNA phosphodiesterase assaysWhole-cell extract (WCE) was prepared by resuspension of1-BR or 850-BR primary fibroblast cell pellets (1times106cells) in100μL lysis buffer (40 mM TrisHCl pH 75 100 mM NaCl01 Tween-20 1 mM DTT 1 mM PMSF 1x EDTA freeprotease cocktail inhibitor) followed by 30 minutes of in-cubation on ice and mild sonication The WCE was clarifiedby centrifugation for 10 minutes at 4degC at 16000g ina microfuge and the protein concentration quantified usingthe bicinchoninic acid (BCA) assay reagent (ThermoFisher)
Figure 1 TDP2 splice site mutation in an individual from the United States with SCAR23
(A) Pedigree analysis White symbolswild-type TDP2 Black symbols ho-mozygosity for the TDP2 splice sitemutation c425+1GgtA (IVS3+1GgtA) inthe proband (ldquo1rdquo) Dotted symbolsheterozygosity for c425+1GgtA (IVS3+1GgtA) (B) Sanger sequencing of theproband demonstrating (left) thehomozygous TDP2 mutation c425+1GgtA (IVS3+1GgtA) and (right) the ab-sence of the ZSCAN9 variantc914AgtG Patient sequences areshown at the bottom and referencesequences are shown at the top The2 nucleotides relevant to the muta-tions are underlined (C) Pathologicfeatures of the Irish and US patients(D) Haplotypes of Irish patients(51670 Irish pedigree) and the cur-rent patient from the United States(1583786) carrying the homozygousc425+1GgtA variant in TDP2 Only thevariants bordering and directly withinthe homozygous regions and witha minor allele frequency of 5 areshown Variants are indicated by theiraccession number in the dbSNP da-tabase and the position of c425+1GgtA is in bold The minimal over-lapping region is delimited byrs9396886 and rs749338 and is 151Mb in size Black bars represent thehomozygous haplotype as inferredfrom exome sequencing data TDP2 =tyrosyl DNA phosphodiesterase 2
4 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
Clarified WCE (15μg total protein) was incubated with 40nM TDP2 substrate (Cy5-59Tyrosine-ssDNA19-BHQ) orTDP1 substrate (BHQ-ssDNA13-39Tyrosine-Cy5) in re-action buffer (50 mM TrisHCl pH80 10 mM MgCl280 mM KCl and 1 mM DTT 005 Tween-20) in a totalvolume of 6μL at room temperature and Cy5 fluorescencewas measured at 640 nm at the indicated time intervals ona BMG PHERAstar plate reader
DSB repair assaysCells were grown on coverslips until confluent and thentreated for 30 minutes with 25μM etoposide or irradiatedwith x-rays (2 Gy) After treatment cells were rinsed andfixed for 10 minutes in PBS containing 4 para-formaldehyde at the indicated time points Cells werepermeabilized (20 minutes in PBS-02 Triton X-100)
blocked (1 hour in PBS-5 BSA) and incubated with anti-γH2AX (Millipore 05-636 12500) and anti-CENP-F(Abcam ab5 12500) antibodies for 3 hours in PBS con-taining 5 BSA Cells were then washed (3 times 5 minutes inPBS containing 01Tween-20) incubated for 1h with thecorresponding Alexa Fluor conjugated secondary antibody(11000 5 BSA) and washed again as described earlierFinally cells were counterstained with DAPI (SigmaGillingham UK) and mounted in VECTASHIELD (VectorLabs Peterborough UK) Images were acquired on anautomated wide-field microscopy Olympus ScanR system(motorized IX83 microscope) with ScanR Image Acquisi-tion and Analysis Software 20times045 (LUCPLFLN20timesPH) dry objectives and Hamamatsu ORCA-R2 digitalCCD camera C10600 For the analysis of G0G1 cells inpatient primary fibroblasts only CENP-F-negative cells
Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
(A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts as measured by immunoblotting (B)TDP1 and TDP2 biochemical assay Hydrolysis of a 39-phosphotyrosyl (TDP1 assay) or 59-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus resulting in elevated fluorescence at 640 nm (C) Real-time (0ndash30minutes)measurements of 59-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) orWCE (15μg protein)from 1-BR normal or 850-BR patient primary fibroblasts using the single-stranded oligonucleotide (40 nM) with a 59-phosphotyrosyl-linked Cy5 fluorophoreand 39-BHQ (D) Real-time measurements (0ndash30 minutes) of 39-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 39-phospho-tyrosyl-linked Cy5 fluorophore and 59-BHQ Data are mean of 3 independent experiments plusmnSEM Statistical significance was determined by two way analysisof variance (ANOVA) p lt 0001 BHQ = black hole quencher TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cell extract
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 5
were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
6 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
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is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
sequencing on an Illumina HiSeq sequencing system with100bp paired-end reads Bidirectional sequence was assembledaligned to reference gene sequences based on human genomebuild GRCh37UCSC hg19 and analyzed for sequence var-iants in the selected genes or regions of interest using a custom-developed analysis tool (XomeAnalyzer) Capillary sequencingwas used to confirm all potentially pathogenic variants identi-fied in this individual Sequence alterations were reportedaccording to the nomenclature guidelines of the HumanGenome Variation Society The WES identified a homozygoussplice site mutation (c425+1GgtA) in the TDP2 gene Forcomparison of the current patient with the Irish pedigreepreviously reported6 we conducted haplotype analysis Var-iants were considered for homozygosity if they were (1) cov-ered by at least 4 reads or more (2) present in 80 of all readsor more (3) designated as a substitution (4) uniquely posi-tioned in the human genome and (5) present in the exomedata of both individuals Homozygous regions were de-termined using a sliding window accepting 2 or less homozy-gous variants per 10 variants assessed
