ARTICLE OPEN ACCESS

Homoplasmic mitochondrial tRNAPro mutationcausing exercise-induced muscle swelling andfatigueKarine Aure MD PhD Guillemette Fayet MD PhD Ivan Chicherin PhD Benoit Rucheton PharmD

Sandrine Filaut Anne-Marie Heckel Julie Eichler MSc Florence Caillon MD Yann Pereon MD PhD

Nina Entelis PhD Ivan Tarassov PhD and Anne Lombes MD PhD

Neurol Genet 20206e480 doi101212NXG0000000000000480

Correspondence

Dr Lombes

annelombesinsermfr

or Dr Tarassov

itarassovunistrafr

AbstractObjectiveTo demonstrate the causal role in disease of the MT-TP m15992AgtT mutation observed inpatients from 5 independent families

MethodsLactate measurement muscle histology and mitochondrial activities in patients PCR-basedanalyses of the size amount and sequence of muscle mitochondrial DNA (mtDNA) andproportion of the mutation respiration mitochondrial activities proteins translation transferRNA (tRNA) levels and base modification state in skin fibroblasts and cybrids and reactiveoxygen species production proliferation in the absence of glucose and plasma membranepotential in cybrids

ResultsAll patients presented with severe exercise intolerance and hyperlactatemia They were asso-ciated with prominent exercise-induced muscle swelling conspicuous in masseter muscles (2families) andor with congenital cataract (2 families) MRI confirmed exercise-induced muscleedema Muscle disclosed severe combined respiratory defect Muscle mtDNA had normal sizeand amount Its sequence was almost identical in all patients defining the haplotype as J1c10and sharing 31 variants only 1 of which MT-TP m15992AgtT was likely pathogenic Themutation was homoplasmic in all tissues and family members Fibroblasts and cybrids withhomoplasmic mutation had defective respiration low complex III activity and decreasedtRNAPro amount Their respiratory complexes amount and tRNAPro aminoacylation appearednormal Low proliferation in the absence of glucose demonstrated the relevance of the defectson cybrid biology while abnormal loss of cell volume when faced to plasma membrane de-polarization provided a link to the muscle edema observed in patients

ConclusionsThe homoplasmic MT-TP m15992AgtT mutation in the J1c10 haplotype causes exercise-induced muscle swelling and fatigue

From the Inserm U1016 Institut Cochin (KA BR AL) INSERM Paris Department of Neurophysiology (KA) Foch Hospital Suresnes Centre de Reference Maladies Neuro-musculaires Hotel-Dieu AOC (GF YP) CHU Nantes CNRS UMR 7156 GMGM (IC A-MH JE NE IT) University of Strasbourg Service de Biochimie Metabolique CHU Pitie-Salpetriere (BR SF) AP-HP Paris Service de Radiologie et Imagerie Medicale Hotel-Dieu (FC) CHU Nantes CNRS UMR 8104 (AL) Universite Paris-Descartes-Paris5 (AL) ParisFrance and Present Address MV Lomonossov State University (IC) Moscow Russia

Go to NeurologyorgNG for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

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

Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Mitochondrial diseases due to oxidative phosphorylation de-fects represent an extraordinary diagnostic challenge becauseof their diversity The hundreds genes involved located oneither the mitochondrial or the nuclear DNA1 or the frequentheteroplasmy of mitochondrial DNA (mtDNA) mutationsie coexistence of wild-type and mutant mtDNA molecules2

likely explain part of that diversity In addition deleterioushomoplasmic mtDNA mutations ie mutations affecting allmtDNA molecules cause diseases with incomplete penetranceand tissue-specific expression that disclose the complexity ofnuclear-mitochondrial interaction3 This was the case withmutations causing Leber hereditary optic neuropathy4 ordeafness5 It was particularly striking for the homoplasmicMT-TI m4300AgtG mutation whose expression pattern rangedfrom infantile cardiac failure with severe cardiac mitochondrialdefect to apparent health6 Initial identification of these dele-terious homoplasmic mutations thus required either their re-currence with a characteristic phenotype4 or the demonstrationof the mitochondrial defect in the clinically targeted organ6

Among deleterious mtDNA mutations point mutationsaffecting transfer RNA (tRNA) genes are common (seeMitomap at mitomaporg) Their identification relies on apathogenicity scoring system using criteria from clinicalinvestigations database analysis and functional studies7

Among the functional studies cybrids ie cytoplasmichybrids with the patientrsquos mtDNA and the nuclear genomefrom immortal cells8 bring the possibility to demonstratethe mtDNA origin of the mitochondrial defect and are thusconsidered a gold standard for the pathogenicity assessmentof mtDNA mutations7 However as expected with muta-tions causing tissue-specific expression impairment thesecells being a surrogate tissue for the disease often disclosedonly a mild mitochondrial defect910

The impact of tRNAmutations depends on theminimal set of 22mtDNA tRNAs using a nonuniversal genetic code and specificdecoding rules for mitochondrial protein synthesis11 In particularall the codons with any base in the third codon position encodingthe same amino acid are recognized by only 1 tRNA that carries anunmodified U in the wobble position of the anticodon12

We here report the homoplasmic mtDNA m15992AgtTmutation of the wobble position of tRNAPro in 5 families withsevere exercise intolerance and combined respiratory chaindefect in muscle We demonstrate the mutation pathogenicityin cybrids by showing its association with respiratory defectdecreased tRNAPro steady state and altered response to de-polarization reminiscent of the exercise-induced ionic dis-turbances observed in patients

