Quantification of mitochondrial DNA damage and copy number in circulating blood of patients with systemic sclerosis by a qPCR-based assay,

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Anais

Brasileiros

de

Dermatologia

www.anaisdedermatologia.org.br

INVESTIGATION

Quantiﬁcation

of

mitochondrial

DNA

damage

and

copy

number

in

circulating

blood

of

patients

with

systemic

sclerosis

by

a

qPCR-based

assay

夽,夽夽

Shaﬁeh

Movassaghi

a

_,

_Sara

_Jafari

a

_,

_Kowsar

_Falahati

b

_,

_Mitra

_Ataei

b

_,

Mohammad

Hossein

Sanati

b

_,

_Zohreh

_Jadali

c,∗

a_Department_of_{Rheumatology,}_Imam_Khomeini_Hospital,_Tehran_University_of_Medical_Sciences,_Tehran,_Iran

b_Clinical_Genetics_Department,_National_Institute_of_Genetic_Engineering_and_{Biotechnology,}_Tehran,_Iran

c_School_of_Public_Health,_Tehran_University_of_Medical_Sciences,_Tehran,_Iran

Received24April2019;accepted12November2019 Availableonline20March2020

KEYWORDS Autoimmunity; DNA; Mitochondrial; Oxidativestress; Real-timepolymerase chainreaction; Scleroderma, systemic Abstract

Background: Althoughnotfullyunderstood,oxidativestresshasbeenimplicatedinthe

patho-genesisofdifferentautoimmunediseasessuch assystemicsclerosis.Accumulatingevidence indicatesthatoxidativestresscaninducemitochondrialDNA(mtDNA)damageandvariations inmtDNAcopynumber(mtDNAcn).

Objective: TheaimofthisstudywastoexploremtDNAcnandoxidativeDNAdamagebyproducts

inperipheralbloodofpatientswithsystemicsclerosisandhealthycontrols.

Methods: Fortysixpatientswithsystemicsclerosisandfortyninehealthysubjectswere

stud-ied.Quantitativereal-timePCRwasusedtomeasuretherelativemtDNAcnandtheoxidative damage(oxidizedpurines)ofeachsample.

Results: ThemeanmtDNAcnwaslowerinpatientswithsystemicsclerosisthaninhealthy

con-trolswhereasthedegreeofmtDNAdamagewassigniﬁcantlyhigherincasesascomparedto controls. Moreover, therewas a negativecorrelation between mtDNAcn andoxidative DNA damage.

Studylimitations: ThelackofsimultaneousanalysisandquantiﬁcationofDNAoxidative

dam-agemarkersinserumorurineofpatientswithsystemicsclerosisandhealthycontrols.

夽 _How_to_cite_this_article:_Movassaghi_S,_Jafari_S,_Falahati_K,_Ataei_M,_Sanati_MH,_Jadali_Z._{Quantiﬁcation}_of_{mitochondrial}_DNA_damage

andcopynumberincirculatingbloodofpatientswithsystemicsclerosisbyaqPCR-basedassay.AnBrasDermatol.2020;95:314---9.

夽夽_Study_conducted_at_the_Tehran_University_of_Medical_Sciences,_Tehran,_Iran. ∗_{Corresponding}_author.

E-mail:zjadali@yahoo.co.uk(Z.Jadali).

https://doi.org/10.1016/j.abd.2019.11.003

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Conclusion: ThesedatasuggestthatalterationinmtDNAcnandincreasedoxidativeDNAdamage maybeinvolvedinthepathogenesisofsystemicsclerosis.

Introduction

SystemicSclerosis(SSc)isararechronicdiseaseof connec-tivetissue characterizedbya vasculopathyand ﬁbrosisin theskinandvariousinternalorgans.1,2_The _exact_cause_of

diseaseisunknown,butitsoccurrencecanbeinﬂuencedby acomplicatedinterplayofgenetic,epigeneticand environ-mentalfactors.3_Recently,_great_attention_has_been_devoted

totheroleofoxidativestressinthepathogenesisofSSc.It isthoughttobeinvolvedinthepathophysiologicalprocesses of increased synthesis and accumulation of extracellular matrix,abnormalimmuneactivationandvascular endothe-lialdamage,thethreeoutstandingfeaturesofdisease.4,5

Increasedoxidativestressreflectsanimbalancebetween pro-oxidant/antioxidanthomeostasisandleadstoincreased levels of toxic Reactive Oxygen Species (ROS). There are two main pathways of ROS generation, the endoge-nous and exogenous pathways. Mitochondria are the major endogenous source of ROS. They have their own genome[mitochondrialDNA(mtDNA)]andareindispensable organellesfor normal cellularphysiology.mtDNA is multi-copy, and its abundance is regulated in a tissue specific manner.mtDNAcnfluctuatesinresponsetothe physiologi-calenvironmentsurroundingthecell,butremainsrelatively stablewithincellsunderphysiologicalconditions.6_In

patho-logical circumstances, the number of mtDNAcn can vary depending ondifferent factors, including altered cellular redox balance. mtDNA is also prone to oxidative stress damagebecause of itshigh exposureto theROS that are producedbymitochondria.7

Guanine oxidationproducts areimportantindicatorsof oxidative DNA damage. These chemical markers can be removedbyFpg(formamidopyrimidine[fapy]-DNA glycosy-lase),whichisabaseexcisionrepair enzymeandreleases purinelesionsfromboth nuclearandmtDNA.7,8_Fpg

cleav-age ofoxidatively damaged DNAcan reduce ampliﬁcation of enzyme-digested target DNA by Taq DNA polymerase.9

Therefore,thismethodhasbeenwidelyusedasameasure ofoxidativedamagetoDNA.Theaimsofpresentstudywere todeterminewhethermtDNAcnvariationanditsoxidative damageindex---mtDNA(CT);canbedetectedinpatients withSSc.

Methods

The study has receivedapproval fromthe ethics commit-teeof University of MedicalSciences, andwasperformed inaccordancetotheethicsprinciplesintheDeclarationof Helsinki.Allparticipantsvoluntarilyagreedtoparticipatein thestudyandgavefullyinformedconsent.

Studypopulation

Forty six patients with SSc and 49 age and sex matched healthyindividualswithnohistoryofsclerodermaorother

chronic and autoimmune diseases were included in the study.ThepatientswithSScwerediagnosedaccordingtothe AmericanCollegeofRheumatology(ACR)/EuropeanLeague AgainstRheumatism(EULAR)2013 criteria.10,11 _Among _the

enrolledpatients,6 (13.04%) hadlimitedformof SScand 40(86.96%)haddiffusesystemicsclerosis;12(26.09%)had interstitiallungdisease(ILD),3(6.52%)hadPulmonary Arte-rial Hypertension (PAH) and 3 (6.52%) had both ILD and PAH. The mean age of the patients with SSc (6 men, 40 women) and the controls (5 men, 44 women) was 46.09 and41.90years,respectively.Themeandurationofdisease was7.61±6.13 yearswith a range of 1---40 years. Thirty eightpatientswithdiffusesystemicsclerosisand6patients with limited cutaneous SSc had received steroid and/or immunosuppressanttreatmentsbeforesampling.Drugdose anddurationdiffersdependingontheseverityofthelesions andtheaffectedorgans.Noneofthepatientshad inﬂamma-tory/autoimmunedisordersother thanSScandallofthem wereinaninactivestateatthetimeofvisit.Table1shows demographicandclinicalcharacteristicsinSScsubjectsand controls.

DNAextraction

Bloodsamples were collected into 5mL tubes containing ethylenediamino tetraaceticacid (EDTA). DNAextraction from whole blood was performed using DNA isolation kit (GeneAll, Seoul, Korea), according to the manufacturer’s instructions.

The concentration and puriﬁcation of the extracted DNA were measured by using agarose gel electrophore-sis (1.5%) and spectrophotometry with NanoDrop 2000c (ThermoFisherScientiﬁc,USA).AllDNAsampleswere sub-sequentlystoredat−20◦Cuntiluse.

PreparationofDNAtemplate

Forthedetectionofoxidativedamage,100ngofsampleDNA (2␮Lofstocksolution)wasincubatedfor1hat37◦Cin10␮L ofreactionmixturecontaining1unitsofFPGenzyme(New England BioLabs, UK), 10mM Bis Tris Propane-HCl, 10mM MgCl2,1mMDTTand0.1mg/mLbovineserumalbumin.For

detectionofmtDNAcn,sampleDNAwaspreparedinsimilar way,butintheabsenceofFPG.

QuantitativedeterminationofmtDNAcopynumber Reactionsforquantitativereal-timePCRassay(qPCR)were performedwith SYBRPremixEX TaqII kit(Takara, Japan) usinga Rotor-Gene 6000 apparatus(Corbett Life Science, Australia).PCR wasperformed for 42cycles,witha 1min initialdenaturationat 95◦Cfor theﬁrstcycleandall sub-sequent cycles consisted of denaturing at 95◦C for 4s, annealingat 62◦Cfor30s,andextensionat 72◦Cfor15s, aswellasaﬁnalextensionstepof15sat72◦C.

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Table1 Demographicandclinicalfeaturesofpatientswithsystemicsclerosisandhealthycontrols.

Features Patients Controls

Number 46 49

Age(years) 46.09±12.70 41.90±12.23

Durationofdisease(years) 7.61_±6.13

Gender(female/male) 40/6 44/5 IcSSc/dcSSc,n(%) 6(13.04%)/40(86.96%) 0 Organinvolvement Skin 30 0 Lung(ILD) 17 0 Heart(PAH) 10 0

Renal(historyofrenalcrsis) 1 0

Gastrointestinal 20 0

Joint 5 0

Treatment

Typeofdisease Drugname Numberofpatients

Limitedcutaneous systematicsclerosis Methotrexate 4 Azatioprin 1 Mycophenolate 1 Diffusesystemic sclerosis Methotrexate 6 Azatioprin 10 Mycophenolate 14 Prednisolone 1 Azatioprin+prednisolone 7 None 2

PCRreactionswereperformedinatotalvolumeof10␮L with1␮L ofgenomic DNA, 0.5␮Lof each primer, 5␮Lof SYBRPremixand3␮Lofdouble-distilledwater.

FormtDNA quantiﬁcation,onesetof primerstargeting themitochondriallyencodedNADHdehydrogenasesubunit2 (ND2)andfornuclearDNAquantiﬁcation,onesetofprimers targeting␤-actingenewereselected.Allprimerwere pur-chased fromGene FanavaranCompany (Tehran, Iran) and theirexactsequenceswasdescribedpreviously.12_The_ratio

ofmtDNAtonuclearDNAineachsamplewascalculatedas anestimateforthenumberofmtDNA.

QuantitativedeterminationofmtDNAdamage Thecontentofguanineoxidationproductswasdetermined by qPCR-based technique. The level of oxidative damage inmitochondrialDNAisreﬂectedbyincreasedquantitiesof oxidizedpurinenucleotidesincluding8-OHdG.FPGdigestion wasusedforthedetectionofoxidativeDNAdamage prod-ucts.LevelsofoxidativemtDNAdamagewereshownasCt, whichisthedifferencebetweenthresholdcycle(Ct)values betweenenzyme-treated and enzyme-nontreatedsamples (Ct-TandCt-N,respectively).ThehigherCtvalue corre-spondstoahigherlevelsofoxidativestressinmitochondria.

Statisticalanalysis

Thenormallyandnon-normallydistributeddatawere ana-lyzedbyindependent-samplest-testandtheMann---Whitney U test, respectively. The levels of linear dependence between two variables were calculated by the Pearson

1 0 n = 46 n = 49 Patients Controls P = 0.001 mitochondr ial DNA/n uclear DNA (relativ e r atio)

Figure 1 The mean mitochondrial copy number in periph-eral blood of patients with systemic sclerosis and healthy subjects. Copy number of mtDNA was quantiﬁed using the delta Ct (Ct) of average Ct of mtDNA and nucler DNA (Ct=CtmtDNA+Ct␤actin).

correlationcoefﬁcient.Thistestissuitablefornormal con-tinuousvariables;p-valuesoflessthan0.05wereconsidered as threshold of statistical signiﬁcance. The experimental resultswereshownasmean±standarddeviation.SPSS soft-ware (v11.0; SPSS, Inc. Chicago, IL, USA) was used to performstatistical operationsand interpretthe results of research.

Results

Decreased mitochondrial DNA copy number in peripheral bloodofpatientswithSSc.

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7 5 3 1 -1 -3 n = 46 Δ CT n = 49 Patients P = 0.045 Controls

Figure2 IncreasedmtDNAdamageinpatientswithsystemic sclerosisascomparedtonormalcontrols.TheCtisthe differ-encebetweenCtvaluefromDNAsampletreatedwithFPGand CtvaluefromDNAsamplewithoutFPGtreatment.Pleasenote thata higherCtvalue correspondsto acomparably higher levelsofoxidativethesedamage.

As shown in fig. 1, mtDNAcn of peripheral blood was significantly (p=0.001) decreased in the patient group (0.49±0.02) compared with the HC group (0.52±0.04). Therewasnoassociation betweenmtDNAcnandage,sex, diseaseduration.Moreover,therewasnosignificant differ-enceinmtDNAcn amongpatientswithlimitedanddiffuse subsetofdisease(p>0.05).

IncreasedoxidativeDNAdamageinpatientswith SSc

To study the existence of oxidative damage, the pres-enceofoxidizedpurinebases(mostly8-oxodGuo)thatare FPG-sensitive sites weredetermined in DNAof peripheral blood samples from patients with SSc and normal con-trols. The results showed(Fig. 2)that the meanvalue of oxidative DNAdamage in patients (0.99±1.40)was signi-ﬁcantly increased (p=0.045) compared to healthy group (0.42±1.37).Moreover,noassociationwasfoundbetween oxidativeDNAdamageandage,sex, anddiseaseduration. Statistical analysisalsorevealednosigniﬁcant differences betweenpatients withlimited anddiffuseSScin termsof oxidativeDNAdamage(p>0.05).Interestingly,therewasa negative correlation between the mtDNAcn and oxidative DNAdamage(r=−0.392,p=0.007).

Discussion

ThereisgeneralagreementthatSScisanautoimmune dis-ease and its immnopathogenesis is similar in complexity tothatof other autoimmune diseases.13 _The _exact

mech-anismwhichcausesdiseaseisunclear.Nonetheless,recent studies have indicated the significanceof oxidative stress influenceindiseaseprocesses.Forinstance,Bourjiand col-leagues have shown that fibrotic and nonfibrotic skins of patients with SSc have higher levels of ROS than healthy controls.TheyalsofoundthatSScpatientshadlower vita-minC(whichhaspowerfulantioxidantactivity)levelsthan healthycontrols.14 _Increased_production _of_ROS _by

ﬁbrob-lasts, endothelialand T-cellsthatarekeycellularplayers

inthepathogenesisofSScsuggestsanotherevidencelinking oxidativestresswiththemaineventsofthediseasesuchas ﬁbrosisandvasculardamage.15

There are many different pathways for generation of ROS.MitochondriaaremainsourcesofROSandatthesame time are the main targets of their unfavorable effects. ThemajorbulkofmitochondrialROSarisingfromthe elec-trontransportchain,asabyproductofaerobicrespiration process.16_ROS_act_as_a_double-edged_sword_because

uncon-trolled generation of ROS promotes oxidative stress that causes severe injury to basic kinds of biological macro-moleculesincludingDNA,protein,carbohydratesandlipid. Alternatively,ROScanparticipateinavarietyofbiological processes in normal cells including cell signaling path-ways and immune responses against pathogens or foreign substances.17

In recent years, several studies have examined the impactofquantitativeandqualitativevariationofmtDNAcn on human diseases and have shown remarkable changes in mtDNAcn in various diseases.12,18 _The _copy _number _of

mitochondrial DNA can be modiﬁed by oxidative stress. Furthermore, increased production of ROS can result in oxidative damage tocellular componentsincluding DNA.6

Therefore, evaluation of the mtDNAcn quantity and its oxidative damage index will improve our understanding aboutthepathogenesisofoxidativestress-relateddiseases suchasSSc.

The results of present study indicated that the mean value of mtDNAcn was signiﬁcantly lower than those in correspondingnormal.To the bestof ourknowledge, this report is the ﬁrst of its kind to measure alterations of mtDNAcn in peripheral blood of patients with SSc. Cur-rently,thereislittleinformationregardingtherelationship betweenbiochemical,structural,andfunctionalchangesin mitochondriaandSSc.

One report by Feldmann and colleagues indicates the presence of giant mitochondria in the hepatocytes of patientswithsystemicscleroderma.Buttheydidnotprovide anyexplanation concerning the mechanism(s)behind this cellularevent.19 _Some_researchers_have _also_reported_the

presenceofAntimitochondrialAntibodies(AMAs)inthesera ofpatientswithSSc.Theseantibodiesarefoundinupto25% ofpatients20 _and_have _activity _against_components_of _the

multi-enzyme Complex Pyruvate Dehydrogenase (PDHC)21

which is located in the inner mitochondrial matrix and produces reduced substrates for the electron transport chain.22_AMAs_are_considered_the_serum_hallmark_of

autoim-munediseasessuchasPrimaryBiliaryCirrhosis(PBC).23_and

the clinical motivation behind the assessment of AMAs in patientswithSScwasbasedontheobservationthatalarge proportionofthesepatientsshowedcoexistingautoimmune conditionincludingPBC.24

Severalother autoantibodies,includingAnticentromere (ACA),Anti-topoisomerase(ATA),andanti-RNApolymerase III(anti-RNAP)antibodies, havebeendescribedinpatients with SSc that could be considered as good biomarkers for distinct clinical manifestations and prognosticsubsets ofSSc.25

There is alsosome evidence for a positive association betweentheseantibodies andAMA. Forinstance, Wielose etal.ﬁndsthattheprevalenceofACAsissigniﬁcantlyhigher inSScpatients,whohavepositiveAMAtiters,comparedwith

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thosewithnegativeAMAtiters.26_This_study_also_revealed_a

strongassociationbetweenAMAsandACAsinpatientswith limited cutaneous SSc, which is consistent with previous reports.27

Theseﬁndingsindirectlysuggestthatmitochondriamay be involved in SSc, as well as suggesting a possible role for oxidative stress. This notion is strengthened by other observations.Forinstance,analysisoftheBronchoalveolar LavageFluid(BALF)ofSScpatientswithlungﬁbrosis(SScFib+)

indicated a signiﬁcant upregulation of mitochondrialDNA topoisomerase1(mtDNATOP1),28_an_enzyme_that_has_a

func-tionalroleinmtDNAmaintenanceandtopology.29_Moreover,

downregulationof GlutathioneS-Transferase P(GSTP)and Superoxide Dismutase (SOD) were observed in BALF from patientswithSScFib+.28_It_must_be_mentioned_that_SOD_and

GSTParetwocomponentsoftheanti-oxidantdefence sys-temthatprotectcellsandtissuesfromoxidants especially inthelung.30,31

OxidativeDNAbasedamageproductsalsoserveas mark-ersofoxidativestressandamongthefournitrogenousbases inDNA;guanine isthe mosteasily oxidizable nucleicacid base. Therefore guanine is a primary target of oxidative modiﬁcation32_and_measurement_of_oxidized_guanine_species

can provide a useful tool for assessing oxidative stress-induced DNA damage in both nuclear and mitochondrial compartments.

Inthecurrentstudy,weusedqPCRassaytoevaluateDNA baseoxidation,inperipheralbloodcellsfromSScpatients andhealthycontrols.

The results indicated that there is statistically a sig-niﬁcantdifferencebetweentheanalyzedgroupsregarding oxidativemtDNA damage.Moreover,asigniﬁcant negative correlationwasobservedbetweenmtDNAcnandthedegree ofoxidativemtDNA(r=−0.392,p=0.007).

These results are in line with previous studies that reported increased oxidative stress and DNA damage in ﬁbroblasts obtained from patients with systemic sclerosis (scleroderma).33,34

Although theexact molecular mechanismof DNA dam-age remains to be elucidated, it seems that SSc-patients derived autoantibodies can stimulate multiple intracellu-lar signaling pathways (such as Wnt and Ras pathways) whichcanbeinﬂuencedbyROSandcan havealsoimpact on the ROS production. In this scenario, uncontrolled changes in ROS production and signal transduction may resultinaccumulationof DNAdamage,activationof ROS-dependentgenesandhighsusceptibilitytoapoptosis.These events arethoughtto participatein the formation of tis-sueﬁbrosisasconsequencesofapoptosisanddepositionof collagen.35

Conclusion

Insummary, the present studyassessed the mtDNAcnand mtDNA damage by using a quantitative PCR-based assay. Our results show that patients with SSc reduced mtD-NAcnandgreatermitochondrialdamagewhichisconsistent withmitochondrial dysfunction.Therefore, in addition to traditionalexperimentsaimedatunravelingthe pathophys-iologyofSSc,futurestudiesarenecessarytoconﬁrmthese ﬁndings.

Financial

support

This project was conducted with ﬁnancial support (con-tractn◦ 24743)ofTehranUniversityofMedicalSciencesfor Research.

Authors’

contributions

Shaﬁeh Movassaghi: Approval of the ﬁnal version of the manuscript; intellectual participationin the propaedeutic and/ortherapeuticconductofthestudiedcases.

Sara Jafari: Approval of the ﬁnal version of the manuscript; intellectual participationin the propaedeutic and/ortherapeuticconductofthestudiedcases.

Kowsar Falahati: Approval of the ﬁnal version of the manuscript; obtaining, analysis,and interpretation of the data.

Mitra Ataei: Approval of the ﬁnal version of the manuscript; obtaining, analysis,and interpretation of the data.

MohammadHosseinSanati:Approvaloftheﬁnalversion ofthemanuscript;criticalreviewofthemanuscript.

Zohreh Jadali: Statistic analysis; approval of the ﬁnal versionofthemanuscript;conception andplanning ofthe study;elaborationandwritingofthemanuscript;obtaining, analysis,andinterpretation ofthe data;effective partici-pationinresearchorientation;intellectualparticipationin thepropaedeuticand/ortherapeuticconductofthestudied cases;criticalreviewoftheliterature;criticalreviewofthe manuscript.

Conﬂicts

of

interest

Nonedeclared.

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