Mitochondrial preparation and subcellularfractionationMitochondria were prepared as described previously7 with fewmodifications HeLa cells and fibroblasts (control and patient)were harvested resuspended in homogenization buffer (HB[06 M mannitol 10 mM Tris-HCl pH 74 1 mM (ethyleneglycol-bis(β-aminoethyl ether)-NNN9N9-tetraacetic acid)(EGTA) 01 bovine serum albumin (BSA) (wtvol]) andsubjected to differential centrifugation Mitochondria were pel-leted at 11000g for 10 minutes at 4degC and resuspended in HBthe postmitochondrial supernatant was retained after centrifu-gation (ldquopost-mito spinrdquo) For submitochondrial fraction prepa-ration HeLa cell mitochondria (300 μg) were treated with 16 μgof proteinase K on ice for 30minutes followed by the addition of5 mM phenylmethanesulfonyl fluoride (PMSF) This fractionwas pelleted at 11000g for 10minutes at 4degC and resuspended inHB Mitoplasts were obtained by resuspending PK-treated mi-tochondria in 9 volumes of 10 mM Tris-HCl (pH 74) andtreated with PK as described earlier Inner mitochondrialmembrane proteins were extracted in the presence of 100 mMNa2CO3 followed by centrifugation at 100000g for 15 minutesat 4degC Proteins (30 μg) from each fraction were loaded onto12 SDS-PAGE gel transferred to the polyvinylidene difluoride(PVDF) membrane and analyzed by immunoblotting usingprimary antibodies to apoptosis inducing factor (AIF) (NEB)eIF4E (Cell Signalling) EF-Tu (custom made) NDUFB8(Mitosciences) and TDP2 (see Western Blotting below)
Cell culture and vectorsHuman A549 cells were grown in Dulbecco Modified EagleMedium (Gibco ThermoFisher Waltham MA) containing10 fetal calf serum (FCS) 2 mM glutamine penicillin (100unitsmL) and streptomycin (100 μgmL) Human fibroblastswere grown in Minimum Essential Media (Gibco) containing15 FCS 2 mM glutamine penicillin (100 unitsmL) andstreptomycin (100 μgmL) All cells were grown at 5 CO2 at
37degC TDP2-mutated patient primary human fibroblasts wereestablished from a patientrsquos skin biopsy and were denoted 850-BR The control human fibroblast cell line 1-BR3 (denotedhere for simplicity as 1-BR) was previously derived from anunrelated normal individual and has been described pre-viously8 For complementation experiments we used 1-BRcells that were immortalized previously with hTERT (denotedas 1-BR hTERT) and a derivative of 850-BR immortalized inthe current study by retroviral-mediated hTERT expressionand selected in a medium containing 1 μgml puromycin(denoted as 850-BR hTERT) For complementation withhuman TDP2 850-BR hTERT cells were transfected with ei-ther empty eGFP-N1 vector or eGFP-N1 construct encodingGFP-tagged human TDP2 (denoted as TDP2-GFP-N1) andstable transfectants selected for 21 days by growth in a mediumcontaining 05mgmLG418 (Gibco ThermoFisherWalthamMA) TDP2-GFP-N1 was generated by PCR amplificationof the human TDP2 ORF using the primers TDP2_FW(59-AAAGAATTCATGGAGTTGGGGAGTTGCCTG-39)and TDP2_RV (59-AAAGGATCCAATATTATATCTAAGTTGCACAGAAGACC-39) and subcloning the PCRproduct into the EcoRIBamHI sites of eGFP-N1
CRISPRCas9 gene editingA549 TDP2minusminus cells were created as previously reported9 Inbrief we used a 17-bp (minus the PAM) RNA sequencetargeting TDP2 exon 4 (59-GTAGAAATATCACATCT-39)which was selected using the tool E-CRISP (e-crisporgE-CRISP) and cloned into the guide RNA vector 41824(AddGene) The TDP2 guide construct was cotransfectedwith hCas9 expressed from plasmid 41815 (AddGene) usingAmaxa Nucleofector plataform (Lonza Basel Switzerland)Kit T program X-001 Transfected cells were enriched byselection in 1 mgmL G418 (Thermofisher) for 5 days beforeisolation of single clones and screening for loss of TDP2expression by Western blotting
Sanger sequencingDNA was extracted from 850-BR cells using the DNeasyBlood amp Tissue kit (Qiagen Manchester UK) PCRreactions used Phusion HF-DNA Polymerase (NEB) andthe following primers TDP2_FW GCCAGTGTT-GACCTAACCAATGAAGA TDP2_RV CTGTAGAAA-TATCACATCTGGGCTGTACC ZSCAN9_FWATGAAGTAACCAAGACTGAGGACAGAGAG and ZSCAN9_RVAGACCAGCTCAGCCACTGTGTGGATCT PCR productswere purified before sequencing using a QIAquick PCR purifi-cation kit (Qiagen)
Western blottingAnti-TDP2 antibody was used at 15000 in Western blottingand has been described previously10 Anti-Actin (SigmaA4700) was used at 12000 Western blot assessment ofOXPHOS components in patient primary fibroblasts wasconducted as described previously11 using antibodies againstNDUFB8 (Abcam cat ab110242) SDHA (Abcam catab14715) UQCRC2 (Abcam cat ab14745) COXI (Abcam
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 3
cat ab14705) ATP5A (Abcam cat ab14748) SDHB(Abcam cat ab14714) COX II (Abcam cat 110258) andVDAC1 (Abcam cat ab14734)
Clonogenic survival assays1-BR and 850-BR primary fibroblasts cells were plated ontofeeder layers (see below) and 3 hours later treated with indicatedconcentrations of etoposide for 21 days to allow the formation ofmacroscopic colonies which were rinsed in phosphate bufferedsaline (PBS) and fixedstained in 70 ethanol1 methyleneblue A549 and TDP2minusminus A549 were plated 4 hours beforetreatment with the indicated concentrations of etoposide for 12days and stained as described earlier The surviving fraction ateach dose was calculated by dividing the average number of
colonies (gt50 cells) in treated dishes by the average number inuntreated dishes For feeder layers 1-BR cells were irradiated (35Gy) and plated 24 hours before use at 5 times 104 cells10 cm dish