Patients and methodsPatientsPatient 1 (III-10 in family 1 in figure 1) presented with swellingof the temporal and masseter muscles during feeding since theneonatal period Exercise intolerance occurred during earlychildhood never associated with rhabdomyolysis At age 22years she sought medical advice for myalgia and exhaustionafter walking 500 m followed with nausea vomiting andheadache if effort was continued Physical examination wasnormal at rest Lactatemia was 21 mM in the fasting state andhigh after nonischemic forearm exercise test13

Patient 2 (III-13 in family 1) sought medical advice at age 14years for clinical and biological signs similar to patient 1

Patient 3 (II-2 in family 2) presented at age 14 years withfatigability and muscle pain after moderate exercise (1 kmcycling) He also presented with bilateral cataract which waspresent in his mother His physical examination was normalapart from short stature (minus25 SD) Lactatemia was 6 mM inthe postprandial state and high after moderate effort

Patient 4 (III-6 in family 3) complained of myalgia and fatiga-bility since early childhood with vomiting if effort was contin-ued Physical examination only disclosed short stature (minus3 SD)Lactatemia was 63 mM in the fasting state and high after effort

Patient 5 (III-5 in family 4) complained of myalgia andmusclefatigue since age 3 years Vomiting and malaise after pro-longed efforts started after age 6 years At age 13 years shecould only walk 200 m Lactatemia was high in the fastingstate (33 mM) and after nonischemic forearm exercise test13

Patient 6 (III-5 in family 5 in figure 1) presented with congenitalcataract Severe exercise intolerance occurred in childhood andwas associated with masseter swelling At age 38 years she couldonly walk 500 m because of myalgia and muscular exhaustionLactatemia was normal in the fasting state but high after exercise

Methods

Patientsrsquo investigationsAll patients gave their written informed consent for their analysesfor diagnostic investigations and for their use in clinical researchand publication according to our institutional ethics board Thestudy received approval from our Institutional Review Board

Brain imaging used a 15 T General Electric MRI with a headand neck coil including coronal and axial T1 and short-tauinversion recovery (STIR) sequences

GlossarymtDNA = mitochondrial DNA mt-tRNA = mitochondrial transfer RNA STIR = short-tau inversion recovery TMRE =tetramethylrhodamine ethyl esther

2 Neurology Genetics | Volume 6 Number 4 | August 2020 NeurologyorgNG

Muscle fragments were immediately frozen and stored atminus80degC until use Standard procedures provided blood buccalmucosa cells urinary sediment and cultured fibroblasts de-rived from a forearm skin biopsy (patients 1 2 3 and 4)Muscle histology followed standardized protocols1415

Molecular biologyExtraction of DNA from muscle and fibroblasts used standardmethods based on proteinase K and SDS digestion it usedQIAamp DNA Mini Kit extraction (Qiagen) for blood cellsfrom buccal mucosa or urinary sediment Extraction of RNAused the miRNeasy Mini Kit (Qiagen) or TRIzol (ThermoFisher Scientific)

Long-range PCR screened for large-scale deletions while quan-titative PCR evaluated the mtDNA copy number16 The Sangermethod provided mtDNA sequence Mispairing PCR restrictionquantified heteroplasmy using the DdeI site created by the mu-tation Primers are in table e-1 linkslwwcomNXGA282

Quantification of mitochondrial tRNAs used Northern blot-ting of 8 polyacrylamide 8 M urea (Tris-borate) gels onHybond N+ membranes and T4 polynucleotide kinasemdash59-end 32P-labeled probes (table e-1 linkslwwcomNXG

A282) Radioactive signals were quantified using TyphoonTrio and ImageQuant software (GE Healthcare)

Acid-denaturating gel separation and Northern blot analyzedthe tRNA aminoacylation state17 while [35S]-methionine invivo labeling in the presence of emetin a specific inhibitor ofcytosolic ribosomes assessed mitochondrial translation18 andreverse transciption followed by PCR (RT-PCR) with ex-tended oligonucleotides and sequencing with shorter primers(table e-1 linkslwwcomNXGA282) searched for the pres-ence of inosine at the wobble position of tRNA anticodon19

Cell biologyCytoplasmic hybrids (cybrids) were obtained from patients 1and 4 fibroblasts8

Cell respiration was analyzed in Oroboros high-resolutionrespirometer20 whereas mitochondrial activities in muscle andfibroblasts used standardized spectrophotometric protocols21

Mitochondrial production of superoxide ion was assessed byflow cytometry using 5 μM MitoSOX Red (Invitrogen)22

Mitochondrial pellet preparation and Western blot after bluenative polyacrylamide gel electrophoresis were as described in

Figure 1 Family trees of the patients with the m15992AgtT mutation

Arrows indicate the proband in each family stars = subjectswith samples for molecular studies white forms = subjectsasymptomatic or without sufficient information black forms= patients with severe exercise intolerance ie disablingdaily life gray forms = moderate exercise intolerance ienot disabling daily life but clearly experienced strippedforms = patients with isolated chewing-induced masseterswelling question marks indicate unknown clinical status ofthe subject In families 1 and 5 severe intolerance to exer-cise was constantly associated with chewing-induced mas-seters swelling but the reverse was not true with severalpatients in family 5 presenting only chewing-induced mas-seters swelling

