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AssociationAssoci

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WILEY_

    BLACKWELLUsP26hnplotypesnndmntehqrtilityinthemeninT”twnn,China

    ;DOI:10.1111,j.17457262.2008.0H0439.x

    

    ;0r’

    ;Associati0nofUSP26hap1otypesinmeninTaiwan,China

    ;withseverespermatogenicdefectW1tnSeVereSpermatogenlCCLeteCt ;IWenLee,LongChingKuan,ChienHungLHsien-AnPan,ChaoChinHsu4,Yun

    gChiehTsai,Pa0LinKuo,

    ;YenNiTeng.

    ;D,nD6fPfc&IyncDZDI;affo,zfC,2ggfV,cDffgD

    cfnP,zfn,z7D,zw,(

    ;DprPf00ffrc,zdGvcrJf0DGP,zP,ZDpf,Zf,z7DD,fw,z,(:

    ;3InstitHteofBas{cSciences.NCi

    ;Abstract

    ;Aim:Tl0cOmp1etecomDrehensivehaplotVpeanalVsisOfSP26f0rbothfertileandinferti1emen.MethOds:Two

    ;hundredinfenilemenwithsevereo?gospermiaornonobstructiveazoosperrniaweresub

    jectedtosequenceana1ysis

    ;f0rtheentirecOdingsequencesOftheP26gene.TwOhundredmenwithprOvenfertiljtyweregenOtypedby

    ;primerextensionmethods.Al1ele/gen0tvpefrequencies,linkagedisequilibrium(LD)characteristicsandhaplotypesof

    ;fertilemenwerecomparedwithinfertilemen.Results:Theallelefrequencies0ffivesinglenuc1eOtidepOlymOr

    ;phisms(37037linsACA,494T>C,576G>A,ss6202791C>T,l737G>A)weresignificant1yhigherininfenilepatients

    ;thancontrolsubects.Themaiorhap1otvpesininfertilemenwere11ACCGA(28%ofthepopulati0n),TGCCGA

    ;f15%),TACCAA(8%),TGCCAA(6%),TATCAA(5%)andCATCAA(5%).Thema{orhapl0typesforthecontrol

    ;sub{ectswereTACCGA(58%ofthepopulation),CACCGA(7%),CATCGA(6%)andTGCCGA(5%).Haplotvpes

    ;TGCCGA,TATCAA,CATCAA,CATCGC,TACCAAandTGCCAAwereOver_transmittedinpatientswichspematO

    ;genicdefect,whereashaplOtVpesTACCGA,CACCGA,andCATCGAwereunder.transmittedinthesepatients.

    ;ConcIusiOn:SomeP26allelesandhaplotVpesareassociatedwithspematogenicdefectintheHannationa1itVin

;raiwan,China.nA,f2DD8?DJD8969D)

    ;Keyw0rds:singlenucleotidepolymorphism;26gene;spem1at0genicdefect;linkagedise

    qui1ibrium

    ;Correspondenceto:Prof.YenNireng,DeDartmentofBiote

    ;chnol0gv.ChiaN.dnUniversicvofPhar|ndcvandScience,60ErhJen

    ;Road,Sec.1.Pa0An,JenTeHsiang,Tainan717,Tlaiwan,China. ;I1el:+886626649l1ext.2549Fax:+88662662135

    ;Emai1:tengyennI1@yahO0.com.tw

    ;Received20080220AcceDted20080703

    ;896?

    ;1Intr0ducti0n

    ;Inflerti1ityaflfectsOnein10cOupleswOrldwide.In ;rOughlyhalf0fthesecOuples,themalefactOris ;imp1icated.Althoutherearemanyfactorsthatcon

    ;tributet0maleinferti1ity,thecausecannotbeidentified ;florthemajorityofinfenilemen1].Inrecentyears,there

    ?20O8,AsianJourna1ofAndrology.SIMMandSJTU.AllrightsreserVed

    6):8969O4 ;AsianJAndrot2o08:lo

    ;hasbeenanintensivesearchf_0rgeneticcauses0fmale ;inferti1ity.ScreeningwithmarkersOnthelOngarmOf ;mehumanYchr0mOsOmehasdetectedYlamicrOdeletiOns ;in5%15%0fmaleswithspermatogenicdefect.Itis ;believedthatmanygenesIocatedonthelong(q)annof ;theYchr0mOsOmearerequiredf0rspem1atOgenesis.This ;regiOn,bandinginql1.23OftheYchr0mOsOme,includes eazoospermiafact0r(A2:F)locus,whichcontajnsagene ;orgenesthatarerequiredf0rnorn1alspematogenesis2,

    ;3.0thergeneticfactorsthathavebeenreponedtobe ;invO1vedinspermatogenicdeflectincludemutatiOnsat ;themitOchOndrialDNApOlymeraselOcus,apOlVmOr

    ;phismOfthecytOchrOmeP4501A1gene,mutatiOnsOf ;thefolliclestimuIatinghonnonereceptorgene(,,the ;deletedinazoospern1ialikegene(),thesvnaptone

    ;malcomplexprotein3gene(l,CP),humanBD,

    ;caseinkinase2alphagenes,dystrophiamVOtonicaprO

    ;teinkinasegene(DP,DNApo1vmerasegamma

    ;(PDLG),methv1enetetrahvdmfl0latereductaserFR)

    ;,dndf0llicle.scimulatinghorn1onereceptor()4l61.

    26appearst0beanattractivecandidatef0ra ;sterilegenecOnsideringitsuniqueexpressiOnpattern. ;E,26cOn1prisesasingleexOnandisIOcatedOnXq26.2. ;ItsmessengerRNA(mRNA)sequenceis2794bplong ;anditsproteinconsistsof913aminoacidsfGenbank: ;NM031907.1).Itsmousehomologwasf0undtobe

;onlyexpressedinspermatogonia[17].P26belongs

    ;toalaefamilyofdeubiquitinacingenzymes(DUB)[18.

    ;Deubiquitinationofmacr0moleculesbyDUB,including ;ubiquitinprOteases,canrescuemacrOmOleculesfrOm

    specifjc,Ntermina1. ;degradatiOnthrOughsubstrate

    ;dependent,enzymaticreaction[19,201.Previousstudies ;haveaddressedthein1pOrtanceOftheubiquitinpath

    ;wayduringmammalianflenilizatiOn,includingacmsOmaI ;functionandspematozoazonapeUucida(ZP)penetfa

    ;tion[2l,22].Duringspematogenesis,deubiquitination ;enzVmeshavebeenshOwntobeinvOlvedintheregula

    ;tionofproteinturnover(e.g.,replacementofhistones ;byprOtamine,germcellapOptOsis,mitOticpr0liferatiOn ;anddiff_erentiationofspennatogonialstemcells)23

    ;28].Severa1papershavebeenpub1ishedontheroleof ;26inmaleinfertity.Stouflfsf,.291analvzed

    ;patientswimvariOushistOlOgicalpattemsOfspeatO

    ;genicdeflect(Sertolicel1.onlysyndromeandmaturation ;arrest)fOrthepresenceofmutationsinP26_Thev

    ;foundsomeSertolice11.onlysyndrome(SCOS)patients ;hadtIlegenotype0f370371insACA(rs3840975),49411>C ;andl423C>T(rs41299088)[29.AstudybvPaduch

    ;Te|:+86-21.5492-2824;Fax:+86-21_5492?2825;shanghai,China

    ;alsOprOvidedevidencelinking,5P26mutatiOntOmale

    ;infenility[30].Althoughthesetwostudiessuggestedthat ;aspecincgeneticcluster(37O37linsACA,494T>Cand

    ;1423C>T)mightbeassociatedwithtesticular ;dysf_unctiOn,tw0OtherstudiesshOwedthatthisgenetic ;clusterwasnotrestrictedtOmenwithtesticulardvs

    ;function[31,32.Inarecentpublicationon26,the

    ;authOrsidentifiedseveralnOvelmutatiOnsinthe(P26 ;genetnat?ugntcausespennatOgenes1slmpa1ment1na ;smallgroupofinfertilemen(,2=41)f33].However, ;meta—analysisofprevi0uscase.contro1studies”ntota1.

    ;544patientsand1705contro1s)revealednosignificant ;assOciai0n0fthe370371insACA.494T>CaJ1dl423C>T ;genocypewithmaleinfertility[14,2932].Furtherstudies

    ;basedOnalaregrOupOfpatientswithdiversifledethnic ;backgrOundswillbevaluabletObrjngmOreinsightintO ;therO1eOf)26inmaleinfertilitv.

    ;1nthepresentstudy,wesetOuttOanalVzegenetic ;variantsandhaplotypesOfLP26f0rbOthfertileand ;in1.eItilemenbysequencea1a1Vsisf0rtheentirecOdingre.

    ;giOnOfP26.W_efl0undsOme.5P26geneticvants ;andhaplOtypeswereOverttransmittedandsomewere

    ;undertransmittedinpatientswithseverespematOgenic ;defectintheHannatjOnalitvin11aiwan.China.W_ealsO ;identifjedsomenOve1geneticvariants0rmutationsin ;patients.Ourresu1tsupportsimpOnantr0lesOfP26 ;inhumanspermatOgenesis.

    ;2MaterialsandmethOds

    ;2.jSHbjects

    ;ThesmdywasapprOvedbymeInstitutiOna1Review ;BOardOfNaciOna1ChengKungUniversityMedica1Centre ;(raiwaJ1,China).Flr0mJanuarV2001toDecember2005. ;200infenilemenpresentingwimsevereoligOzOOspe?nia

    ;(spermatozoacount<5×l06/mL)ornonobstructive

    ;azoospemiaand200menwithpmvenfertilitv(control ;group)wereenrolled.AUofthecontrolsubiectshadfa

    ;theredatleasttwOchildrenwithin5vearswithOutas

    ;sistedreproductivetechnOlogies.Thepatemalr.elation

    ;shipbetweencontrolsubiectsandtheiroffspringwas ;cOnfirmedusingtheAmpF/STR.PrOfilerPluszVgOsitV

    ;deteminationsystem(App1iedBiosystems,FosterCitv, ;CA,USA).AUpatientsandcontrolsubjectsbelongedto ;theHannationaljtyinTaiwan,memajorethI1icgroupin ;China(makingupmorethan95%ofthisprovexalninati0n, ;atleasttwosemenanalyses,hormoneassays(1uinizing ;h0rmone[LH],f0uiclestimulatinghorn1one[FSH],pro

    ;1actinPRLandtestosterone[T),karyotypingandamo

    ;leculartestfl0rchr0mosoma1deleti0ns.Patientssus

    ;pectedtOhavenOn?Obsn1lctiVeazOOspeI111jawerleadVised ;tOundergobilateraltesticularbiOpsies.Serumlevels0f ;FSH,LH,PRLandTweremeasuredusingcOmmercial ;radiOimmunOassaVts:COatA.COumFSHIRMACOat

    ;ACOuntLHIRMA.COatACountPRLIRMAand

    ;MMI?ITETotalTest0sterone(DiagnosticProducts,Los ;Angeles,CA,USA1.ChrI)mOsomeanalVsiswasperfbm1ed ;usingtheGTGmethod(G_bandingbvTrvpsinGiemsa

    ;technique).MoleculLrana1vsisofY_chromosoma1dele

    ;tiOnsinc1udedacOmbinatiOn0fl6genebasedprimers,as

    ;describedpreviouslv34].Patientswimanvidentifiable ;causeofma1einfertilitV,includingcongenitalbilatera1ab. ;sence0fvasdeference(CBAVD1,crvotorchidism, ;varicOcele,diabetesmellitusOrhVpertension,0rwithhis

    0ryhatmayaftectspenaogenes1s(e.g.,0rChit1s,?Iauma,

    ;malignancies,.)wereexcludedfromthestudvgr0up.

    ;NOsignicantdifferencewasf0undbetweenage.serum ;LH.estradiOlandTconcentratiOnsOfinfertilemenand ;controlsubiects.SpermcountandFSHconcentrations ;weresignificantlVhigherinpatientscOmparedwiththe ;controlgr0up(P<0.005).0f20Ostudvsubjects,l37 ;hadseveI_eOlig0spemiaand63hadazOOspeImia. ;2.2SequencennnlsiSofpntients

    _plesofpatientsweresubjected ;GenomicDNAsaI1

    ;tOsequenceanalysisfOrtheentirecodingsequencesOf ;theP26gene.GenOmicDNAwasextractedfr0mpe

    ;ripheralblO0dsamplesusingaPuregeneDNAisOlati0n ;kitfGentra,Minneapolis.MN,USA).26genomicse

    ;quence(AF285593)wasobtainedflr0mtheNCB1website ;(http://www.ncbi.nlm.nih.goV).Theentiregene(2794bp) ;wasdiVidedint0fiVeOVerlappingfagmentsrangingf0m ;409to600bpin1ength.Tbamp1if_ypanja1fragmentsof ;meP26exon,polvmerasechainreaction(PCR)reac

    ;tiOnswereperf_0nnedin20LvOlumescontaining200ng ;ofgen0micDNA,10mmol/LTrisHCl(pH8.3),

    ;50mm0l/LKC1.0.1%TritonX100.200LLm0l/LdeOx

    ;vribonucleotidetriph0sphates(dNTP),l00pmol0feach ;primerand1UqDNApo1ymerase(Pr0megaCorp., ;Madison,WIUSA).TheprimersetsforP26are

    ;describedin(Table1).PCRamplificationwasper

    ;f0rmedinanautOmatedtherma1cvcler(0mniGene ;ThermalCvc1er:HvbaidLtd.,Ashfl0rdMidd1esex,UK). ;PCRproductsweresequencedtoidentiflVmutations0r ;pOlVmor1)hisms.Sequenceana1Vsiswasperf0rmedwith ;anaut0maticsequencerfABI377.App1iedBi0sVstems/ ;PE1.

    ;2.GP0,.rcDnf,

    ;Gen0micDNAsamplesofthecontro1subiectswere ;subjectedtogen0typingbyprimerextensionmethods. ;TheamplicOnswereamp1medinamultip1exfashi0nand ;each20ULreactionc0nsistedOf5OngOfgenomicDNA, ;l0pmolOfprimers,4.0mm0l/LMgCl20.2mm0I/L ;dNTPand0.5UOfAmpliTaqGOldDNApOlVmerase ;(AppliedBiosvstems).Thecyclingc0nditi0nswere:950C ;f0r5min,f01lowedbv35cvclesat95.CfOr1min,55.C ;f0r1minand72.Cf0r1min.withafina1extensiOnf0r ;rab1e1.Primersetsusedf0rPCRandDNAsequencingofthe

    P26gene.ThenucleotidesequencesisaccordingtoAF285593.The ;1engmsOfpolymerasechainreactionpr0ductsize ;.

;8P:Amersham

    ;PharmaciaBiotech,Uppsala,Sweden)and2Uofexo

    ;nuc1easeI(BMBiochemica,Mannheim,Germanv1ina ;10Il1LreactionvolumefOr1hat37.C.SNaPshOtmu1

    ;tiplexPCRwasperformedina10LreactiOnvOlume ;containing1.25ULofSNaPshotMu1tiD1exReadvReac. ;tionMix(AppliedBi0systems),4ULofSAP/exonuclease ;ItreatedPCRproducts,and1to3pmO1Ofeach

     ;SNaPshOtprimer.Them1a1cVclingfOrSNaPshOtreac

    ;tionscOnsistedOf25cvclesat96.Cforl0s.50.Cfor ;5sand60.Cf0r30s.TheSNaPshotmultiDlexreaction ;wasthentreatedbvSAPinanl1uLreactionvo1ume ;cOntainingl0LOfSNaPshOtmultiplexPCRprOduct ;and1U0fSAP.ThesampleswereanalVzedusingan ;ABI3l00geneticanalyzer(App1iedBi0systems)andthe ;al1e1edetenninationwascarriedOutwiththeGenOtyper ;3.7pr0gram(AppliedBi0svstems).

    ;2.4Stntisttcnlnnnl,,sis

    ;TestsforassOciationwithsingIemarkersand ;haplotypesincOntrO1sampleswereperfOnnedusingthe ;test.P<0.05wasconsideredstatistica11vsignificant. ;ThereIativeriskofspermat0genicdeflectwasestimated ;ftomIogistic0ddsratiosf0R)and95%confidencein

    ;terVals(CI)inmuItiVariateanalysis.11estsf0rhaplotype ;assOciatiOnwithspermat0genicdefectwereperformed ;usingEHPLUSsofcware[35,andstatisticalsignicance

    ;wasestimatedusingthepennutationtestPMPLUS361.

    ;AssOciatiOnsbetweenhaplotVpesandmaleinfenilitvwere ;analVzedusinglinearregressionmodels.The1inkagedis

    equilibrium(LD)coefficient(D’)betweeneachpairof

    ;sing1enucleotidepolymoT1)hisms(SNPs)wasca1culated ;usingtheldmaxprogramwithintheGOLDsoftware ;package[37].

    ;3Results

    .PD,vo,f,z,”,z,z7w,z.(

    ;ThecOdingsequencesOftheUP26genewereex

    ;aminedbVsequencinganalysisflorthepresenceOfmuta. ;tiOnsofsequencevariantsininfertilemen.Thesequence ;fromGenBank(AF2855931wasusedasthereference ;seauenceandtheAofATGOfthestartcOdonwasnum

    ;bered+1.IntotaI.sevengeneticvantsweI.eobsef,ed,

    ;includingOneinsertionvariant370371insACA

    ‘rel:+86-21?5492-2824;Fax:+86-215492?2825;shanghaichina ;(rs3840975),causingathreonineinseni0ninaminoacid

    ;pOsitiOnl21,andsixSNPs,494T>C.576G>A ;frs413O454O),ss62O279lC>T,1423C>T(rs41299O88), ;1737G>Aand2202A>C.A10fthesegeneticvariants

    ;wereavaableinthewebsitefhtcD://www-ncbi-nlm-nih. ;goV/)andpeer.reviewarticles29321.Onesubstituti0n,

    ;576G>AfEl92),doesnotaltertheaminoacidsequence: ;aglutaInicacidatpositionl92.TheotherfiveSNPwere ;pI.edictedtOc!leateaminOacidalteraiOn:494T>Cchanges

    ;aleucineintoaserine(Ll65S1;ss620279lC>Tchanges ;aserineintoapheny1alaninerS600F):1423C>Tsubsti. ;tutesahistidinef0ratvr0sine(H475Y):1737G>Asub ;stitutesamethionineforaisoleucine(M579I,:and ;2202A>Csubstitutesa1ysinef0raasparagine(K734N). ;ThefO11OwingsubstitutiOnsOccurredOn1v0nceinthe ;patientsgrOupbutnotinthefertilecOntrOlsgroup: ;393A>T(Sl31S);468T>G(O156H):l976C>T ;rT659M):2144A>TfN7l5I):2182A>TfI128F):2195T> ;C(F732S);2204T>G(V735G);2239A>T(I747F): ;2247A>C(E749D);2250A>G(Q750Q);and2271T>C ;(P757P).Exceptfor1976C>Tr]r659M),thesesubsti

    ;tutionshavenotbeenreported301.Thesubstitutionof

    ;520T>Gand565T>GispredictedtOcausepremature ;stOpcOdOnatcOdonl75and189,respectivelV.HaplO. ;tVpeana1ysesnu1edOutthatpatientscaI1.yingthesevari

    ;antsitesOccurredOnlV0nceintheinfertilitVgrOup. ;3.2Allelicnndgenopicquenciesinr1ileandin

    rfe,”en

    ;Al1elicflrequenciesandoddsratiosf_0reachsequence ;variantOfP26areshOwninTlable2.0ftheseven ;variants.37037linsACA.494T>C,576G>A.

    ;ss6202791C>Tandl737G>Aaresignificantlvassoci

    ;atedwithspermatogenicdefect.The.va1uef0rthese

    ;fivevariantswaslessthan0.05andmeoddsratioswere ;largerthan1.FOrexample,thePvaluesfOr370

    ;37linsACAand576G>Aarelessthan0.0001andthe ;ORare35.86and4.095fTable21.

    ;3.3Reproducteeffectofthe7o37nnsACA.494T>C

    ;1423C>T2eneticclsternnditsderivation ;TheancestralP26clusterf37037linsACA.

    ;494T>C,1423C>T1anditsfirstderjvationonlvoccur ;inthegroupofjnfertilemen.IncOntrast,thesec0nd ;derivationonlvoccursinthegroufenilemen.A1lelefrequencieswerecOmparedbetweeni

    nfenile

    ;menandcontrolsubjects.sNPs,singlenucleotidepolym0rphisms;0R,oddsratio;cI,cofidc

enrVal

    le3.Thegenotypefrequencyofthe37O

    37nnsACA,494T>Candl423C>Tcluster,itsfirstandsecondderiVatiVeinfenileand

    ;inilemen.Ana1yzedforstatjStjca1signmcancebyFisher’sexacttest.Accordinglo

    .)R,oddratjo 37

    ;3.Ln足以ffrM,fLD)Pfved,?nf;l

    (Table41showsthepairwiseLD(D’)betweendif_

    ;ferentalleles.FOrpatientswithspermatOgenicdefect, ;2202A>CwasfoundtobeincOmp1eteLDwith370

    ;371insACA494T>C,576G>Aandl423C>TfD.=1). ;494T>ChadstrOngLDwith370371insACAand

    1423C>T(D’=0.829and0.804respective1y).Forc0n

    r0lsubcts,37O371insACAw

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