Tyrosyl DNA phosphodiesterase assaysWhole-cell extract (WCE) was prepared by resuspension of1-BR or 850-BR primary fibroblast cell pellets (1times106cells) in100μL lysis buffer (40 mM TrisHCl pH 75 100 mM NaCl01 Tween-20 1 mM DTT 1 mM PMSF 1x EDTA freeprotease cocktail inhibitor) followed by 30 minutes of in-cubation on ice and mild sonication The WCE was clarifiedby centrifugation for 10 minutes at 4degC at 16000g ina microfuge and the protein concentration quantified usingthe bicinchoninic acid (BCA) assay reagent (ThermoFisher)
Figure 1 TDP2 splice site mutation in an individual from the United States with SCAR23
(A) Pedigree analysis White symbolswild-type TDP2 Black symbols ho-mozygosity for the TDP2 splice sitemutation c425+1GgtA (IVS3+1GgtA) inthe proband (ldquo1rdquo) Dotted symbolsheterozygosity for c425+1GgtA (IVS3+1GgtA) (B) Sanger sequencing of theproband demonstrating (left) thehomozygous TDP2 mutation c425+1GgtA (IVS3+1GgtA) and (right) the ab-sence of the ZSCAN9 variantc914AgtG Patient sequences areshown at the bottom and referencesequences are shown at the top The2 nucleotides relevant to the muta-tions are underlined (C) Pathologicfeatures of the Irish and US patients(D) Haplotypes of Irish patients(51670 Irish pedigree) and the cur-rent patient from the United States(1583786) carrying the homozygousc425+1GgtA variant in TDP2 Only thevariants bordering and directly withinthe homozygous regions and witha minor allele frequency of 5 areshown Variants are indicated by theiraccession number in the dbSNP da-tabase and the position of c425+1GgtA is in bold The minimal over-lapping region is delimited byrs9396886 and rs749338 and is 151Mb in size Black bars represent thehomozygous haplotype as inferredfrom exome sequencing data TDP2 =tyrosyl DNA phosphodiesterase 2
4 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
Clarified WCE (15μg total protein) was incubated with 40nM TDP2 substrate (Cy5-59Tyrosine-ssDNA19-BHQ) orTDP1 substrate (BHQ-ssDNA13-39Tyrosine-Cy5) in re-action buffer (50 mM TrisHCl pH80 10 mM MgCl280 mM KCl and 1 mM DTT 005 Tween-20) in a totalvolume of 6μL at room temperature and Cy5 fluorescencewas measured at 640 nm at the indicated time intervals ona BMG PHERAstar plate reader
DSB repair assaysCells were grown on coverslips until confluent and thentreated for 30 minutes with 25μM etoposide or irradiatedwith x-rays (2 Gy) After treatment cells were rinsed andfixed for 10 minutes in PBS containing 4 para-formaldehyde at the indicated time points Cells werepermeabilized (20 minutes in PBS-02 Triton X-100)
blocked (1 hour in PBS-5 BSA) and incubated with anti-γH2AX (Millipore 05-636 12500) and anti-CENP-F(Abcam ab5 12500) antibodies for 3 hours in PBS con-taining 5 BSA Cells were then washed (3 times 5 minutes inPBS containing 01Tween-20) incubated for 1h with thecorresponding Alexa Fluor conjugated secondary antibody(11000 5 BSA) and washed again as described earlierFinally cells were counterstained with DAPI (SigmaGillingham UK) and mounted in VECTASHIELD (VectorLabs Peterborough UK) Images were acquired on anautomated wide-field microscopy Olympus ScanR system(motorized IX83 microscope) with ScanR Image Acquisi-tion and Analysis Software 20times045 (LUCPLFLN20timesPH) dry objectives and Hamamatsu ORCA-R2 digitalCCD camera C10600 For the analysis of G0G1 cells inpatient primary fibroblasts only CENP-F-negative cells
Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
(A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts as measured by immunoblotting (B)TDP1 and TDP2 biochemical assay Hydrolysis of a 39-phosphotyrosyl (TDP1 assay) or 59-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus resulting in elevated fluorescence at 640 nm (C) Real-time (0ndash30minutes)measurements of 59-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) orWCE (15μg protein)from 1-BR normal or 850-BR patient primary fibroblasts using the single-stranded oligonucleotide (40 nM) with a 59-phosphotyrosyl-linked Cy5 fluorophoreand 39-BHQ (D) Real-time measurements (0ndash30 minutes) of 39-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 39-phospho-tyrosyl-linked Cy5 fluorophore and 59-BHQ Data are mean of 3 independent experiments plusmnSEM Statistical significance was determined by two way analysisof variance (ANOVA) p lt 0001 BHQ = black hole quencher TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cell extract
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 5
were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
6 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
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cat ab14705) ATP5A (Abcam cat ab14748) SDHB(Abcam cat ab14714) COX II (Abcam cat 110258) andVDAC1 (Abcam cat ab14734)
Clonogenic survival assays1-BR and 850-BR primary fibroblasts cells were plated ontofeeder layers (see below) and 3 hours later treated with indicatedconcentrations of etoposide for 21 days to allow the formation ofmacroscopic colonies which were rinsed in phosphate bufferedsaline (PBS) and fixedstained in 70 ethanol1 methyleneblue A549 and TDP2minusminus A549 were plated 4 hours beforetreatment with the indicated concentrations of etoposide for 12days and stained as described earlier The surviving fraction ateach dose was calculated by dividing the average number of
colonies (gt50 cells) in treated dishes by the average number inuntreated dishes For feeder layers 1-BR cells were irradiated (35Gy) and plated 24 hours before use at 5 times 104 cells10 cm dish
Tyrosyl DNA phosphodiesterase assaysWhole-cell extract (WCE) was prepared by resuspension of1-BR or 850-BR primary fibroblast cell pellets (1times106cells) in100μL lysis buffer (40 mM TrisHCl pH 75 100 mM NaCl01 Tween-20 1 mM DTT 1 mM PMSF 1x EDTA freeprotease cocktail inhibitor) followed by 30 minutes of in-cubation on ice and mild sonication The WCE was clarifiedby centrifugation for 10 minutes at 4degC at 16000g ina microfuge and the protein concentration quantified usingthe bicinchoninic acid (BCA) assay reagent (ThermoFisher)
Figure 1 TDP2 splice site mutation in an individual from the United States with SCAR23
(A) Pedigree analysis White symbolswild-type TDP2 Black symbols ho-mozygosity for the TDP2 splice sitemutation c425+1GgtA (IVS3+1GgtA) inthe proband (ldquo1rdquo) Dotted symbolsheterozygosity for c425+1GgtA (IVS3+1GgtA) (B) Sanger sequencing of theproband demonstrating (left) thehomozygous TDP2 mutation c425+1GgtA (IVS3+1GgtA) and (right) the ab-sence of the ZSCAN9 variantc914AgtG Patient sequences areshown at the bottom and referencesequences are shown at the top The2 nucleotides relevant to the muta-tions are underlined (C) Pathologicfeatures of the Irish and US patients(D) Haplotypes of Irish patients(51670 Irish pedigree) and the cur-rent patient from the United States(1583786) carrying the homozygousc425+1GgtA variant in TDP2 Only thevariants bordering and directly withinthe homozygous regions and witha minor allele frequency of 5 areshown Variants are indicated by theiraccession number in the dbSNP da-tabase and the position of c425+1GgtA is in bold The minimal over-lapping region is delimited byrs9396886 and rs749338 and is 151Mb in size Black bars represent thehomozygous haplotype as inferredfrom exome sequencing data TDP2 =tyrosyl DNA phosphodiesterase 2
4 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
Clarified WCE (15μg total protein) was incubated with 40nM TDP2 substrate (Cy5-59Tyrosine-ssDNA19-BHQ) orTDP1 substrate (BHQ-ssDNA13-39Tyrosine-Cy5) in re-action buffer (50 mM TrisHCl pH80 10 mM MgCl280 mM KCl and 1 mM DTT 005 Tween-20) in a totalvolume of 6μL at room temperature and Cy5 fluorescencewas measured at 640 nm at the indicated time intervals ona BMG PHERAstar plate reader
DSB repair assaysCells were grown on coverslips until confluent and thentreated for 30 minutes with 25μM etoposide or irradiatedwith x-rays (2 Gy) After treatment cells were rinsed andfixed for 10 minutes in PBS containing 4 para-formaldehyde at the indicated time points Cells werepermeabilized (20 minutes in PBS-02 Triton X-100)
blocked (1 hour in PBS-5 BSA) and incubated with anti-γH2AX (Millipore 05-636 12500) and anti-CENP-F(Abcam ab5 12500) antibodies for 3 hours in PBS con-taining 5 BSA Cells were then washed (3 times 5 minutes inPBS containing 01Tween-20) incubated for 1h with thecorresponding Alexa Fluor conjugated secondary antibody(11000 5 BSA) and washed again as described earlierFinally cells were counterstained with DAPI (SigmaGillingham UK) and mounted in VECTASHIELD (VectorLabs Peterborough UK) Images were acquired on anautomated wide-field microscopy Olympus ScanR system(motorized IX83 microscope) with ScanR Image Acquisi-tion and Analysis Software 20times045 (LUCPLFLN20timesPH) dry objectives and Hamamatsu ORCA-R2 digitalCCD camera C10600 For the analysis of G0G1 cells inpatient primary fibroblasts only CENP-F-negative cells
Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
(A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts as measured by immunoblotting (B)TDP1 and TDP2 biochemical assay Hydrolysis of a 39-phosphotyrosyl (TDP1 assay) or 59-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus resulting in elevated fluorescence at 640 nm (C) Real-time (0ndash30minutes)measurements of 59-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) orWCE (15μg protein)from 1-BR normal or 850-BR patient primary fibroblasts using the single-stranded oligonucleotide (40 nM) with a 59-phosphotyrosyl-linked Cy5 fluorophoreand 39-BHQ (D) Real-time measurements (0ndash30 minutes) of 39-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 39-phospho-tyrosyl-linked Cy5 fluorophore and 59-BHQ Data are mean of 3 independent experiments plusmnSEM Statistical significance was determined by two way analysisof variance (ANOVA) p lt 0001 BHQ = black hole quencher TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cell extract
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 5
were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
6 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
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is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
Clarified WCE (15μg total protein) was incubated with 40nM TDP2 substrate (Cy5-59Tyrosine-ssDNA19-BHQ) orTDP1 substrate (BHQ-ssDNA13-39Tyrosine-Cy5) in re-action buffer (50 mM TrisHCl pH80 10 mM MgCl280 mM KCl and 1 mM DTT 005 Tween-20) in a totalvolume of 6μL at room temperature and Cy5 fluorescencewas measured at 640 nm at the indicated time intervals ona BMG PHERAstar plate reader
DSB repair assaysCells were grown on coverslips until confluent and thentreated for 30 minutes with 25μM etoposide or irradiatedwith x-rays (2 Gy) After treatment cells were rinsed andfixed for 10 minutes in PBS containing 4 para-formaldehyde at the indicated time points Cells werepermeabilized (20 minutes in PBS-02 Triton X-100)
blocked (1 hour in PBS-5 BSA) and incubated with anti-γH2AX (Millipore 05-636 12500) and anti-CENP-F(Abcam ab5 12500) antibodies for 3 hours in PBS con-taining 5 BSA Cells were then washed (3 times 5 minutes inPBS containing 01Tween-20) incubated for 1h with thecorresponding Alexa Fluor conjugated secondary antibody(11000 5 BSA) and washed again as described earlierFinally cells were counterstained with DAPI (SigmaGillingham UK) and mounted in VECTASHIELD (VectorLabs Peterborough UK) Images were acquired on anautomated wide-field microscopy Olympus ScanR system(motorized IX83 microscope) with ScanR Image Acquisi-tion and Analysis Software 20times045 (LUCPLFLN20timesPH) dry objectives and Hamamatsu ORCA-R2 digitalCCD camera C10600 For the analysis of G0G1 cells inpatient primary fibroblasts only CENP-F-negative cells
Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
(A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts as measured by immunoblotting (B)TDP1 and TDP2 biochemical assay Hydrolysis of a 39-phosphotyrosyl (TDP1 assay) or 59-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus resulting in elevated fluorescence at 640 nm (C) Real-time (0ndash30minutes)measurements of 59-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) orWCE (15μg protein)from 1-BR normal or 850-BR patient primary fibroblasts using the single-stranded oligonucleotide (40 nM) with a 59-phosphotyrosyl-linked Cy5 fluorophoreand 39-BHQ (D) Real-time measurements (0ndash30 minutes) of 39-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 39-phospho-tyrosyl-linked Cy5 fluorophore and 59-BHQ Data are mean of 3 independent experiments plusmnSEM Statistical significance was determined by two way analysisof variance (ANOVA) p lt 0001 BHQ = black hole quencher TDP2 = tyrosyl DNA phosphodiesterase 2 WCE = whole-cell extract
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 5
were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
6 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
were scored For A549 and A549 TDP2minusminus cells were gatedto the G1 population according to the DAPI profile ofScanR Image Analysis Software For patient fibroblastcomplementation experiments cells were gated accord-ingly to eGFP expression on ScanR Analysis Software inaddition to the G1 population according to the DAPIcontent
ResultsWe recently described mutations in TDP2 in three Irishpatients from the same family with intellectual disabilityseizures and ataxia a disease now denoted as spinocerebellarataxia 23 (SCAR23)6 Here we describe a 6-year-old patientin the United States with very similar pathology includingdevelopmental delay epilepsy and ataxia and in whom weidentified by whole-exome and Sanger sequencing possessesthe same homozygous splice site mutation in TDP2 (c425+1GgtA) (figure 1 AndashC) Whether there is a connection be-tween the current patient and the Irish family is not clear buta comparison of the WES data from the two families revealedthat the two apparently unrelated affected individuals ofwhom their exome was sequenced share the same homozy-gous haplotype of 151 Mb (figure 1D) It is important tonote that in contrast to the Irish patients however the currentpatient lacks the mutation c914AgtG (pHis305Arg) inZNF193 a zinc finger protein of unknown function (figure1B right) We previously concluded that this variant was not
disease causing in the Irish patients6 and the absence of thisvariant in the current patient confirms that this and also thatTDP2 mutation is the cause of SCAR23
To enable additional molecular and cellular analyses ofSCAR23 we generated primary fibroblasts (denoted 850-BR)from a skin biopsy kindly provided by the current patientWestern blotting failed to detect TDP2 protein in the 850-BRpatient fibroblasts (figure 2A) consistent with the lack ofdetectable TDP2 protein previously reported in lympho-blastoid cells from the Irish patients6 The absence of de-tectable TDP2 protein in the lymphoblastoid cell lines wasexplained by the impact of the TDP2 mutation on splicingwhich resulted in nonsense-mediated decay and greatly re-duced (lt20) levels of TDP2 mRNA6 To confirm the im-pact of the splice site mutation on TDP2 activity we useda highly sensitive tyrosyl DNA phosphodiesterase bio-chemical assay This assay uses a single-stranded oligonucle-otide substrate in which hydrolytic release of a Cy5-labeledtyrosine moiety present on one terminus of the oligonucle-otide results in increased fluorescence due to evasion ofa black hole quencher present on the opposite terminus(figure 2B) By situating the fluorescent-labeled tyrosine oneither the 39 or 59 terminus this assay can detect TDP1 orTDP2 activity respectively Although whole-cell extract fromnormal 1-BR human fibroblasts exhibited robust 59-tyrosylDNA phosphodiesterase activity 850-BR patient fibroblastsdid not (figure 2C) The small amount of activity observed in
Figure 3 Reduced DSB repair in TDP2-mutated patient fibroblasts and A549 cells after topoisomerase 2-induced DNAdamage
(A) DSBs were measured by γH2AXimmunostaining in normal 1-BR andpatient 850-BR primary fibroblasts be-fore and after treatment for 30 minuteswith DMSO vehicle or 25 μM etoposide(ldquo0rdquo) followed by subsequent in-cubation in a drug-free medium for theindicated repair periods (B) DSBs weremeasured as above in wild-type A549cells and in A549 cells in which TDP2wasdeleted by CRISPRCas9 gene editing(TDP2minusminus A549) The level of TDP2 andactin (loading control) in the cell linesused for these experiments is shown byWestern blotting on the right (C) DSBswere measured as above in hTERT-im-mortalized 1-BR fibroblasts and 850-BRfibroblasts and in the latter cells aftertransfection with the empty GFP vectoror vector encoding recombinant humanTDP2-GFP The level of TDP2 and actin(loading control) in the cell lines used forthese experiments is shown byWesternblotting on the right Data are mean plusmnSEMof 3 independent experiments andstatistically significant differences weredetermined by the t test (ns = not sig-nificant p lt 005 p lt 001 p lt0001) DSB = double-strand breakGFP = green fluorescent protein TDP2 =tyrosyl DNA phosphodiesterase 2
6 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