NeurologyorgNG Neurology Genetics | Volume 6 Number 4 | August 2020 3

Ref 23 Primary antibodies were polyclonal antibodies againstthe F1 domain of bovine complex V UQCRC2 MT-ND1 orMT-CO2 (respectively produced by Pr Joel Lunardi Gre-noble by Dr Catherine Godinot Lyon or our group24) ormonoclonal antibodies against the respiratory complex IISDHA subunit SDHA (Abcam) incubated overnight at0degndash4degC Secondary antibodies were peroxidase conjugated(Sigma-Aldrich) and visualized with Piercetrade ECL WesternBlotting Substrate (Life Technologies) Quantification usedvolumes and basal adjustment with rolling ball by Fusion FX(Vilber Company)

To analyze cell proliferation 3000 cells per well were culturedin four 24-well plates and Dulbecco Modified Eagle Mediumwith 1 mM pyruvate and either 1 gL glucose or 200 μMglutamine without glucose25 Cell numbering was performedin triplicates for each condition every 24 hours (T0 T24T48 and T72 plates) using the neutral red method26 andexpressed as fold increase relative to T0

To evaluate plasma membrane 100000 cybrid cells wereincubated in 96-well plate with 75 μM tetramethylrhod-amine ethyl esther (TMRE) and 5 μM verapamil an in-hibitor of the multidrug resistancendashassociated proteinsBecause resting membrane potential essentially dependson K diffusion gradient progressive increase of external Kconcentration (from 10 to 80 mM) induced progressivedepolarization Osmolarity was kept at 300 mOsmL After120-minute incubation fluorescence was quantified by theAccuritrade C6 flow cytometer

StatisticsNormality evaluation used the Shapiro-Wilk test Dependingon the distribution of data comparison between 2 groupsused theMann-Whitney or t test Differences were consideredsignificant when p lt 005

Data availabilityAny data not published within the article will be shared asanonymized data by request from any qualified investigator

ResultsThe chewing-induced masticatory muscleswelling was due to edemaNuclear MRI of facial muscles analyzed the exercise-inducedmuscle swelling in 2 members of family 5 Chewing a sand-wich during 15 minutes induced a 20ndash30 increase inthickness of the temporal and masseter muscles of patient 6(figure 2 A and B) and her aunt (II-5 in family 5) (figure 2 Cand D) Mild adipose infiltration of the masseter and lateralpterygoid muscles was present in patient 6rsquos aunt Prolongededema was indicated by hypersignal in STIR fat-suppressionimages in all masticatory muscles of patient 6 (figure 2 E andF) and in the masseter temporal and lateral pterygoidmuscles of her aunt (figure 2 G and H)

Mitochondrial myopathy underlay themuscle symptomsMuscle histology disclosedmild alterations in patients 2 3 4 and5 including moderate lipidosis and increased subsarcolemmal

Figure 2 Chewing-induced edema of the facial muscles in 2 patients with the m15992AgtT mutation

Coronal T1-weighted NMRI of thehead of patient 6 (A and B) and heraunt (C and D) coronal STIR NMRI ofpatient 6 (E and F) and her aunt (Gand H) images obtained at rest (A CE and G) and 15 minutes afterchewing a sandwich (B D F and H)The measures show that after chew-ing the diameter of temporal muscleincreased by 22 in patient 6 (A vs B)and by 34 in her aunt who in-creased the diameter of hermassetermuscle by 37 (C vs D) NMRI = nu-clear magnetic resonance imagingSTIR = short-tau inversion recovery

4 Neurology Genetics | Volume 6 Number 4 | August 2020 NeurologyorgNG

mitochondria (figure e-1A linkslwwcomNXGA279) It wasconsidered normal for patient 6

Spectrophotometric assays of the respiratory chain revealed se-vere combined defect of respiratory complexes I III and IV withincreased citrate synthase in the muscle biopsies from all pro-bands (table 1) Respiratory complex II the only complexwithout mtDNA-encoded subunit was either normal or elevated

All patients shared the same homoplasmicMT-TP m15992AgtT mutationLong-range PCR excluded the presence of large size rear-rangement whereas quantitative PCR ruled out depletionSequencing of the whole mtDNA sequence revealed that allpatients had the samemtDNA sharing 31 of 32 variants (tablee-2 linkslwwcomNXGA283) Thirty-one of these 32variants were highly likely polymorphisms 28 reported atleast 1462 times in GenBank database a MT-RNR1 variantreported 37 times in different ethnic backgrounds and 2synonymous mutations (MTND2 c5024CgtT and MT-CO1m7028CgtT) (mitomaporg) These polymorphisms definedthe mtDNA haplotype as J1c10 for all the families The lastvariant common to all patients was the m15992AgtT mu-tation in the MT-TP gene Several criteria indicated its po-tential pathogenicity (1) it was reported only once in morethan 49000 GenBank full-length mtDNA sequences (2) itmodified the strictly phylogenetically conserved wobble basein the tRNA anticodon (see MitotRNAdbMamit-tRNAwebsite at mttrnabioinfuni-leipzigde) and (3) alteration ofthe wobble position in mt-tRNA anticodon is a major dele-terious factor in several mt-tRNA confirmed mutations27

The mutation appeared homoplasmic (figure 3) with PCRrestriction in the DNA samples from muscle of all the

probands from blood of all patients marked with a star infigure 1 and from urinary sediment and buccal cells of pa-tients 1 and 2 their mother and grandmother

Transfer of the m15992AgtT mutation intocybrids demonstrated its deleterious potentialon OXPHOS activitiesFibroblasts derived from a skin biopsy of patients 1 2 3 and 4 had100 mutation (figure 3) They disclosed a mild but significantdecrease of their basal respiration and respiration linked to adeno-sine triphosphate (ATP) production (figure 4A) Citrate synthaseactivity was significantly increased (figure 4B) Complexes I III IVand V appeared decreased reaching significance for complex IIIafter normalization to citrate synthase (figure 4 B and C)

Cybrid clones derived from patient 2 and patient 4 fibroblastshad homoplasmic m15992AgtT mutation (figure 3) Theyshowed decreased respiration either basal linked to ATP pro-duction or maximal (figure 4A) As in fibroblasts cybrids haddecreased activity for all the mtDNA-depending complexesreaching significance for respiratory complex III (figure 4 B andC) The amount of the respiratory complexes appeared normalwhen analyzed by Western blot after blue native polyacrylamidegel electrophoresis (figure e-1B linkslwwcomNXGA279)

Mutant cells disclosed significantly slower proliferation thancontrol cells in the absence of glucose whereas their pro-liferation rate was identical in its presence (figure 5A) Themild oxidative phosphorylation pathway (OXPHOS) defectwas therefore relevant affecting the cell proliferation capacityin a medium without glucose252829 It had no apparent im-pact on the mitochondrial production of superoxide ionwhich was similar in mutant and control cybrids in the basalstate (figure 5B)30

Table 1 Mitochondrial activities in the muscle of patients with homoplasmic m15992AgtT mutation

P2 P3 P4 C1 (n = 200) P5 P6 C2 (n = 140)

CI total 11a 8a 7a 25 plusmn 7 17a 21a 48 plusmn 15

CI rot sens 0a 6a 0a 22 plusmn 7 12a 18a 42 plusmn 14

CII 39 90 30 31 plusmn 8 77 136 62 plusmn 19

CIII total 80a 26a 23a 124 plusmn 274 70a 63a 235 plusmn 64

CIII am sens 22a 11a 5a 107 plusmn 26 6a 23a 160 plusmn 59

II + CIII 3a 5a 2a 18 plusmn 7 14a 13a 50 plusmn 15

CIV 43a 63a 9a 56 plusmn 17 109a 89a 199 plusmn 57

CS 314 1005 248 153 plusmn 35 276 470 209 plusmn 64

Abbreviations CI total = NADH ubiquinone oxidoreductase activity CI rot sens = CI fraction sensitive to rotenone inhibition ie specific respiratory complex Iactivity CII = succinate ubiquinone oxidoreductase activity ie respiratory complex II activity CIII total = ubiquinol cytochrome c oxidoreductase activity CIIIam sens = CIII fraction sensitive to antimycin inhibition ie specific respiratory complex III activity CIV = cytochrome c oxidase activity ie respiratorycomplex IV activity CS = activity of citrate synthase a Krebs cycle enzyme considered as representing themitochondrialmass n = number of different controlsanalyzed in each seriesActivities in bold font indicate values below the 10th centile of control values activities in italics font indicate values above the 90th centile of control valuesa After the respiratory activities indicate values below the 10th centile of control values after their normalization to citrate synthase activity P2 P3 P4 P5 andP6 = results obtained in themuscle biopsy from patients 2 3 4 5 and 6 C1 C2 = successive control series due to the change of spectrophotometric assays in200921

NeurologyorgNG Neurology Genetics | Volume 6 Number 4 | August 2020 5

The mutant tRNAPro had a lower steady-statebut normal aminoacylationNorthern blot analysis in several cell lines revealed significantdecrease of the amount of the mitochondrial tRNAPro in all themutant cell lines fibroblasts or cybrids (figure e-2A linkslwwcomNXGA280) Cybrids also disclosed an increase oftRNAVal which could suggest an increase in the mitochondrialribosomes because tRNAVal is one of their integralcomponents31

In accordance mitochondrial translation studied in 2 patientsrsquofibroblast lines by [35S]-methionine pulse-chase incorporationshowed a moderately decreased translation of most mtDNA-encoded proteins (figure e-2B linkslwwcomNXGA280)

As expected because the mutated base was not known as in-volved in the prolyl-tRNA synthetases recognition of thetRNAPro the in vivo aminoacylation state of the mutant tRNAappeared as normal17 (figure e-3A linkslwwcomNXGA281)

Because tRNAPro is encoded in the light mtDNA strand itssequence is in the reverse sense Mutation m159992AgtTthus replaces the wild-type uridine in the tRNA sequence byan adenine at the wobble position of the anticodon Anonmodified adenine in the wobble position of tRNA is veryrare and is almost always deaminated into inosine to improvethe decoding process32 We investigated whether that wasthe case for the mutant mitochondrial tRNAPro using thefact that inosine is read as guanosine during the RT-PCRreaction19 In both fibroblasts and cybrids we could notdetect any trace of inosine in the mutant mitochondrialtRNAPro (figure e-3B linkslwwcomNXGA281) Thisresult fitted with the absence of the needed enzymatic ap-paratus in mitochondria33

Abnormal decrease of the cell volume inmutant cybrids on depolarization by externalpotassium was reminiscent of the exercise-induced muscle edema observed in patientsIonic disturbances were a striking observation in 2 families withthe m15992AgtT mutation They were reminiscent of ionicdisturbances observed in patients with homoplasmicMT-ATP6mutations and recurrent paralysis episodes associated with sig-nificant plasma membrane depolarization in fibroblasts22 Corefacilities providing patch-clamp electrophysiologic analyses arescarce Therefore to analyze the plasma membrane potential incybrids we used an indirect determination based on TMRE afluorescent probe that follows Nernst equation and thus may beused to analyze membrane potentials34 Because 75 μMTMREsignificantly decreases respiration34 the mitochondrial mem-brane potential does not influence the TMRE fluorescencesignal which thus essentially represents both the cell size andplasma membrane potential As expected increasing in isos-motic conditions the external K concentration from 5 to 10 2030 40 and 80 mM led to progressive decrease of TMREfluorescent signal to 91 plusmn 9 78 plusmn 12 73 plusmn 7 61 plusmn 9 and 38 plusmn10 of its initial value respectively (analysis of 33 independentcell populations mutant and wild type grouped) At the basalstate the size (evaluated by the forward scatter) and the TMREfluorescence (evaluated by the fluorescence area FLA) did notdiffer between wild-type and mutant cybrids with either them15992AgtTmutation or the m9185TgtCmutation previouslyassociated with permanent plasma membrane depolarization22

(figure 5 C and D) In contrast at 80 mM external K con-centration both types of mutant cybrids had significantly de-creased their size whereas wild-type cybrids had maintained asize similar to the basal state (figure 5C) All 3 cybrid types had

Figure 3 Homoplasmy of the m15992AgtT MT-TP mutation

(A) Strategy of the mispairing PCR for restriction analysis of the m15992AgtTmutation proportion Italic characters indicate the primers used to amplify the200-bp-longmtDNA fragment fromposition 15820 to 16019 DdeI restriction siteis highlighted in cyan blue the nucleotide position 15992 in underlined bold bluecharacter the mispairing position in the backward primer is in underlined boldred (B) Restriction fragments of mtDNA fragments cut with DdeI P1 P2 P3 P4P5 and P6 = patients 1 2 3 4 5 and 6 respectively C = wild-type controls M =DNA from muscle L = DNA from blood leukocytes DNA U = DNA from urinarysediment B = DNA frombuccal cells F = DNA from cultured skin fibroblasts Theneed for mispairing is shown in the upper panel with the control sample (C)amplified with and without mispairing and then cut with DdeI Without mis-pairing the WT restriction fragment run only slightly above the mutated frag-ments in agreement with their length being only 5 bp longer than the mutantfragments With mispairing abolishing the DdeI site at position 15996 all sam-ples amplified from patientsrsquo tissues run homogeneously at a longer distancethan wild-type control mtDNA fragment in agreement with their 28 bp shorterlength The upper panel also shows that themutation appeared homoplasmic inevery tissue tested in several members of family 1 The rest of the panels showthat the mutation appeared homoplasmic in every member analyzed in the 2families where DNA from blood leukocytes was available (figure 1) It alsoappeared homoplasmic in cultured fibroblasts derived from a skin biopsy ofpatients 1 2 3 and 4 and in the cybrid clones obtained from either patient 2 orpatient 4 fibroblasts mtDNA = mitochondrial DNA WT = wild type

6 Neurology Genetics | Volume 6 Number 4 | August 2020 NeurologyorgNG

decreased their TMRE signal in accordance with the induceddecrease of plasmamembrane potential but mutant cybrids to alevel significantly lower than wild-type cybrids

DiscussionIn this article we demonstrate the pathogenicity of anoriginal homoplasmic MT-TP mutation targeting skeletalmuscle In muscle the severe combined respiratory chaindefect demonstrated the mtDNA origin of the diseaseHowever we could not exclude a nuclear DNA alterationsolely affecting muscle mtDNA expression because the ab-sence of paternal transmission by the few affected fathers wasinsufficient to demonstrate maternal inheritance Therefore

we had to provide demonstration of the mutation deleteri-ous potential

As observed with other deleterious homoplasmic mtDNAmutations with restricted clinical expression fibroblasts andcybrids presented with very mild enzymatic defect635 How-ever that defect was statistically significant with unbiasednonparametric statistical tests In addition it induced signifi-cant reduction of the mutant cells proliferation in the absenceof glucose showing its relevance to cell physiology36 Becausemost organs essentially comprise postmitotic cells cell pro-liferation is not relevant for clinical symptoms Therefore weaddressed the ionic disturbances that were a striking aspect ofthe disease prominent in 2 families When faced to an isos-motic high potassium concentration inducing plasma

Figure 4 Mitochondrial activities in cells with the m15992AgtT mutation

White bars = values obtained in control cells (C)black bars = values obtained in mutant cells (MT-TP) p lt 005 p lt 001 compared with thecontrol group using the Mann-Whitney test or ttest when data had a normal distributionaccording to the Shapiro-Wilk test (A) Respira-tion rates measured in an Oroboros high-reso-lution respirometer and expressed aspmolmiddotO2middotsec

minus1middotmillion cellsminus1 Data shown asmean and SEM of 12 independent measure-ments for mutant fibroblasts (4 mutant linesderived frompatients 1 2 3 and 4 each assayed3 times) 50 for control fibroblasts (14 differentcontrol lines) 8 for mutant cybrids (6 differentmutant lines derived from 2 different fibroblastslines) and 15 for control cybrids (derived from 6different control fibroblasts lines) basal = basalrespiration calculated by subtracting the non-respiratory oxygen consumption (with 1 mMKCN) from the respiration in culture mediumwith 1gL glucose ATP = respiration linked to ATPproduction calculated by subtracting the respi-ration under complete inhibition of ATP synthaseby 1 μM oligomycin from the respiration in cul-ture medium with 1 gL glucose Max = maximalrespiratory capacity calculated by subtractingthe nonrespiratory oxygen consumption (with1 mM KCN) from the maximal respiration ob-served after uncoupling with successive addi-tions of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (B) Maximal velocitiesanalyzed by spectrophotometric assays on fro-zen mitochondrial pellets and expressed asnanomolesmiddotminminus1middotmg cellular proteinsminus1 Datashown as mean and SEM of 8 independentmeasurements for mutant fibroblasts (4 mutantlines each assayed twice) 9 for control fibroblasts(6 control lines) 7 for mutant cybrids (5 mutantlines) and 7 for control cybrids (6 different controllines) CI = respiratory complex I (rotenone sensi-tiveNADHubiquinoneoxidoreductaseactivity) CII= respiratory complex II (succinate ubiquinoneoxidoreductase activity) CIII = respiratory com-plex III (antimycin sensitive ubiquinol cytochromec oxidoreductase activity) CIV = respiratory com-plex IV (cytochrome c oxidase activity) CV = ATPsynthase (oligomycin-sensitive ATP hydrolysis ac-tivity) CS = citrate synthase (C) Oxidative phos-phorylation pathway (OXPHOS) maximal velocitiesnormalized to the citrate synthase activity of thesample

NeurologyorgNG Neurology Genetics | Volume 6 Number 4 | August 2020 7

membrane depolarization cybrids with the MT-TP mutationdiffered from wild-type cybrids by losing part of their cell vol-ume most probably through loss of internal fluid That be-havior fitted with the edema observed in patients using muscleimaging after 15-minute chewing effort

At the molecular level the mutant mitochondrial tRNAPro

had a reduced steady-state level in cells It bore an unmodifiedadenosine at the wobble position of its anticodon which thusshould optimally recognize only codon CCU that representsless than 25 of the total mtDNA proline codons Specialmitochondrial decoding rules probably allowed the mutanttRNA to recognize all the 4 proline codons but less efficiently

These 2 anomalies (reduced tRNA steady-state and decodingproblems) might have a cumulative effect on mitochondrialtranslation Why the impact on OXPHOS was mild in fibro-blasts and cybrids but drastic in muscle remained hypotheticalin the absence of appropriate muscle fragments

The role of the J1c10 haplotype in themutation pathophysiologywas disputable That haplotype was constant in the 5 familieshere reported and in the sole GenBank report of the mutation Itis not pathogenic in itself being observed in 25ndash5 of thepeople living in the western half of France but it is a significantmodifier in Leber hereditary optic neuropathy3738 and in thecybrid resistance to rotenone toxicity39

Figure 5 Functional impact of the m15992AgtT mutation outside OXPHOS pathway

WT = wild-type cybrids MT-TP = cybrids with100 m15992AgtT mutation p lt 005 p lt001 p lt 0001 compared with the controlgroup using the Mann-Whitney test or t testwhen data had a normal distribution accordingto the Shapiro-Wilk test (A) Proliferation assay inthe presence of 1 gL glucose (glu) or in its ab-sence but in the presence of 200 μM glutamine(no glu) Number of experiments 9 independentassays on 3 different control cybrid lines and 7independent assays on 6 different mutant cybridlines (B) Evaluation of the superoxide ion pro-duction using the slope ofMitoSOX fluorescenceResults shown as a box plot with themedian thequartile and the centile as well as internal dotsnumber of experiments 10 independent assayson 5 different control cybrid lines and 5 in-dependent measurements on 5 different cybridlines with 100 m15992AgtT mutation (C) Cellsize evaluation using the FSC given by the Accuri6 flow cytometer The FSC values in the basalstate (ie in normal culture medium with TMREand verapamil as explained in the methods sec-tion) are given in million for the sake of read-ability The FSC values in the presence of 80 mMexternal K are given as of the cells FSC in thebasal state (D) Evaluation of the plasma poten-tial using the fluorescence signal of TMRE mea-sured in channel A (FLA) of the Accuri 6 flowcytometer The FLA values in the basal state aregiven in hundreds thousands for the sake ofreadability The FLA values in the presence of80 mM external K are given as of the cells FLAin the basal state FSC = forward scatter TMRE =tetramethylrhodamine ethyl esther

8 Neurology Genetics | Volume 6 Number 4 | August 2020 NeurologyorgNG

According to the pathogenicity scoring system for tRNAmutationsupdated in 20117mutationm159992AgtThas a score of 13whichindicated a mutation definitely pathogenic (2 for independentpedigrees 2 for phylogenetic conservation 2 for muscle histology2 for biochemical defects and 5 for cybrid analysis)

The homoplasmic m15992AgtT MT-TP mutation in theJ1c10 haplotype causes severe intolerance to exercise oftenassociated with exercise-induced muscle edema

AcknowledgmentThe authors thank Frederic Bouillaud INSERMU1016 for hisinsightful discussion of the data Dr Armelle Magot Centre deReference Maladies Neuromusculaires AOCHotel-Dieu CHUNantes for providing the results of patient 6 muscle biopsy andRafael Boucher Clement Vaz and Caroline LrsquoHermitte-SteadINSERM U1016 for their efficient technical help

Study fundingNo targeted funding reported

DisclosureA Lombes and I Tarassov are financially supported byINSERM and CNRS A Lombes received grants from theFondation pour la Recherche Medicale (FRM) (grantDPM20121125550) the AFM Telethon and the AMMi(Association contre les Maladies Mitochondriales) I Tar-assov received financial support from the University ofStrasbourg the LabEx MitoCross and the Graduate SchoolIMCBio (National Program PIA Programme InvestissementdrsquoAvenir) Go to NeurologyorgNG for full disclosures

Publication historyReceived by Neurology Genetics December 4 2019 Accepted in finalform June 2 2020

References1 Craven L Alston CL Taylor RW Turnbull DM Recent advances in mitochondrial

disease Annu Rev Genomics Hum Genet 201718257ndash2752 Shanske S Moraes CT Lombes A et al Widespread tissue distribution of mi-

tochondrial DNA deletions in Kearns-Sayre syndrome Neurology 19904024ndash28

3 Carelli V Giordano C drsquoAmati G Pathogenic expression of homoplasmic mtDNAmutations needs a complex nuclear-mitochondrial interaction Trends Genet 200319257ndash262

4 Wallace DC Singh G Lott MT et al Mitochondrial DNA mutation associated withLeberrsquos hereditary optic neuropathy Science 19882421427ndash1430

5 Prezant TR Agapian JV BohlmanMC et al Mitochondrial ribosomal RNAmutationassociated with both antibiotic-induced and non-syndromic deafness Nat Genet19934289ndash294

6 Taylor RW Giordano C Davidson MM et al A homoplasmic mitochondrial transferribonucleic acid mutation as a cause of maternally inherited hypertrophic cardio-myopathy J Am Coll Cardiol 2003411786ndash1796

7 Yarham JW Al-Dosary M Blakely EL et al A comparative analysis approach todetermining the pathogenicity of mitochondrial tRNA mutations Hum Mutat 2011321319ndash1325

8 King MP Attardi G Human cells lacking mtDNA repopulation with exogenousmitochondria by complementation Science 1989246500ndash503

9 Jun AS Trounce IA Brown MD Shoffner JM Wallace DC Use of trans-mitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459that causes leber hereditary optic neuropathy and dystonia Mol Cell Biol 199616771ndash777

10 Toompuu M Tiranti V Zeviani M Jacobs HT Molecular phenotype of the np 7472deafness-associated mitochondrial mutation in osteosarcoma cell cybrids Hum MolGenet 199982275ndash2283

11 Fender A Sissler M Florentz C Giege R Functional idiosyncrasies of tRNA iso-acceptors in cognate and noncognate aminoacylation systems Biochimie 20048621ndash29

12 Watanabe K Unique features of animal mitochondrial translation systems The non-universal genetic code unusual features of the translational apparatus and their rel-evance to human mitochondrial diseases Proc Jpn Acad Ser B Phys Biol Sci 20108611ndash39

Appendix 1 Authors

Name Location Contribution

Karine AureMD PhD

Department ofNeurophysiology FochHospital Suresnes France

Acquisition of dataanalysis of data anddrafted and revised themanuscript

GuillemetteFayet MDPhD

Centre de ReferenceMaladiesNeuromusculaires Hotel-Dieu CHU Nantes France

Acquisition of dataanalysis of data anddrafted and revised themanuscript

IvanChicherinPhD

MV Lomonossov StateUniversity Moscow Russia

Acquisition of data andanalysis of data

BenoitRuchetonPharmD

Service de BiochimieMetabolique CHU Pitie-Salpetriere AP-HP ParisFrance

Acquisition of dataanalysis of data anddrafted and revised themanuscript

SandrineFilaut

Service de BiochimieMetabolique CHU Pitie-Salpetriere AP-HP ParisFrance

Acquisition of data andanalysis of data

Appendix 1 (continued)

Name Location Contribution

Julie EichlerMSc

CNRS UMR 7156 GMGMUniversity of StrasbourgFrance

Acquisition of data andanalysis of data

Anne-MarieHeckel

CNRS UMR 7156 GMGMUniversity of StrasbourgFrance

Acquisition of data andanalysis of data

FlorenceCaillon MD

Service de Radiologie etImagerie Medicale Hotel-Dieu CHU Nantes France

Acquisition of dataanalysis of data anddrafted and revised themanuscript

YannPereon MDPhD

Centre de ReferenceMaladiesNeuromusculaires Hotel-Dieu AOC CHU NantesFrance

Acquisition of dataanalysis of data anddrafted and revised themanuscript

Nina EntelisPhD

CNRS UMR 7156 GMGMUniversity of StrasbourgFrance

Acquisition of dataanalysis of data anddrafted and revised themanuscript

IvanTarassovPhD

CNRS UMR 7156 GMGMUniversity of StrasbourgFrance

Designed andconceptualized the studyacquisition of dataanalysis of data anddrafted and revised themanuscript

AnneLombesMD PhD

Inserm U1016 InstitutCochin INSERM ParisFrance

Designed andconceptualized the studyacquisition of dataanalysis of data anddrafted and revised themanuscript

NeurologyorgNG Neurology Genetics | Volume 6 Number 4 | August 2020 9

13 Hogrel JY Laforet P Ben Yaou R Chevrot M Eymard B Lombes A A non-ischemicforearm exercise test for the screening of patients with exercise intolerance Neurology2001561733ndash1738

14 Dubowitz V Sewry CA Oldfors A Muscle Biopsy A Practical Approach Phila-delphia PA Saunders Elsevier 2013

15 Possekel S Lombes A Ogier de Baulny H et al Immunohistochemical analysis ofmuscle cytochrome c oxidase deficiency in children Histochem 199510359ndash68

16 Agier V Oliviero P Laine J et al Defective mitochondrial fusion altered respiratoryfunction and distorted cristae structure in skin fibroblasts with heterozygous OPA1mutations Biochim Biophys Acta 201218221570ndash1580

17 Varshney U Lee CP RajBhandary UL Direct analysis of aminoacylation levels oftRNAs in vivo Application to studying recognition of Escherichia coli initiator tRNAmutants by glutaminyl-tRNA synthetase J Biol Chem 199126624712ndash24718

18 Karicheva OZ Kolesnikova OA Schirtz T et al Correction of the consequences ofmitochondrial 3243AgtG mutation in the MT-TL1 gene causing the MELAS syn-drome by tRNA import into mitochondria Nucleic Acids Res 2011398173ndash8186

19 Kawahara Y Quantification of adenosine-to-inosine editing of microRNAs using aconventional method Nat Protoc 201271426ndash1437

20 Pesta D Gnaiger E High-resolution respirometry OXPHOS protocols for humancells and permeabilized fibers from small biopsies of human muscle Methods MolBiol 201281025ndash58

21 Medja F Allouche S Frachon P et al Development and implementation of stan-dardized respiratory chain spectrophotometric assays for clinical diagnosis Mito-chondrion 20099331ndash339

22 Aure K Dubourg O Jardel C et al Episodic weakness due to mitochondrial DNAMT-ATP68 mutations Neurology 2013811810ndash1818

23 Haraux F Lombes A Kinetic analysis of ATP hydrolysis by complex V in four murinetissues towards an assay suitable for clinical diagnosis PLoS One 201914e0221886

24 Aure K Fayet G Leroy JP Lacene E RomeroNB Lombes A Apoptosis inmitochondrialmyopathies is linked to mitochondrial proliferation Brain 20061291249ndash1259

25 Aure KMamchaoui K Frachon P Butler-BrowneGS Lombes AMouly V Impact onoxidative phosphorylation of immortalization with the telomerase gene NeuromusculDisord 200717368ndash375

26 Repetto G del Peso A Zurita JL Neutral red uptake assay for the estimation of cellviabilitycytotoxicity Nat Protoc 200831125ndash1131

27 Yasukawa T Suzuki T Ishii N Ohta S Watanabe K Wobble modification defect intRNA disturbs codon-anticodon interaction in a mitochondrial disease EMBO J2001204794ndash4802

28 Reitzer LJ Wice BM Kennell D Evidence that glutamine not sugar is the majorenergy source for cultured HeLa cells J Biol Chem 19792542669ndash2676

29 Will Y Dykens JMitochondrial toxicity assessment in industrymdasha decade of technologydevelopment and insight Expert Opin Drug Metab Toxicol 2014101061ndash1067

30 Quinlan CL Gerencser AA Treberg JR Brand MD The mechanism of superoxideproduction by the antimycin-inhibited mitochondrial Q-cycle J Biol Chem 201128631361ndash31372

31 Rorbach J Gao F Powell CA et al Human mitochondrial ribosomes can switch theirstructural RNA composition Proc Natl Acad Sci U S A 201611312198ndash12201

32 Grosjean H de Crecy-Lagard V Marck C Deciphering synonymous codons in thethree domains of life co-evolution with specific tRNA modification enzymes FEBSLett 2010584252ndash264

33 de Crecy-Lagard V Boccaletto P Mangleburg CG et al Matching tRNA modifica-tions in humans to their known and predicted enzymes Nucleic Acids Res 2019472143ndash2159

34 Scaduto RC Jr Grotyohann LWMeasurement of mitochondrial membrane potentialusing fluorescent rhodamine derivatives Biophys J 199976469ndash477

35 Howell N Leber hereditary optic neuropathy how do mitochondrial DNA mu-tations cause degeneration of the optic nerve J Bioenerg Biomembr 199729165ndash173

36 Rossignol R Letellier T Malgat M Rocher C Mazat JP Tissue variation in thecontrol of oxidative phosphorylation implication for mitochondrial diseases Bio-chem J 2000347(pt 1)45ndash53

37 Carelli V Achilli A Valentino ML et al Haplogroup effects and recombination ofmitochondrial DNA novel clues from the analysis of Leber hereditary optic neu-ropathy pedigrees Am J Hum Genet 200678564ndash574

38 Hudson G Carelli V Spruijt L et al Clinical expression of Leber hereditary opticneuropathy is affected by the mitochondrial DNA-haplogroup background Am JHum Genet 200781228ndash233

39 Strobbe D Caporali L Iommarini L et al Haplogroup J mitogenomes are the mostsensitive to the pesticide rotenone relevance for human diseases Neurobiol Dis 2018114129ndash139

10 Neurology Genetics | Volume 6 Number 4 | August 2020 NeurologyorgNG

DOI 101212NXG000000000000048020206 Neurol Genet

Karine Aureacute Guillemette Fayet Ivan Chicherin et al swelling and fatigue

mutation causing exercise-induced muscleProHomoplasmic mitochondrial tRNA

This information is current as of July 15 2020

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 2020 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent64e480fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent64e480fullhtmlref-list-1

This article cites 38 articles 10 of which you can access for free at

Subspecialty Collections

httpngneurologyorgcgicollectionmuscle_diseaseMuscle disease

netics-mitochondrial_disordershttpngneurologyorgcgicollectionmitochondrial_disorders_see_geMitochondrial disorders see GeneticsMitochondrial disorders

httpngneurologyorgcgicollectionmitochondrial_disordersMitochondrial disorders

httpngneurologyorgcgicollectionmetabolic_disease_inheritedMetabolic disease (inherited)following 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 2020 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent64e480fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent64e480fullhtmlref-list-1

This article cites 38 articles 10 of which you can access for free at

Subspecialty Collections

httpngneurologyorgcgicollectionmuscle_diseaseMuscle disease

netics-mitochondrial_disordershttpngneurologyorgcgicollectionmitochondrial_disorders_see_geMitochondrial disorders see GeneticsMitochondrial disorders

httpngneurologyorgcgicollectionmitochondrial_disordersMitochondrial disorders

httpngneurologyorgcgicollectionmetabolic_disease_inheritedMetabolic disease (inherited)following 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 2020 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet