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:(20002A213);(7032029):100034();():(NR3C1),(),NR3C1396764DNA,PCRNR3C129,(DHPLC)PCR,DNA170DNA,DHPLC12,DNA,3([198GA+200GA],[1374AG+IVSG268-IVSG263delAAAAAA+IVSH29CG+2382CT],[1896CT+2166CT+2430TC])2,(10.3%15.4%)(1.5%7.5%),2OR7.542.26NR3C112;2,;();;;,Analysisonassociationofglucocorticoidreceptorgenepolymorphismwithsteroid2resistanceinidiopathicnephroticsyndromeofchildrenYEJian2wei,DINGJie,HUANGJian2ping,CHENYan,YAOYong,XIAOHui2jie,YANGJi2yun,SHENYing,MENGQun.DepartmentofPediatrics,FirstHospital,PekingUniversity,Beijing100034,ChinaAbstractObjectiveThenephroticsyndromeisdefinedbyheavyproteinuria,edema,hypoalbuminemia,andhyperlipidemia.Idiopathicnephroticsyndrome(INS)mainlyoccursinchildren,whichisgenerallytreatedwithglucocorticoids.Themajorityofpatientsaresteroid2sensitive(SSINS)whilesteroid2resistanceoccursinasubsetofNSchildren(SRINS).AlthoughintensiveeffortshavebeenundertakentostudytheassociationsbetweenSRINSandrenalpathologicalchanges,pharmacokinetics,andtheGRdensityandbindingaffinity,themechanismsunderlyingsteroid2resistancearestillnotelucidatedentirely.Theauthorshypothesizedthatitmightbeassociatedwithpolymorphismsintheglucocorticoidreceptorgene(NR3C1).ThestudyaimedtoscreentheNR3C1geneforpolymorphismsingenomicDNAsamplesfromSRINS,SSINSchildrenandcontrolgroup,andtoanalyzetheassociationofthepolymorphismsintheNR3C1geneandSRINSofchildren.MethodsTotally39SRINSand67SSINSchildren(81malesand25femaleswiththemeanageof7years)wereinvolvedinthestudy.Umbilicalcordbloodof62normalneonatesandperipheralbloodof2healthyvolunteerswereselectedascontrols.GenomicDNAwasisolatedfromperipheralbloodlymphocytesofallsubjects.AlltheNR3C12codingexonsandintron2flankingportionswereamplifiedbypolymerasechainreaction(PCR).Forpolymorphismscreen,PCRproductswereanalyzedbydenaturinghighperformanceliquidchromatography(DHPLC).DNAfragmentswithaberrantelutionprofileswerere2amplifiedandsequenceddirectly.ResultsTwelveaberrantelutionprofileswereidentifiedwithDHPLCinSRINS,SSINSandcontrols.Amongthem,6previouslyreportedpolymorphismsand6novelpolymorphismswereconfirmedbysequencing(198GA,200GA,IVSD216GT,1896CT,2166CT,2430TC;novel,1206CT,1374AG,IVSG268-IVSG263delAAAAAA,2193TG,IVSH29C16620039419ChinJPediatr,September2003,Vol41,No.9©1995-2003TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.G,2382CT),and3groupsofSNPswereincompletelinkagedisequilibrium,whichresultedin3differenthaplotypes([198GA+200GA],[1374AG+IVSG268-IVSG263delAAAAAA+IVSH29CG+2382CT],[1896CT+2166CT+2430TC]).Thelasttwogenotypeswerefirstreported.Thegenotypefrequenciesofthe2novelhaplotypeswere10.3%vs1.5%inSRINSandSSINS,and15.4%vs7.5%inSRINSandSSINS,respectively.Otherpolymorphismswererelativelyraredetectablebothinpatientsandcontrols.ConclusionTwelvepolymorphismsintheNR3C1geneweredetectedwiththetechniqueofDHPLC,ofwhichsixpolymorphismswereidentifiedatthefirsttime.Twotypesofnewlyfoundhaplotypeswereassociatedwithsteroid2resistantidiopathicnephroticsyndromeofchildren,whichmightberesponsibleforsteroid2resistanceinpartialidiopathicnephroticsyndromeofchildren.KeywordsReceptors,glucocorticoid;Polymorphism(genetics);Resistance;Nephroticsyndrome;Chromatography,highpressureliquid(idiopathicnephroticsyndrome,INS),1/4INS[1][2][3],,(glucocorticoidreceptor,GR)[4],NR3C1(nuclearreceptorsubfamily3,groupC,member1),GR,NR3C1,GRNR3C1,,NR3C11.INS106,20011200212,54,50,281,25,7(215,117);(steroid2sensitiveINS,SSINS)67,(steroid2resistanceINS,SRINS)39,2001[5]2.200121062,2,,1.DNA:510ml,1%[6]DNA2.:Koper[7],NR3C1(29)(1)1NR3C1(53)(bp)()Exon221F:GATTCGGAGTTAACTAAAAGR:ATCCCAGGTCATTTCCCATC44653Exon222F:CCAAGCAGCGAAGACTTTTGR:TACCTGGGGACCCAGAAGAA45053Exon223F:CCACAGAGAAGGAGTTTCCAR:TTGCCTGACAGTAAACTGTG46253Exon224F:CCAGTAATGTAACACTGCCCCR:TTCGACCAGGGAAGTTCAGA35758Exon225F:AGTACCTCTGGAGGACAGATR:GTCCATTCTTAAGAAACAGG25053Exon3F:AGTTCACTGTGAGCATTCTGR:CGTGAGAAATAAAACCAAGT32453Exon4F:GACAGAAGGCTGTCCTTATAR:CATTATGCGTATCAAGCATA32353Exon5F:GAATAAACTGTGTAGCGCAGR:TAGTCCCCAGAACTAAGAGA47053Exon6F:GATCTTCTGAAGAGTGTTGCR:GGGAAAATGACACACATACA21853Exon7F:GAAAGTTCTCCAAAATTCTGR:TTGGTGTCACTTACTGTGCC46953Exon8F:GACACAGTGAGACCCTATCTR:CACCAACATCCACAAACTGG37153Exon9F:GGAATTCCAGTGAGATTGGTR:TATAAACCACATGTAGTGCG4285026620039419ChinJPediatr,September2003,Vol41,No.9©1995-2003TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.3.PCR:25lPCR,DNA50ng,012mol/L,dNTPs100mol/L,MgCl2115mmol/L,Taq(Perkin2Elmer)1125UPCR:955min,941min,115min(1),72115min,35,725min(GeneAmp2400,PE,)PCR1l110g/L,4.:PCR953min,-015/min65,;245.(DHPLC):2PCRWAVE(Transgenomic,)DHPLC[8]5l,100ngDNA,WAVEMakerñ(411142)DNA,019ml/minDHPLCNR3C1:(170)134(SSINS45,SRINS25,64),DHPLC(35,),PCR(DNA),2,DHPLC,DHPLC:36/106DNA(SSINS22,SRINS14),PCRDHPLC()10%6.DNA:DHPLC,,,ABI2PRISMTM377DNAPCR()NR3C11.:PCR,;PCR1l,2.DHPLCDNA:134DNA,DHPLC12,DNA,2193TG6:1206CT,1374AG,IVSG268-IVSG263delAAAAAA,2193TG,IVSH29CG,2382CT,6:198GA,200GA,IVSD216GT,1896CT,2166CT,2430TC(denDunnen[9]);,3([198GA+200GA],[1374AG+IVSG268-IVSG263delAAAAAA+IVSH29CG+2382CT],[1896CT+2166CT+2430TC]),2,126,::[198GA+200GA],