850-BR cell extract compared with reactions supplementedwith reaction buffer alone reflects nucleases in the cell extractrather than residual TDP2 activity because similar results wereobserved when we used cell extract from human cells in whichTDP2 was deleted by CRISPRCas9 (unpublished observa-tions) The lack of TDP2 activity in patient cells did notreflect differences in the technical quality of cell extracts be-cause the level of TDP1 activity in these extracts was similar tothat in normal human 1-BR fibroblasts (figure 2D) Weconclude from these data that the TDP2 splice site mutationgreatly reduces and most likely ablates both TDP2 proteinand activity in 850-BR patient fibroblasts
Next we addressed the impact of the TDP2 splice site mu-tation on nuclear DSB repair rates in 850-BR patient fibro-blasts using immunofluorescent detection of γH2AX as anindirect measure of DSBs12 Although similar levels of nuclearγH2AX foci were present in normal and patient fibroblastsimmediately after treatment with etoposide for 30 minutesthe levels of these γH2AX foci decreased far more slowly inthe patient fibroblasts during subsequent incubation in a drug-free medium (figure 3A) However DSB repair was com-pleted in patient cells within 8ndash24 hours consistent with theestablished existence of alternative nuclease-dependent
mechanisms for repair of TOP2-induced DSBs in humancells41314 Similar results were observed in human A549 cellsin which TDP2 was mutated by CRISPRCas9 gene editingconfirming the importance of TDP2 for repair of TOP2-induced DSBs (figure 3B) More importantly complemen-tation of 850-BR cells with expression construct encodingrecombinant human TDP2 restored normal rates of nuclearDSB repair confirming that the DNA repair defect in thepatient fibroblasts was the result of theTDP2mutation (figure3C) This defect in DSB repair was accompanied by cellularhypersensitivity to TOP2-induced DSBs because both 850-BR patient fibroblasts and TDP2minusminus A549 cells were hyper-sensitive to etoposide in clonogenic survival assays (figure 4 Aand B) In contrast to etoposide-induced DSBs both DSBrepair rates and levels of cell survival were normal in 850-BRpatient fibroblasts after treatment with ionizing radiation(figure 4 C and D) confirming the specificity of the DNArepair defect in the patientrsquos cells for DNA breaks induced byTOP2
Finally because analyses of muscle biopsy from the patient(see the case report) suggested the presence of possibledefects in the ETC we examined 850-BR patient fibroblastsfor defects in mitochondrial function However the patient
Figure 4Hypersensitivity of TDP2-mutated patient fibroblasts andA549 cells to DNAdamage induced by etoposide but notγ-rays
(A) Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts in a medium containing the in-dicated concentrations of etoposide(B) Clonogenic survival of wild-typeA549 and TDP2minusminus A549 cells in a me-dium containing the indicated con-centrations of etoposide (C)Clonogenic survival of 1-BR normaland 850-BR patient primary fibro-blasts as above following γ-irradia-tion (D) DSBs were measured byγH2AX immunostaining in normal 1-BR and patient 850-BR primary fibro-blasts before and after the indicatedperiods after γ-irradiation (2 Gy) Dataare mean plusmn SEM of 3 independentexperiments and statistically signifi-cant differences were determined bytwo-way ANOVA (ns = not significantp lt 0001) TDP2 = tyrosyl DNAphosphodiesterase 2
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 7
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
fibroblasts failed to exhibit significant defects in respiratorychain complexes in biochemical assays compared witha range of different normal (control) human fibroblasts(figure 5Aa) Similar results were observed when wecompared wild-type A549 cells with A549 cells in which
TDP2 was mutated by gene editing (figure 5Ab) We alsofailed to identify any impact of the TDP2 mutation or de-letion in 850-BR fibroblasts and A549 cells respectively inthe level of respiratory chain proteins as measured by im-munoblotting (figure 5B)
Figure 5 Mitochondrial functionality in normal and TDP2-mutated cells
(A) Activity ofmitochondrial respiratorycomplexes in normal (red bars) andpatient 850-BR (blue bars) primaryhuman fibroblasts (Aa) and in wild-type human A549 (red bars) andTDP2minusminus A549 cells (blue bars) (Ab) wasdetermined as previously described15
The mean enzyme activities in controlcells (n = 8) are set to 100 and errorbars represent SD (B) Levels of mito-chondrial respiratory complex pro-teins in age-matched controlfibroblasts (C1 andC2) andpatient 850-BR fibroblasts (Ba) and in wild-typeA549 cells and TDP2minusminus A549 cells (Bb)as measured by immunoblotting forsubunits of CI (NDUFB8) CII (SDHA) CIII(UQCRC2) CIV (COXI) and CV (ATP5A)and using VDAC1 as a mitochondrialloading control (C) Subcellular locali-zation of TDP2 in HeLa cells (Ca) and innormal (1-BR) and patient (850-BR)primary human fibroblasts (Cb) Leftcell-equivalent amounts of HeLa totalcell lysate (30 g total protein lane 1)cell lysate depleted of mitochondria(ldquopost-mito spinrdquo lane 2) mitochondriatreated with proteinase K (to removeproteins associated with the outermembrane lane 3) mitoplasts (mito-chondrial matrix plus inner membraneproteins lane 4) mitoplasts treatedwith proteinase K (lane 5) and innermembrane mitochondrial proteins(extractedwith sodium carbonate lane6) were immunoblotted for TDP2 andfor protein markers of the cytosol(eIF4E) and each of the mitochondrialcompartments intermembrane space(AIF) mitochondrial matrix (EF-Tu) andinner mitochondrial membrane(NDUFB8) Right cell-equivalentamounts of 1-BR or 850-BR fibroblasttotal cell lysates (30μg protein) de-pleted of mitochondria (ldquopost-mitospinrdquo) and of mitoplasts were immu-noblotted for TDP2 as above The po-sition of full-length TDP2 (arrow) anda nonspecific band detected by theantibody (asterisk ) are indicatedTDP2 = tyrosyl DNA phosphodiester-ase 2
8 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
We next fractionated HeLa cells and both 1-BR and 850-BRfibroblasts into subcellular components including intact mi-tochondria and mitoplasts to examine whether TDP2 is lo-cated within the mitochondria and thus in the correct locationto repair mitochondrial DNA (figure 5C) Most of the ma-terial detected by the anti-TDP2 antibody and whichmigratedto the position expected for TDP2 was present as expected inthe cellular fractions containing cytosolic and nuclear proteins(figure 5Ca lanes 1 and 2) However a very small amount ofthis material was detected in intact mitochondria and mito-plasts (figure 5Ca lanes 3ndash5) In 1-BR normal humanfibroblasts most of the material detected by anti-TDP2 an-tibody was again present in the cytosolicnuclear fraction andthis material was TDP2 because it was absent from parallelpreparations from patient 850-BR fibroblasts (figure 5Cb)However if TDP2 was present in mitoplasts prepared from1-BR cells it was below the level of detection in our experi-ments (figure 5Cb arrow) We did note the presence ofa slower migrating band that was detected by the TDP2 an-tibody in both HeLa and 1-BR mitoplasts but this band wasalso present in parallel preparations from 850-BR patientfibroblasts indicating that it was not TDP2 (figure 5Cbasterisk) Collectively although we cannot rule out the pres-ence of a small amount of endogenous TDP2 in mitochon-dria we cannot detect defects in mitochondrial function inSCAR23 patient fibroblasts Consequently we suggest thatthe neurologic disease pathology in SCAR23 is most likely theresult of a defect in nuclear DSB repair
DiscussionWe recently identified TDP2 mutations in a recessive hered-itary genetic disorder associated with intellectual disabilityseizures and ataxia now denoted as spinocerebellar ataxiaautosomal recessive 23 (SCAR23)6 In the three Irish siblingsoriginally described we also identified the mutation c914AG(pHis305Arg) in ZNF193 a zinc finger protein of unknownfunction which we concluded was not disease causing butwhich we could not exclude as a contributor to the disease6
However in contrast to the Irish patients the new patientidentified in the United States and reported here harbors thesame TDP2-associated haplotype but lacks the mutation inZNF193 ruling out a contribution of the latter and confirmingthe TDP2 mutation as the cause of SCAR23
The TDP2-mutated patient fibroblasts established here are thefirst such reported and have provided valuable informationconcerning the molecular and cellular defect in SCAR23Consistent with TDP2 defects in other cell types we observedreduced rates of DSB repair and elevated cellular sensitivityafter treatment with the TOP2 poison etoposide These phe-notypes were the result of the TDP2 mutation in the patientfibroblasts because they were phenocopied in TDP2minusminus A549cells created by gene editing and were complemented by thereintroduction of the wild-type human TDP2 transgene Ofinterest the magnitude of these phenotypes is greater in patient
fibroblasts and gene-edited A549 cells than what we have seenpreviously with our previous gene-edited cell lines9 We believethat this is because the cell lines reported here are effectivelyTDP2 null whereas our previously generated TDP2 gene-edited human cells retained a minor isoform of TDP2 that isexpressed at low levels and is primarily cytoplasmic
It is notable that TOP2 poisons are used widely in the clinic totreat a variety of cancers and we point out that the use of theseagents in patients with TDP2 mutations should be avoided orat the very least treated with extreme caution AlthoughTDP2-defective cancer cells will be hypersensitive to this typeof chemotherapy normal cells will also be hypersensitive asillustrated in the current work by the etoposide sensitivityobserved in both TDP2minusminus A549 lung cancer cells andSCAR23 primary fibroblasts
The presence of mitochondrial dysfunction in the SCAR23patient described here is suggested by ETC studies and the highlactate and lactatepyruvate ratio in muscle (see the PatientCase Study in the Methods section) In addition the currentpatient exhibits phenotypes consistent with mitochondrialdysfunction such as hypotonia low energy and fatigability It isunclear whether phenotypes consistent with mitochondrialdefects are also present in the Irish patients although nonewere originally noted6 Unfortunately neither fibroblasts normuscle biopsy are available from these patients to address thispossibility The lack of detectable mitochondrial defect in pri-mary skin fibroblasts from the current patient and in gene-edited A549 cells does not support amajor role for TDP2 in therepair of mitochondrial DNA However the absence of mito-chondrial defects in skin fibroblasts from patients with mito-chondrial defects in muscle is not uncommon15 Neverthelessgiven the dramatic impact of the TDP2 mutation on nuclearDNA repair we suggest that any association of SCAR23 withmitochondrial disease is most likely an indirect or secondarydysfunction resulting from a nuclear disorder16
Author contributionsG Zagnoli-Vieira F Bruni K Thompson and L He designedand conducted mitochondrial experiments S Walkerdesigned TDP assay R Taylor supervised mitochondrialexperiments APM de Brouwer conducted haplotype anal-ysis D Niyazov identified and assessed the patient andidentified the TDP2 mutation KW Caldecott designed di-rected and coordinated the project and wrote the manuscript
AcknowledgmentThe authors thank the patient and the patientrsquos family andLimei JuElena Korneeva for generation of primary 850-BRpatient fibroblasts and hTERT-immortalized derivatives
Study fundingThis study was funded by Programme grants to KWC fromthe MRC (MRP0101211) CRUK (C6563A16771) ERC(SIDSCA Advanced Grant 694996) and by an MRC PhDStudentship for GZV (MRNN50189X1)
NeurologyorgNG Neurology Genetics | Volume 4 Number 4 | August 2018 9
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
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httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
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httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
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httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
DisclosureG Zagnoli-Vieira has no financial disclosures to reportF Bruni serves on the editorial boards of Biomarkersjournal and Brain and Nervous System Current Research andreceives research support from ANVUR K ThompsonL He S Walker R Taylor and APM de Brouwer reportno disclosures D Niyazov has served on the scientificadvisory boards of Alexion Pharmaceuticals and Mal-linckrodt ARD Inc has received funding for travelspeaker honoraria from Sanofi Genzyme Corporationserves on the editorial board of Molecular Syndromologyhas consulted with the Phase II clinical trial of Elamipre-tide in Mitochondrial Myopathy is a current member ofthe Sanofi Genzyme Corporation Speakersrsquo Bureau andhas served as an expert witness for a legal case regardingvaccine injury in a child with mitochondrial diseaseKW Caldecott reports no disclosures Full disclosureform information provided by the authors is available withthe full text of this article at NeurologyorgNG
Received December 20 2017 Accepted in final form May 24 2018
References1 Lindahl T Instability and decay of the primary structure of DNA Nature 1993362
709ndash7152 Goodarzi AA Jeggo PA The repair and signaling responses to DNA double-strand
breaks Adv Genet Elsevier 2013821ndash45
3 Cortes Ledesma F El-Khamisy SF ZumaMCOsborn K Caldecott KW A human 59-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damageNature 2009461674ndash678
4 Gomez-Herreros F Romero-Granados R Zeng Z et al TDP2-Dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks andgenome instability in cells and Vivo Plos Genet 20139e1003226
5 Zeng Z Cortes Ledesma F El-Khamisy SF Caldecott KW TDP2TTRAP is themajor 59-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular re-sistance to topoisomerase II-induced DNA damage J Biol Chem 2011286403ndash409
6 Gomez-Herreros F Schuurs-Hoeijmakers JHM McCormack M et al TDP2 protectstranscription from abortive topoisomerase activity and is required for normal neuralfunction Nat Genet 201446516ndash521
7 Bruni F Gramegna P Oliveira JMA Lightowlers RN Chrzanowska-LightowlersZMA REXO2 is an oligoribonuclease active in human mitochondria PLoS One20138e64670
8 Hoch NC Hanzlikova H Rulten SL et al XRCC1mutation is associated with PARP1hyperactivation and cerebellar ataxia Nature 201654187ndash91
9 Gomez-Herreros F Zagnoli-Vieira G Ntai I et al TDP2 suppresses chromosomaltranslocations induced by DNA topoisomerase II during gene transcription NatCommun 20178233
10 Thomson G Watson A Caldecott K et al Generation of assays and antibodies tofacilitate the study of human 59-tyrosyl DNA phosphodiesterase Anal Biochem 2013436145ndash150
11 Thompson K Majd H Dallabona C et al Recurrent de novo dominant mutations inSLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrialDNA copy number Am J Hum Genet 201699860ndash876
12 Rogakou EP Pilch DR Orr AH Ivanova VS Bonner WM DNA double-stranded breaksinduce histone H2AX phosphorylation on serine 139 J Biol Chem 19982735858ndash5868
13 Hoa NN Shimizu T Zhou Z-W et al Mre11 Is Essential for the Removal of LethalTopoisomerase 2 Covalent Cleavage Complexes Mol Cell 201664580ndash592
14 Zagnoli-Vieira G Caldecott KW TDP2 TOP2 and SUMO what is ZATT aboutCell Res 201717182ndash1406
15 Kirby DM Thorburn DR Turnbull DM Taylor RW Biochemical assays of re-spiratory chain complex activity Methods Cell Biol 20078093ndash119
16 Niyazov DM Kahler SG Frye RE Primary mitochondrial disease and secondarymitochondrial dysfunction importance of distinction for diagnosis and treatmentMol Syndromol 20167122ndash137
10 Neurology Genetics | Volume 4 Number 4 | August 2018 NeurologyorgNG
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet
DOI 101212NXG000000000000026220184 Neurol Genet
Guido Zagnoli-Vieira Francesco Bruni Kyle Thompson et al Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23)
This information is current as of August 1 2018
ServicesUpdated Information amp
httpngneurologyorgcontent44e262fullhtmlincluding high resolution figures can be found at
References httpngneurologyorgcontent44e262fullhtmlref-list-1
This article cites 16 articles 2 of which you can access for free at
Citations httpngneurologyorgcontent44e262fullhtmlotherarticles
This article has been cited by 2 HighWire-hosted articles
Subspecialty Collections
httpngneurologyorgcgicollectionspinocerebellar_ataxiaSpinocerebellar ataxia
httpngneurologyorgcgicollectiongait_disorders_ataxiaGait disordersataxia
httpngneurologyorgcgicollectionall_epilepsy_seizuresAll EpilepsySeizuresfollowing collection(s) This article along with others on similar topics appears in the
Permissions amp Licensing
httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in
Reprints
httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online
reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2018 The Author(s)
is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet