Association
WILEY_
；BLACKWELLUsP26hnplotypesnndmntehqrtilityinthemeninT”twnn,China
；DOI:10.1111,j.1745—7262.2008.0H0439.x
；‘
；0厂r’
；Associati0nofUSP26hap1otypesinmeninTaiwan,China
；withseverespermatogenicdefectW1tnSeVereSpermatogenlCCLeteCt ；I—WenLee,Long—ChingKuan,Chien—HungLHsien-AnPan,Chao—ChinHsu4,Yun
g—ChiehTsai,Pa0一LinKuo,
；Yen—NiTeng.
；D以,伽nD6fPf—c&IyncDZDI;affo,zfC,2gg己fV,以cDffgD厂
cfnP,zfn,z7D,zw,(以
；Dp以rPf0厂0易ffrc,zdGvcrJf0DGP,zP,ZDp口f,Zf,z以7DD,fw,z,(:乜
；3InstitHteofBas{cSciences.NC矗i口
；Abstract
；Aim:Tl0cOmp1etecomDrehensivehaplotVpeanalVsisOfSP26f0rbothfertileandinferti1emen.MethOds:Two
；hundredinfenilemenwithsevereo?gospermiaornon—obstructiveazoosperrniaweresub
jectedtosequenceana1ysis
；f0rtheentirecOdingsequencesOftheP26gene.TwOhundredmenwithprOvenfertiljtyweregenOtypedby
；primerextensionmethods.Al1ele/gen0tvpefrequencies,linkagedisequilibrium(LD)characteristicsandhaplotypesof
；fertilemenwerecomparedwithinfertilemen.Results:Theallelefrequencies0ffivesinglenuc1eOtidepOlymOr—
；phisms(370—37linsACA,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?DJD896—9D)
；Keyw0rds:singlenucleotidepolymorphism;26gene;spem1at0genicdefect;linkagedise
qui1ibrium
；Correspondenceto:Prof.Yen—Nireng,DeDartmentofBiote—
；chnol0gv.ChiaN.dnUniversicvofPhar|n『dcvandScience,60Erh—Jen
；Road,Sec.1.Pa0一An,Jen—TeHsiang,Tainan717,Tlaiwan,China. ；I1el:+886—6—266—49l1ext.2549Fax:+886—6—266—2135
；E—mai1:tengyennI1@yahO0.com.tw
；Received2008—02—20AcceDted2008—07—03
；896?
；1Intr0ducti0n
；Inflerti1ityaflfectsOnein10cOupleswOrldwide.In ；rOughlyhalf0fthesecOuples,themalefactOris ；imp1icated.Althou曲therearemanyfactorsthatcon—
；tributet0maleinferti1ity,thecausecannotbeidentified ；florthemajorityofinfenilemen【1].Inrecentyears,there
；?20O8,AsianJourna1ofAndrology.SIMMandSJTU.AllrightsreserVed
6):896—9O4 ；AsianJAndrot2o08:lo《
；hasbeenanintensivesearchf_0rgeneticcauses0fmale ；inferti1ity.ScreeningwithmarkersOnthelOngarmOf ；mehumanYchr0mOsOmehasdetectedYlamicrOdeletiOns ；in5%一15%0fmaleswithspermatogenicdefect.Itis ；believedthatmanygenesIocatedonthelong(q)annof ；theYchr0mOsOmearerequiredf0rspem1atOgenesis.This ；regiOn,bandinginql1.23OftheYchr0mOsOme,includes ；山eazoospermiafact0r(A2:F)locus,whichcontajnsagene ；orgenesthatarerequiredf0rnorn1alspematogenesis『2,
；3】.0thergeneticfactorsthathavebeenreponedtobe ；invO1vedinspermatogenicdeflectincludemutatiOnsat ；themitOchOndrialDNApOlymeraselOcus,apOlVmOr—
；phismOfthecytOchrOmeP4501A1gene,mutatiOnsOf ；thefollicle—stimuIatinghonnonereceptorgene(,,the ；deletedinazoospern1ia—likegene(),thesvnaptone—
；malcomplexprotein3gene(l,CP),humanBD眦,
；caseinkinase2alphagenes,dystrophiamVOtonicaprO—
；teinkinasegene(DP,DNApo1vmerasegamma
；(PDLG),methv1enetetrahvdmfl0latereductaser日FR)
；,dndf0llicle.scimulatinghorn1onereceptor()『4一l61.
；尸26appearst0beanattractivecandidatef0ra ；sterilegenecOnsideringitsuniqueexpressiOnpattern. ；E,26cOn1prisesasingleexOnandisIOcatedOnXq26.2. ；ItsmessengerRNA(mRNA)sequenceis2794bplong ；anditsproteinconsistsof913aminoacidsfGenbank: ；NM一031907.1).Itsmousehomologwasf0undtobe
；onlyexpressedinspermatogonia[17].己P26belongs
；toala唱efamilyofdeubiquitinacingenzymes(DUB)[18】.
；Deubiquitinationofmacr0moleculesbyDUB,including ；ubiquitinprOteases,canrescuemacrOmOleculesfrOm
specifjc,N—termina1. ；degradatiOnthrOughsubstrate—
；dependent,enzymaticreaction[19,201.Previousstudies ；haveaddressedthein1pOrtanceOftheubiquitinpath—
；wayduringmammalianflenilizatiOn,includingacmsOmaI ；functionandspematozoa—zonapeUucida(ZP)penetfa—
；tion[2l,22].Duringspematogenesis,deubiquitination ；enzVmeshavebeenshOwntobeinvOlvedintheregula—
；tionofproteinturnover(e.g.,replacementofhistones ；byprOtamine,germcellapOptOsis,mitOticpr0liferatiOn ；anddiff_erentiationofspennatogonialstemcells)『23—
；28].Severa1papershavebeenpub1ishedontheroleof ；26inmaleinfert订ity.Stouflfsf以,.『291analvzed
；patientswimvariOushistOlOgicalpattemsOfspe珊atO—
；genicdeflect(Sertolicel1.onlysyndromeandmaturation ；arrest)fOrthepresenceofmutationsin己P26_Thev
；foundsomeSertolice11.onlysyndrome(SCOS)patients ；hadtIlegenotype0f370—371insACA(rs3840975),49411>C ；andl423C>T(rs41299088)[29】.AstudybvPaduch
；Te|:+86-21.5492-2824;Fax:+86-21_5492?2825;shanghai,China ；一
；alsOprOvidedevidencelinking，,5P26mutatiOntOmale
；infenility[30].Althoughthesetwostudiessuggestedthat ；aspecincgeneticcluster(37O一37linsACA,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 ；assOcia【i0n0fthe370—371insACA.494T>CaJ1dl423C>T ；genocypewithmaleinfertility[14,29—32].Furtherstudies
；basedOnalar譬egrOupOfpatientswithdiversifledethnic ；backgrOundswillbevaluabletObrjngmOreinsightintO ；therO1eOf)26inmaleinfertilitv.
；1nthepresentstudy,wesetOuttOanalVzegenetic ；variantsandhaplotypesOfLP26f0rbOthfertileand ；in1.eItilemenbysequencea』1a1Vsisf0rtheentirecOdingre.
；giOnOfP26.W_efl0undsOme.5P26geneticvants ；andhaplOtypeswereOvert—transmittedandsomewere
；under—transmittedinpatientswithseverespematOgenic ；defectintheHannatjOnalitvin11aiwan.China.W_ealsO ；identifjedsomenOve1geneticvariants0rmutationsin ；patients.Ourresu1tsupportsimpOnantr0lesOfP26 ；inhumanspermatOgenesis.
；2MaterialsandmethOds
；2.jSHbjects
；ThesmdywasapprOvedbymeInstitutiOna1Review ；BOardOfNaciOna1ChengKungUniversityMedica1Centre ；(raiwaJ1,China).Flr0mJanuarV2001toDecember2005. ；200infenilemenpresentingwimsevereoligOzOOspe?nia
；(spermatozoacount<5×l06/mL)ornon—obstructive
；azoospemiaand200menwithpmvenfertilitv(control ；group)wereenrolled.AUofthecontrolsubiectshadfa—
；theredatleasttwOchildrenwithin5vearswithOutas—
；sistedreproductivetechnOlogies.Thepatemalr.elation—
；shipbetweencontrolsubiectsandtheiroffspringwas ；cOnfirmedusingtheAmpF/STR.PrOfiler—PluszVgOsitV
；deteminationsystem(App1iedBiosystems,FosterCitv, ；CA,USA).AUpatientsandcontrolsubjectsbelongedto ；theHannationaljtyinTaiwan,memajorethI1icgroupin ；China(makingupmorethan95%ofthisprovexalninati0n, ；atleasttwosemenanalyses,hormoneassays(1uinizing ；h0rmone[LH],f0uiclestimulatinghorn1one[FSH],pro一
；1actin【PRL】andtestosterone[T】),karyotypingandamo—
；leculartestfl0rchr0mosoma1deleti0ns.Patientssus—
；pectedtOhavenOn?Obsn1lctiVeazOOspeI111jawerleadVised ；tOundergobilateraltesticularbiOpsies.Serumlevels0f ；FSH,LH,PRLandTweremeasuredusingcOmmercial ；radiOimmunOassaVts:COat—A.COumFSHIRMACOat—
；A—COuntLHIRMA.COat—A—CountPRLIRMAand
；MMI?ITETotalTest0sterone(DiagnosticProducts,Los ；Angeles,CA,USA1.ChrI)mOsomeanalVsiswasperfbm1ed ；usingtheGTGmethod(G_bandingbvTrvpsin—Giemsa
；technique).Molecul，Lrana1vsisofY_chromosoma1dele—
；tiOnsinc1udedacOmbinatiOn0fl6gene—basedprimers,as
；describedpreviouslv『34].Patientswimanvidentifiable ；causeofma1einfertilitV,includingcongenitalbilatera1ab. ；sence0fvasdeference(CBAVD1,crvotorchidism, ；varicOcele,diabetesmellitusOrhVpertension,0rwithhis一
；亡0ry亡hatmayaftectspe兀na七ogenes1s(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/LTris—HCl(pH8.3),
；50mm0l/LKC1.0.1%TritonX一100.200LLm0l/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,口
；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吣
P26gene.ThenucleotidesequencesisaccordingtoAF285593.The ；1engmsOfpolymerasechainreactionpr0ductsize ；.
；8P:Amersham
；PharmaciaBiotech,Uppsala,Sweden)and2Uofexo—
；nuc1easeI(BMBiochemica,Mannheim,Germanv1ina ；10Il1LreactionvolumefOr1hat37.C.SNaPshOtmu1一
；tiplexPCRwasperformedina10LreactiOnvOlume ；containing1.25ULofSNaPshotMu1tiD1exReadvReac. ；tionMix(AppliedBi0systems),4ULofSAP/exonuclease ；I—treatedPCRproducts,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
；wasestimatedusingthepennutationtestPMPLUS『361.
；AssOciatiOnsbetweenhaplotVpesandmaleinfenilitvwere ；analVzedusinglinearregressionmodels.The1inkagedis—
；equilibrium(LD)coefficient(D’)betweeneachpairof
；sing1enucleotidepolymoT1)hisms(SNPs)wasca1culated ；usingtheldmaxprogramwithintheGOLDsoftware ；package[37].
；3Results
；.PD,vo,f,z,”,z,z7w以,z.(以
；ThecOdingsequencesOftheUP26genewereex—
；aminedbVsequencinganalysisflorthepresenceOfmuta. ；tiOnsofsequencevariantsininfertilemen.Thesequence ；fromGenBank(AF2855931wasusedasthereference ；seauenceandtheAofATGOfthestartcOdonwasnum—
；bered+1.IntotaI.sevengeneticvantsweI.eobsef弋,ed,
；includingOneinsertionvariant370—371insACA
；‘rel:+86-21?5492-2824;Fax:+86-21—5492?2825;shanghaichina ；(rs3840975),causingathreonineinseni0ninaminoacid
；pOsitiOnl21,andsixSNPs,494T>C.576G>A ；frs413O454O),ss62O279lC>T,1423C>T(rs41299O88), ；1737G>Aand2202A>C.A】10fthesegeneticvariants
；wereava订ableinthewebsitefhtcD://www-ncbi-nlm-nih. ；goV/)andpeer.reviewarticles『29—321.Onesubstituti0n,
；576G>AfEl92),doesnotaltertheaminoacidsequence: ；aglutaInicacidatpositionl92.TheotherfiveSNPwere ；pI.edictedtOc!leateaminOacidaltera【iOn: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—
；tutionshavenotbeenreported『301.Thesubstitutionof
；520T>Gand565T>GispredictedtOcausepremature ；stOpcOdOnatcOdonl75and189,respectivelV.HaplO. ；tVpeana1yses工nu1edOutthatpatientscaI1.yingthesevari—
；antsitesOccurredOnlV0nceintheinfertilitVgrOup. ；3.2Allelicnndgenopicquenciesinr1ileandin一
；rfe,”en
；Al1elicflrequenciesandoddsratiosf_0reachsequence ；variantOfP26areshOwninTlable2.0ftheseven ；variants.370—37linsACA.494T>C,576G>A.
；ss6202791C>Tandl737G>Aaresignificantlvassoci—
；atedwithspermatogenicdefect.The尸.va1uef0rthese
；fivevariantswaslessthan0.05andmeoddsratioswere ；largerthan1.FOrexample,theP—valuesfOr370—
；37linsACAand576G>Aarelessthan0.0001andthe ；ORare35.86and4.095fTable21.
；3.3Reproduct沁eeffectofthe7o一37nnsACA.494T>C
；1423C>T2eneticclsternnditsderivation ；TheancestralP26clusterf370—37linsACA.
；494T>C,1423C>T1anditsfirstderjvationonlvoccur ；inthegroupofjnfertilemen.IncOntrast,thesec0nd ；derivationonlvoccursinthegroufenilemen.A1lelefrequencieswerecOmparedbetweeni
nfenile
；menandcontrolsubjects.sNPs,singlenucleotidepolym0rphisms;0R,oddsratio;cI,cofidc
enrVal
；拍le3.Thegenotypefrequencyofthe37O一
37nnsACA,494T>Candl423C>Tcluster,itsfirstandsecondderiVatiVeinfenileand
；in，ilemen.Ana1yzedforstatjStjca1signmcancebyFisher’sexacttest.Accordinglo
】.)R,odd蔓ratjo 【37
；3.Ln足以ff易rM,fLD)Pfved疗,?nf;l尸
；(Table41showsthepairwiseLD(D’)betweendif_
；ferentalleles.FOrpatientswithspermatOgenicdefect, ；2202A>CwasfoundtobeincOmp1eteLDwith370—
；371insACA494T>C,576G>Aandl423C>TfD.=1). ；494T>ChadstrOngLDwith370—371insACAand
；1423C>T(D’=0.829and0.804respective1y).Forc0n一
；自r0lsub记cts,37O一371insACAw