Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure respons...

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Title:
Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide
Series Title:
Journal of Translational Medicine
Physical Description:
Book
Language:
English
Creator:
Duarte, Julio D
Zineh, Issam
Burkley, Ben
Gong, Yan
Langaee, Taimour Y.
Turner, Stephen T.
Chapman, Arlene B.
Boerwinkle, Eric
Gums, John G.
Cooper-DeHoff, Rhonda M.
Beitelshees, Amber L.
Bailey, Kent R.
Fillingim, Roger B.
Kone, Bruce C.
Johnson, Julie A.
Publisher:
BioMed Central
Publication Date:

Subjects

Subjects / Keywords:
Pharmacogenomics
Pharmacogenetics
hydrochlorothiazide
hypertension
blood pressure
DOT1L
SIRT1
MLLT3
SGK1
histone methylation

Notes

Abstract:
Background: Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCa subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure. Methods: We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant. Results: In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed. Conclusions: Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.

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Source Institution:
University of Florida
Holding Location:
University of Florida
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All rights reserved by the source institution.
Resource Identifier:
doi - 10.1186/1479-5876-10-56
System ID:
AA00012397:00001

Full Text

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Supplementary Table 1. SNPs genotyped in SGK1 DOT1L SIRT1 and MLLT3 gene regions SGK1 rs2302061 rs3740051 rs10811350 rs2383138 rs7039016 rs9402571 rs8113174 rs10997866 rs10811352 rs2519454 rs7041793 rs7752937 rs8113528 rs12778366 rs10811358 rs2775265 rs7042746 rs1763527 rs7256735 rs752578 rs10811362 rs2780839 rs7046522 rs1057293 rs12459350 rs35671182 rs10811372 rs2780840 rs7047471 rs7755303 rs12462556 MLLT3 rs10811376 rs2780841 rs7874260 rs6911375 rs12611107 rs10964538 rs10964542 rs3764797 rs787490 9 rs3813344 rs12981806 rs4977421 rs10964545 rs3780826 rs10964539 rs2758152 rs12983678 rs10964597 rs10964547 rs3780829 rs16938091 rs2758151 rs1558118 rs6475454 rs10964552 rs3780830 rs3824576 rs2758150 rs886449 rs10121168 rs10964555 rs6475462 rs6475455 rs12663728 SIRT1 rs4977256 rs10964562 rs668703 rs10964600 rs1743966 rs34639502 rs7043548 rs10964566 rs7018627 rs10964605 DOT1L rs1966188 rs1016962 rs10964593 rs7020320 rs10964620 rs7251881 rs2234975 rs10738563 rs16938109 rs7020494 rs10964636 rs1003531 rs35671182 rs10757132 rs17177890 rs7020850 rs1111766 rs2074552 rs3758391 rs10811359 rs1929170 rs7023694 rs11999881 rs2240130 rs7069102 rs10757140 rs2073853 rs7025895 rs12003167 rs2269879 rs7096385 rs10811346 rs2188229 rs7029191 rs12004010 rs2269882 rs3 6107781 rs10811348 rs2188230 rs7029476 rs12004720 rs2286329 rs35706870 rs12685798 rs2301550 rs7036342 rs12005512

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MLLT3 rs12380857 rs1633699 rs3904577 rs623828 rs1411727 rs12006253 rs12554291 rs16925395 rs3934325 rs6475431 rs7866601 rs12335409 rs1268 2679 rs16938035 rs4246840 rs6475442 rs7866741 rs12337265 rs12683597 rs16938039 rs4448374 rs6475444 rs7868378 rs12342870 rs1291635 rs16938042 rs4977257 rs6475448 rs7870252 rs12344417 rs13289029 rs16938054 rs4977425 rs6475451 rs7873127 rs12346032 rs13299 141 rs16938062 rs4977427 rs734477 rs7874260 rs12350051 rs1360378 rs16938065 rs4977430 rs734772 rs7037941 rs12377027 rs1537069 rs3892039 rs4621895 rs7854241 rs12006034 rs12379192 rs1548375 rs3897271 rs5021140 rs7858673


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epdcx:valueString Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide
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Abstract
Background
Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure.
Methods
We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant.
Results
In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed.
Conclusions
Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.
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Duarte, Julio D
Zineh, Issam
Burkley, Ben
Gong, Yan
Langaee, Taimour Y
Turner, Stephen T
Chapman, Arlene B
Boerwinkle, Eric
Gums, John G
Cooper-DeHoff, Rhonda M
Beitelshees, Amber L
Bailey, Kent R
Fillingim, Roger B
Kone, Bruce C
Johnson, Julie A
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RESEARCH OpenAccessEffectsofgeneticvariationinH3K79methylation regulatorygenesonclinicalbloodpressureand bloodpressureresponsetohydrochlorothiazideJulioDDuarte1,2*,IssamZineh1,3,BenBurkley1,YanGong1,TaimourYLangaee1,StephenTTurner4, ArleneBChapman5,EricBoerwinkle6,JohnGGums1,RhondaMCooper-DeHoff1,AmberLBeitelshees7, KentRBailey8,RogerBFillingim9,BruceCKone10,11andJulieAJohnson1AbstractBackground: Nearlyone-thirdoftheUnitedStatesadultpopulationsuffersfromhypertension.Hydrochlorothiazide (HCTZ),oneofthemostcommonlyusedmedicationstotreathypertension,hasvariableefficacy.Therenal epithelialsodiumchannel(ENaC)providesamechanismforfine-tuningsodiumexcretion,andisamajorregulator ofbloodpressurehomeostasis. DOT1L,MLLT3,SIRT1,and SGK1 encodegenesinapathwaythatcontrols methylationofthehistoneH3globulardomainatlysine79(H3K79),therebymodulatingexpressionoftheENaC a subunit.Thisstudyaimedtodeterminetheroleofvariationintheseregulatorygenesonbloodpressureresponse toHCTZ,andsecondarily,untreatedbloodpressure. Methods: WeinvestigatedassociationsbetweengeneticvariationsinthiscandidatepathwayandHCTZblood pressureresponseintwoseparatehypertensivecohorts(clinicaltrials.govNCT00246519andNCT00005520).Ina secondary,exploratoryanalysis,wemeasuredassociationsbetweenthesesamegeneticvariationsanduntreated bloodpressure.Associationsweremeasuredbylinearregression,withonlyassociationswith P 0.01inone cohortandreplicationby P 0.05intheothercohortconsideredsignificant. Results: Inonecohort,apolymorphismin DOT1L (rs2269879)wasstronglyassociatedwithgreatersystolic( P = 0.0002)anddiastolic( P =0.0016)bloodpressureresponsetohydrochlorothiazideinCaucasians.However,this associationwasnotreplicatedintheothercohort.Whenuntreatedbloodpressurelevelswereanalyzed,wefound directionallysimilarassociationsbetweenapolymorphismin MLLT3 (rs12350051)andgreateruntreatedsystolic( P <0.01inbothcohorts)anddiastolic( P <0.05inbothcohorts)bloodpressurelevelsinbothcohorts.However, whenfurtherreplicationwasattemptedinathirdhypertensivecohortandinsmaller,normotensivesamples, significantassociationswerenotobserved. Conclusions: Ourdatasuggestpolymorphismsin DOT1L,MLLT3,SIRT1 ,and SGK1 arenotlikelyassociatedwith bloodpressureresponsetoHCTZ.However,apossibilityexiststhatrs2269879in DOT1L couldbeassociatedwith HCTZresponseinCaucasians.Additionally,exploratoryanalysessuggestrs12350051in MLLT3 maybeassociated withuntreatedbloodpressureinAfrican-Americans.Replicationeffortsareneededtoverifyrolesforthese polymorphismsinhumanbloodpressureregulation. Keywords: Pharmacogenomics,Pharmacogenetics,hydrochlorothiazide,hypertension,bloodpressure, DOT1L SIRT1, MLLT3 SGK1 ,histonemethylation *Correspondence:juliod@uic.edu1CenterforPharmacogenomicsandDepartmentofPharmacotherapyand TranslationalResearch,UniversityofFlorida,Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleDuarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 2012Duarteetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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BackgroundHydrochlorothiazide(HCTZ)isoneofthemostcommonlyprescribedantihypertensivedrugintheUS,with approximately118millionprescriptionsdispensedin 2010,eitheraloneorcombinedwithanotherantihypertensive[1,2].HCTZandotherthiazidediureticsare recommendedbycurrenthypertensiontreatmentguidelinesintheUnitedStatesasfirst-linetreatmentfor mostpatientswithuncomplicatedessentialhypertension,andarestronglyrecommendedforallpatients requiringtwoormoreantihypertensivesforbloodpressurecontrol[3]. Patientresponsetothiazidesvarieswidely,withdifferentialresponsesbetweenandwithinraces[4].Because ofthis,clinicianshavedifficultypredictingwhich patientswillachievegoodbloodpressureresponsewith thiazidetreatment.Pharmacogeneticstudiescannot onlyhelpexplainthisvariabilityindrugresponse,but canalsoprovidefurtherinformationonthemechanistic basisofthiazides. Thiazidesachievetheirini tialdiureticactionbypreventingrenalsodiumreabsorptionviainhibitionofthe Na+/Cl-cotransporter(NCC)inthedistalconvoluted tubule[5-7].However,themechanismbywhichthiazideschronicallylowerbloodpressureremainspoorly understood.Alsoinvolvedinsodiumreabsorptionisthe distally-locatedepithelialsodiumchannel(ENaC). AlthoughENaCcontributestothereabsorptionof approximately5%oftotalfilteredsodiumload,itprovidesafine-tuningmechanismforsodium,bodyfluid volume,and,ultimately,bloodpressurehomeostasis[8]. BecauseENaCisdistaltoNCCinthenephron,inhibitionofNCC,suchasoccurswiththiazidetherapy, resultsinalteredionconcentrationsinthetubular lumen,particularlyincreasedsodiumconcentrationsat ENaC-expressedregions.Consequently,theclinical effectofvariationsinENaCexpressioncouldbemagnifiedinthiazide-treatedpatients.Infact,evidencealready existsshowingassociationbetweenvariationin NEDD4L ,ageneinvolvedinENaCregulation,and bloodpressureresponsetodiuretics[9].Inaddition, pharmacogeneticresearchhaspreviouslyimplicated ENaCinthiazideresponse,aspolymorphismsin SCNN1G (whichencodestheENaC g subunit)havebeen associatedwithHCTZresponse[10,11].Inaddition, ENaCisexpressedinthevascularsmoothmuscleand mayalsoplaysomeroleinregulatingvascularresistance [12]. Anepigeneticpathwaywasrecentlydiscoveredthat regulatesENaC a expressioninthekidneybymethylationofhistoneproteinH3atlysine79(H3K79)[13-15]. Atthecenterofthispathwayisacomplexincludingthe methyltransferaseDisruptoroftelomericsilencing1 (Dot1)andDNA-bindingproteinALL1fusedgenefrom chromosome9(Af9)[15].Af9(inhumans,encodedby MLLT3 )bindstotheENaC a promoterandlocalizes Dot1fordi-andtri-methylationatH3Lys79,whichis associatedwithENaC a generepression[16].This repressioncanbepreventedbySerum/glucocorticoidinducedkinase(encodedby SGK1 ),whichdisruptsthe assemblyoftheAf9/Dot1complex[14].EvidenceindicatesthatthedeacetylaseSirtuin-1(encodedby SIRT1 ) canalsoformacomplexwithDot1todecreaseENaC a expression.However,themechanismforthisinteraction isstillunclear[17]. Wehypothesizedthatgeneticvariationinthisepigeneticregulatorypathwayplaysaroleintheantihypertensiveeffectsofthiazides,throughitsregulationof ENaC.Secondarily,wehypothesizedthatvariationin thispathwayaffectshumanbloodpressurehomeostasis. Totestthefirsthypothesis,weevaluatedwhetherpolymorphismsin DOT1L,MLLT3,SIRT1 ,and SGK1 affect clinicalbloodpressureresponsetoHCTZinwelldefinedclinicalcohorts.Totestthesecond,weassessed associationsofthesepolymorphismswithuntreated clinicalbloodpressuresasanexploratoryanalysis.MethodsParticipantsStudyparticipantsarosefromthePharmacogenomic EvaluationofAntihypertensiveResponses(PEAR;clinicaltrials.gov#NCT00246519)andtheGeneticEpidemiologyofResponsestoAntihypertensives(GERA; clinicaltrials.gov#NCT00005520).Bothstudieswere approvedbytheinstitutionalreviewboardsateachcenterwheretheywereconducted,andallsubjectsprovidedinformed,writtenconsentbeforebeingscreened forenrollment. PEARwasamulti-centerclinicaltrialexaminingthe roleofgeneticvariabilityonbloodpressureresponseto HCTZand/oratenolol[18].Menandwomenofany racebetweentheagesof17and65withessentialhypertension(clinicdiastolicbloodpressure 90mmHg, 110mmHg)wererecruitedtoparticipate.Afterafourweekantihypertensivewashout,includedparticipants wererandomizedtoreceiveeitherHCTZ12.5mgdaily oratenolol50mgdaily,withmostreceivingdoseescalationsto25mgand100mg,respectivelyforbloodpressuregreaterthan120/70mmHg.Toassureafour-week washoutsufficientlyerasedbloodpressureeffectsofany previousantihypertensivetreatments,weconfirmedthat post-washoutbloodpressurelevelsinpreviously-treated participantswerenearlyidenticaltothosewhohad neverreceivedantihypertensivemedication.Afternine weeks,bloodpressureresponsewasassessedandfor bloodpressuregreaterthan120/70mmHg,theotherDuarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page2of9

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studydrugwasaddedwithanotherdosetitrationand responseassessmentaftersixtonineweeks.Participants werenotgivensodiumrestrictions,butwerecounseled tomaintainconsistentdietaryintakes.Theprimary responsephenotypewashomebloodpressure,which participantswererequiredtotakeintriplicateuponrisingandbeforeretiringatleastfiveofsevendaysprior totheirbloodpressureassessmentvisit.Thecoefficients ofvariancewereapproximately7%forbothsystolicand diastolicmeasurements.Thefirst297self-reportedCaucasiansandAfrican-AmericanstocompleteHCTZ monotherapy(PEARHCTZ)areincludedinthisreport. IntheseparticipantsrandomizedtoHCTZ,greaterthan 90%receiveddoseincreasesto25mg.ForCaucasian andAfrican-Americansrandomizedtoatenolol(PEAR ATEN),374participantswereincludedforuntreated bloodpressureanalyses. GERAwasatwo-centerclinicaltrialdesignedto determinewhetherpolymorphismsinrenin-angiotensinaldosteronesystemgeneswerepredictiveoftheblood pressureresponsetoHCTZ[19].Briefly,participants wereself-reportednon-HispanicCaucasiansandAfrican-Americansbetweentheagesof30and59whohad bloodpressuregreaterthan140/90mmHgoraprevious diagnosisofessentialhypertensionandcurrentantihypertensiveprescription.Previouslytreatedhypertensives hadallantihypertensivedrugsdiscontinuedforfour weeks,thenwereassessedforbloodpressureatbaseline. Ifdiastolicbloodpressureremainedbetween90and110 mmHg,participantswereincludedinthestudyand treatedwithHCTZ25mgdailyforfourweeks.Participantswerecounseledtostabilizesodiumintakeatabout 1mmol/kg/daybeginningatwashoutandcontinuing throughoutthestudyperiod.Allbloodpressuremeasurementsweremadeinaclinicsetting. Twonormotensivesampleswerealsousedinan attempttoreplicateuntreatedbloodpressureassociationsfoundinGERAandPEARAfrican-Americans. ThefirstsampledrawnfromwastheEthnicPainSensitivitytrial,asinglecente rstudydesignedtoexamine ethnicdifferencesinpainsen sitivity[20].206healthy menandwomenbetweentheagesof18-53,representingmultipleethnicgroups,wereenrolledattheUniversityofFlorida.Meanrestingsystolicanddiastolicblood pressure,meanarterialpressure,andmeanrestingheart rateweremeasuredineachparticipant.Forreplication purposes,88availableAfrican-Americanparticipants wereincludedinanalyses.Thesecondsamplewasa hypertensiondatabasefrom theUniversityofFlorida thatenrolled730participants,bothhypertensiveand normotensive.Normotensiveparticipants(systolicblood below140mmHganddiastolicbloodpressurebelow 90mmHg)wereneverdiagnosedwithhighbloodpressureandhadnoparents,siblings,orchildrenwithhigh bloodpressurediagnosedbeforeage65.Forreplication purposes,121availableAfrican-Americannormotensives wereincludedinanalyses.Selectionofpolymorphismsanddeterminationof genotypesAtagSNPapproachwasusedtoassuremaximumcoverageofcommonSNPsineachcandidategeneregion.A tagSNPselectiontoolusingtheMultipop-TagSelect algorithm[21]providedonlinebytheGenomeVariation Serverhttp://gvs.gs.washin gton.edu/GVS/wasqueried forthegeneticregionsof DOT1L,MLLT3,SIRT1 ,and SGK1 intheHapMapYRI(AfricanAncestry)andCEPH (EuropeanAncestry)populations.SNPswithaminor allelefrequencylessthan5%wereexcluded.TheGenomeVariationServerprovidedacomprehensivelistof 144SNPsmeetingthesecriteriawhichtaggedallfour generegions. Tomorepreciselyinvestigatethestrongestcandidate SNPs,putativefunctionalSNPs(pfSNPs)wereaddedto thelistsgeneratedbytheGenomeVariationServer. Thesewerecomputed insilico bytwoseparateprograms,Pupasuite[22]andFastSNP[23].FromthiscombinedlistofpfSNPs,thosewithaminorallelefrequency greaterthan0.05foreitherA fricanorEuropeanancestrywereaddedtothealreadyestablishedtagSNPlist. Theresultingfinallistcontained180SNPstogenotype (Additionalfile1;TableS1). GenotypesinHCTZ-treatedGERAandPEARparticipantsweredeterminedusingacustomGoldenGate AssayfortheBeadXpressReaderSystem(IlluminaInc., SanDiego,CA).Genotypingwascarriedoutaccording tothemanufacturer sprotocol.Rawdataconversion andqualitycontrolwerecompletedinGenomeStudio software(IlluminaInc.,SanDiego,CA).Sampleswere excludediftheirgenotypecallratewasbelow90%.IndividualSNPswereexcludedfromanalysisiftheywere monomorphicinourcohorts,theircallfrequencieswere below75%,ortheirGenTrainscoreswerelessthan0.3. Foruntreatedbloodpressurereplicationanalyses,genotypesweredeterminedusingTaqmanSNPGenotyping AssaysandtheTaqman7900HTRealTimePCRSystem (AppliedBiosystems,FosterCity,CA)accordingtothe manufacturer sprotocol.StatisticalmethodsAssociationsbetweengenotypeandbloodpressure responsestoHCTZweretestedbylinearregression afteradjustmentforcovariates,includinggender,age, anduntreatedbloodpressure.Associationsbetween untreatedsystolicbloodpre ssureanddiastolicblood pressureweretestedinthesamemannerasdescribed forHCTZresponse,exceptcovariateadjustments includedonlygenderandage.StatisticalanalyseswereDuarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page3of9

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completedinJMPGenomics4andSAS9.2(SASInstitute,Cary,NC). BecauseofthelargenumberofSNPstested,adjustmentsformultiplecomparisonswerenecessarytoavoid falsepositives.ABonferro nicorrection,assuming180 independenttests,wouldrequirea P 0.00028forsignificance(0.05/180=0.00028).However,theSNPsanalyzedarestrongbiologicalcandidates,anddonot representindependentte stsduetolinkagedisequilibrium.Additionally,replicationoffindingsisessentialin geneticassociationstudies.Inouranalyses,SNPswere consideredsignificantift heyassociatedwithHCTZ bloodpressureresponseineitherGERAorPEARwith P 0.01,replicatedwith P 0.05intheotherstudy group,andhadmatchingdirectionsofeffect.Withthis replicationstrategy,theoverall P -valuethresholdforsignificanceis0.010.050.5(formatchingdirectionof effect)=0.00025,whichthereforemeetstheBonferroni criterion,assimilarlyjustifiedpreviously[24]. Asaqualitycontrolprocedure,Hardy-Weinbergequilibriumwastestedvia c2analysisseparatelybyraceand studygroup.ThoseSNPswithHardy-Weinbergequilibrium P -valueslessthanaBonferroni-corrected0.00028 wereflaggedandanalyzedundersuspicionofgenotypingerror.ResultsStudycohortsBaselinecharacteristicsweresimilaramongbothstudy cohorts(Table1).Bothno rmotensivereplication cohortsvariedfromthestudycohortsinracialmake-up anduntreatedbloodpressure(bydesign),andalsovariedslightlyfromallothergroupsinageandBMI.In PEAR,HCTZtreatmentd ecreasedbloodpressureby approximately12/7mmHginAfricanAmericansand8/ 4mmHginCaucasians.InGERA,HCTZtreatment decreasedbloodpressurebyapproximately18/9mmHg inAfricanAmericansand11/6mmHginCaucasians. GERA-GeneticEpidemiologyofResponsestoAntihypertensivestrial,PEARHCTZ-Randomizedto HCTZinthePharmacogenomicEvaluationofAntihypertensiveResponsestrial,PEARATEN-Randomized toatenololinthePharmacogenomicEvaluationofAntihypertensiveResponsestrial,EPSAApopulation-AfricanAmericansfromtheEthicPainSensitivitytrial, HTNDBAANormotensives-NormotensiveAfrican AmericansfromaUniversityofFloridahypertension database,BP-bloodpressure,N/A-NotAvailable. Outofatotal180SNPsgenotypedwiththeGoldenGateassay,sixSNPswereexcludedforhavingaSNP callfrequencylessthan75%oraGenTrainscoreless than0.3.AnadditionaltwoSNPsfailedthesequality controlsinGERAonly,sowereanalyzedexclusivelyin PEAR.InGERA,19participantsampleswereexcluded fromanalysisbecauseoflowgenotypecallrates(< 90%).InPEAR,oneparticipantsamplewasexcluded fromanalysesbecauseoflowcallrates.Taqmangenotypingofrs12350051wascompletedinreplicationsampleswithapproximately7%d uplication,revealing98% concordance.Bothrs2269879andrs12350051werein Hardy-Weinbergequilibriuminallgroupsanalyzed.Associationofcandidategenevariationwith hydrochlorothiazideresponseAManhattanplotof-log P -valuesforgeneticassociationswithbloodpressureresponseinGERAandPEAR HCTZ(Figure1)indicatenoSNPassociationswere replicatedaccordingtopredeterminedcriteriaforsignificance.The DOT1L SNPrs2269879cametheclosestto ourcriteriaforasignificantassociation.ThevariantT Table1BaselinedemographicsofGERAandPEARclinicalcohortsGERA PEAR HCTZ PEAR ATEN HTNDBAANormotensivesEPSAAPopulation N 583 297 374 121 88 Age(y) 48.26.750.38.948.69.245.17.1 22.75.1 Sex(%female)47.2 51.2 57.7 52.9 51.1 Race(%) Caucasian 50.3 57.1 58.9 0 0 African-American49.7 42.9 41.1 100 100 BMI(kg/m2) 31.36.030.85.631.06.230.16.8 25.24.3 MeanClinicBP Systolic(mmHg)146.014.4152.613.0151.412.4118.39.1 122.58.9 Diastolic(mmHg)96.15.498.76.198.36.276.76.3 68.45.9 MeanHomeBP Systolic(mmHg)N/A 146.711.2145.29.8N/A N/A Diastolic(mmHg)N/A 94.36.293.56.0N/A N/AValuesarelistedasmeanSDDuarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page4of9

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alleleatrs2269879wasassociatedwitha5.5mmHg greatermeansystolic( P =0.0002)anda3.5mmHg greatermeandiastolic(P =0.0016)responsetoHCTZ inPEARHCTZCaucasians(Figure2),whilenoassociationwasfoundinPEARHCTZAfrican-Americans. BloodpressureresponseassociationsinGERACaucasianspossessedthesamedirectionofeffectasthosein PEARHCTZ(Figure2),butwerenotstatisticallysignificant(systolic P =0.73,diastolic P =0.29).Because GERA P -valuesdidnotmeetourpredeterminedcriteria forsignificance,thisSNPwasnotconsideredareplicatedassociation.Associationofcandidategenevariationwithuntreated bloodpressureInapre-definedsecondaryanalysis,weobservedassociationsbetweenbaseline,untreatedbloodpressureand rs12350051(in MLLT3 )genotypeinbothPEARHCTZ (systolic P =0.005,diastolic P =0.049)andGERAAfrican-Americans(systolic P =0.001,diastolic P =0.010) atthepre-definedthresholdsforassociationandreplication(Figure3).African-AmericanswiththevariantC alleleatrs12350051hadmeanuntreatedsystolicblood pressures6.4mmHghigherinPEARHCTZand6.9 mmHghigherinGERAthanthosewiththeT/Tgenotype(Figure4).Meanuntreateddiastolicbloodpressureswere2.8mmHghigherinPEARHCTZand1.8 mmHghigherinGERA(Figure4).Noassociationwas observedinCaucasians. Sincethisdiscoveredassociationwaswithuntreated bloodpressure,baselinedatacouldbeusedfromAfrican-AmericansrandomizedtoatenololinPEARATEN asafurtherreplication.WhiletheuntreatedbloodpressuresofPEARATENAfrican-Americansfolloweda similartrendtoGERAandPEARHCTZAfrican-Americans,themeansbetweengenotypegroupsdifferedby lessthan1mmHgforbothsystolicanddiastolicblood pressuresandthedifferencewasnotstatisticallysignificant(Figure5).Furtherasso ciationtestingintheAfrican-Americannormotensivereplicationsamples revealednoassociationswithuntreatedbloodpressure (datanotshown).DiscussionWeinvestigatedassociationsinacandidatepathwayfor HCTZbloodpressureresponse,whichcontrolsexpressionofENaC.Ourstudyshowednoassociationbetween commonvariationin DOT1L,MLLT3,SIRT1,orSGK1 andbloodpressureresponsestoHCTZthatmetour pre-definedcriteriaforsignificance.Inanexploratory analysis,wealsoevaluatedtheroleofSNPsinthispathwayonuntreatedbloodpressureandidentified rs12350051in MLLT3 asbeingassociatedwithbaseline bloodpressureinbothGERAandPEARAfrican-Americans.However,inanotherPEARandtwonormotensive cohorts,thisassociationdidnotreplicate. Thestrongestpharmacogeneticassociationwith HCTZresponsewaswithrs2269879inDOT1L,and Figure1 AssociationofcandidateSNPswithbloodpressureresponsetoHCTZinGERAandPEARHCTZcohorts .Thelowerline represents P =0.05,whilethehigherlinerepresents P =0.01.B-AfricanAmerican,W-Caucasian,BL-Untreated,SBP-systolicbloodpressure response,DBP-diastolicbloodpressureresponse. Figure2 Bloodpressurerespons etoHCTZbyrs2269879 genotypeinGERAandPEARinCaucasiansamples .Adjustedfor age,gender,anduntreatedbloodpressure.Errorbarsindicate standarderror.SBP-Systolicbloodpressure;DBP-diastolicblood pressure; P 0.001; P 0.01. Duarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page5of9

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wasonlyobservedinPEARCaucasians.Resultswith systolicanddiastolicresponseinGERAweredirectionallyconsistent,butnonsignificant.Locatedinintron7, rs2269879waschosenforgenotypingasatagSNP. UponreviewintheHapMapCEPHpopulation,theSNP wasfoundtobeinperfectlinkagewithrs8113528(r2= 1.0),inintron3.FastSNPshowedthevariantAalleleat rs8113528createsapossiblebindingsiteforp300,transcriptionalco-activatorthatfunctionsasahistoneacetyltransferase.Additionally,UCSCGenomeBrowser http://genome.ucsc.edu/indicatesrs8113528existsinan areasurroundedbymoderatehistoneacetylation. Becausetheassociationonlymetthesignificance thresholdinPEAR,anddidnotreplicateinGERA,we cannotruleoutthatthisisachancefinding.Onereason foralackofreplicationinGERAmaybethattheeffect ofthisSNPcanonlybedetectedusinghomeblood pressure.PEARwastheonlystudywiththehomeblood pressurephenotypeavailable.Wedecided apriorito useitastheresponsephenotypeinPEARbecausehome bloodpressureisamoreaccu ratephenotype,ashome bloodpressurepredictscardi ovascularriskbetterthan officebloodpressure[25,26].Inaddition,wepreviously foundambulatorybloodpressuremeasurement,another potentiallybetterpredictorofcardiovascularrisk,correlatedwithhomebloodpressuremorethanwithoffice bloodpressureinasubsetofPEARparticipants[27]. PEARhomebloodpressureentrieswereaveragesof multiplemeasurementsspanningatleastfivedays,thus theylikelygiveabetterestimateofparticipants actual bloodpressures.Homebloodpressureisalsoamore precisephenotype,asevidencedbythesmallerstandard deviationsinhomesystolicbloodpressuremeasurementsweobservedinPEARcomparedwithofficemeasurements(Table1systolic: P <0.001,diastolic: P = 0.689).Officemeasurements,theonlybloodpressure phenotypesavailableinGERA,maynotpossesshigh enoughfidelitytodetectthisassociationwith rs2269879.Supportingthistheory,weobservedsimilar, Figure3 AssociationofcandidateSNPswithuntreatedbloodpressureinGERAandPEARHCTZcohorts .Thelowerlinerepresents P = 0.05,whilethehigherlinerepresents P =0.01.B-AfricanAmerican,W-Caucasian,BL-Untreated,SBP-systolicbloodpressure,DBP-diastolic bloodpressure. Figure4 Untreatedbloodpressurebyrs12350051genotypein GERAandPEARHCTZAfrican-Americansamples .Adjustedfor ageandgender.Errorbarsindicatestandarderror.SBP-Systolic bloodpressure;DBP-diastolicbloodpressure; P (trend) 0.01;* P (trend) 0.05. Figure5 Untreatedbloodpressurebyrs12350051genotypein PEARATENAfrican-Americansample .Adjustedforageand gender.Errorbarsindicatestandarderror.SBP-Systolicblood pressure;DBP-diastolicbloodpressure. Duarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page6of9

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butmuchweakerassociationswithofficebloodpressure responseinPEAR.Perhapsifanotherlargehypertensive cohort,prospectivelytr eatedwithHCTZ,becomes obtainableforanalysisofhomebloodpressure responses,theassociationwefoundcouldbetested againforreplication. ThelackofassociationwefoundwithHCTZresponse suggeststhatgenotypingpolymorphismsinthispathway wouldlikelynothelppredictpatientresponsetothiazidediuretics.ThelikelihoodthatcommonSNPassociationsweremissedwith DOT1L,MLLT3,SIRT1,SGK1 andbloodpressureassociationsislow.TagSNPswithin 5000basesofeachcandidategenewereselectedtotry todetectanypossible cis -regulatoryregions.Greateffort wasspentonidentifyingpfSNPs insilico foreachcandidategene,whichwerenotrequiredtobeinthepredefinedgeneregionfortagSNPdevelopment.However, onlySNPswithaminorallelefrequencyof0.05were consideredforgenotyping,soourstudycannotruleout veryrareSNPsinthecandidategeneswithlargeeffect sizesaffectingbloodpressureresponse.Additionally,our datadonotruleoutwhetherornotthispathwayplays anyrolethiazideresponse.IfHCTZdidhavesome smalleffectonH3K79methylation,redundancyin ENaC a regulation[28]couldconceivablyovercomethe changesinH3K79methylationandleavebehindno measurablechangeinpatientbloodpressureresponse. LittleisknownabouttheeffectofthishistoneH3K79 methylationpathwayonbloodpressureregulationin humans,soexploratoryanalysestestingassociationsin untreatedbloodpressurephenotypescouldalsoprovide valuableinformation.TheSNPthatassociatedandreplicatedwithuntreatedbloodpressurewasrs12350051in MLLT3 .ItwaschosenasatagSNP,andislocatedin intron2,withnolinkagetoanyknownfunctionalSNPs. Insilico ,rs12350051wasnotobservedinanyknown miRNAsequences,transcrip tionfactorbindingsites, exonicsplicesites,spliceenhancer,orsilencer sequences.Becausethesamebloodpressureassociation wasnotseeninCaucasians,onepossibilitycouldbe thatthisSNPisinhighlinkagedisequilibriumwithan undiscoveredfunctionalpolymorphisminAfricanAmericans. Onewouldexpectusingapatientpopulationwitha widerangeofbloodpressurestobethebestmethodto detectgeneticassociationswithuntreatedbloodpressure.SothefactthatuntreatedbloodpressureassociationswereseeninPEARandGERAissomewhat surprising,asthesestudiesenrolledhypertensivesspanningarelativelysmallbloodpressurerange.Thiswas oneofthereasonsweattemptedtoreplicatethesefindingsinnormotensivebloodpressurerangesnotrepresentedinPEARandGERA.However,thenormotensive groupsalsohadanarrowbloodpressurerange.Thefact thatnoreplicationwasobservedinnormotensivescould bebecauseofthisnarrowbloodpressurerange,thefact thattheywereyounger,thedifferencesinstudyprotocols,leadingtodifferencesinbloodpressuremeasurementprecision,orthesamplesizesweretoosmalland lackedthepowertodetecttheeffectweobservedinthe largerhypertensivecohorts.Anotherpossibilityisthat perhapstheeffectofthisSNPiseasiertodetectoronly exertsaneffectwithhigherbloodpressures. Ourfindingsarenotthefirsttodetectassociations betweenthisH3K79methylationpathwayandblood pressureregulation. Dot1 conditionalknockoutmice wereshowntoexhibitsaltse nsitivehypertension[29]. Conversely,micenullfor Af17 ,whichhasbeenshown tocompetewithAf9forDot1bindingattheENaCa promoter,[30]exhibitrenalsaltwastingandhypotensionwithdiminishedrenalH3K79methylationand renalENaC a geneexpression[31].Also,Sirtuin-1deacetylationofendothelialnitricoxidesynthase[32]has beensuggestedasapossiblemechanismfortheblood pressurereductionseendur ingcaloricrestriction,a well-knowninducerofSirtuin-1[33].ConclusionsInconclusion,associationslikelydonotexistbetween commonvariationin MLLT3,SIRT1,orSGK1 and bloodpressureresponsestoHCTZinhypertensives. OneSNPin DOT1L (rs2269879)couldplayarolein HCTZresponse,butrequiresfurtherinvestigationto replicatetheassociationfoundinPEAR.Additionally, rs12350051in MLLT3 wasassociatedwithuntreated bloodpressureinAfrican-Americanhypertensiveindividuals.Becausethiswasanexploratoryanalysis,and theassociationwasnotreplicatedinsmallernormotensivesamples,questionsremainastowhetherthis polymorphismisinvolvedin thebloodpressureregulationofnormotensives,andthemechanismbywhich rs12350051exertsaneffectonbloodpressure. Furtherstudyinclinicalpopulationswithbroader bloodpressurerangeswouldhelpanswerthese questions.AdditionalmaterialAdditionalfile1:SupplementaryTable1.SNPsgenotypedin SGK1, DOT1L,SIRT1 ,and MLLT3 generegions. Abbreviations ATEN:atenolol;HCTZ:hydrochlorothiazide;H3K79:histoneH3lysine79;NCC: sodium-chloridecotransporter;ENaC:epithelialsodiumchannel;ENaC : epithelialsodiumchannel subunit;SNP:singlenucleotidepolymorphism; pfSNP:putativefunctionalSNP;GERA:GeneticEpidemiologyofResponsesto Antihypertensives;PEAR:PharmacogenomicEvaluationofAntihypertensive Responses.Duarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page7of9

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Acknowledgements GERA:ThisworkwassupportedbyNIHgrantsHL74735,HL53335,andthe MayoFoundation. PEAR:ThisworkissupportedbyagrantfromtheNationalInstitutesof Health(Bethesda,MD),grantU01GM074492,fundedaspartofthe PharmacogeneticsResearchNetwork.Thisworkisalsosupportedbythe followinggrantsfromtheNIHNationalCenterforResearchResources:grant M01RR00082andUL1RR029890totheUniversityofFlorida,grantsUL1 RR025008andM01RR00039toEmoryUniversity,andUL1RR024150to MayoClinic. ThisresearchwasalsosupportedbyNIHgrantsR01DK075065(B.C.K.),R01 HL064691(J.A.J.),R01NS42754(R.L.F.),K23HL091120(A.L.B.),andT32 DK007518(J.D.D.).WhileDr.ZinehisanemployeeoftheFDA,noofficial FDAendorsementofthismanuscriptisintendednorshouldbeinferred. Authordetails1CenterforPharmacogenomicsandDepartmentofPharmacotherapyand TranslationalResearch,UniversityofFlorida,Gainesville,FL32610,USA.2DepartmentofPharmacyPractice,UniversityofIllinoisatChicago,Chicago, IL60612,USA.3OfficeofClinicalPharmacology,OfficeofTranslational Sciences-CDER,U.S.FoodandDrugAdministration,SilverSpring,MD 20993,USA.4DivisionofNephrologyandHypertension,MayoClinic, Rochester,MN55905,USA.5RenalDivision,DepartmentofMedicine,Emory University,Atlanta,GA30322,USA.6HumanGeneticsCenterandInstituteof MolecularMedicine,UniversityofTexasHealthScienceCenter,Houston,TX 77030,USA.7DivisionofEndocrinology,DiabetesandNutrition,Department ofMedicine,UniversityofMaryland,Baltimore,MD21201,USA.8Divisionof BiomedicalStatisticsandInformatics,MayoClinic,Rochester,MN55905,USA.9DepartmentofCommunityDentistryandBehavioralScience,Universityof Florida,Gainesville,FL32610,USA.10DivisionofNephrology,Hypertension& RenalTransplantation,UniversityofFlorida,Gainesville,FL32610,USA.11DivisionofRenalDiseasesandHypertension,UniversityofTexasHealth ScienceCenter,Houston,TX77030,USA. Authors contributions JDDparticipatedinthestudydesign,participatedingenotyping,performed statisticalanalysesanddraftedthemanuscript.IZparticipatedinthedesign ofthestudyandhelpedtodraftthemanuscript.BBparticipatedinsample processingandgenotypingandhelpedtodraftthemanuscript.KRBassisted withstatisticalanalysis,STTandALBparticipatedinstudycoordinationand helpedtodraftthemanuscript.YG,TYL,ABC,EB,JGG,RMC,RBFparticipated instudycoordination.BCKconceivedthestudy,participatedinitsdesign, andhelpedtodraftthemanuscript.JAJconceivedthestudy,participatedin itsdesign,andhelpedtodraftthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:14November2011Accepted:22March2012 Published:22March2012 References1. 2010Top200genericdrugsbytotalprescriptions. [http://drugtopics. modernmedicine.com/drugtopics/data/articlestandard//drugtopics/252011/ 727243/article.pdf]. 2. 2010Top200brandeddrugsbytotalprescriptions. 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NucleicAcidsRes 2006, 34 :W621-W625. 23.YuanHY,ChiouJJ,TsengWH,LiuCH,LiuCK,LinYJ,WangHH,YaoA, ChenYT,HsuCN: FASTSNP:analwaysup-to-dateandextendableservice forSNPfunctionanalysisandprioritization. NucleicAcidsRes 2006, 34 : W635-W641. 24.McPhersonR,PertsemlidisA,KavaslarN,StewartA,RobertsR,CoxDR, HindsDA,PennacchioLA,Tybjaerg-HansenA,FolsomAR, etal : Acommon alleleonchromosome9associatedwithcoronaryheartdisease. Science 2007, 316 :1488-1491. 25.NiiranenTJ,HanninenMR,JohanssonJ,ReunanenA,JulaAM: Homemeasuredbloodpressureisastrongerpredictorofcardiovascularrisk thanofficebloodpressure:theFinn-Homestudy. Hypertension 2010, 55 :1346-1351. 26.StergiouGS,ArgyrakiKK,MoyssakisI,MastorantonakisSE,AchimastosAD, KaramanosVG,RoussiasLG: Homebloodpressureisasreliableas ambulatorybloodpressureinpredictingtarget-organdamagein hypertension. AmJHypertens 2007, 20 :616-621.Duarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page8of9

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27.BeitelsheesAL,GongY,BaileyKR,TurnerST,ChapmanAB,SchwartzGL, GumsJG,BoerwinkleE,JohnsonJA: Comparisonofoffice,ambulatory, andhomebloodpressureantihypertensiveresponsetoatenololand hydrochlorthiazide. JClinHypertens(Greenwich) 2010, 12 :14-21. 28.PearceD,KleymanTR: Salt,sodiumchannels,andSGK1. JClinInvest 2007, 117 :592-595. 29.ZhangW: ConditionaltargetingofhistoneH3Lys79methyltransferase Dot1geneinmice. JAmSocNephrol 2008, 19 :380A. 30.ReisenauerMR,AndersonM,HuangL,ZhangZ,ZhouQ,KoneBC, MorrisAP,LesageGD,DryerSE,ZhangW: AF17competeswithAF9for bindingtoDot1atoup-regulatetranscriptionofepithelialNa+channel JBiolChem 2009, 284 :35659-35669. 31.ChenL,WuH,PochynyukOM,ReisenauerMR,ZhangZ,HuangL,ZaikaOL, MamenkoM,ZhangW,ZhouQ, etal : Af17deficiencyincreasessodium excretionanddecreasesbloodpressure. JAmSocNephrol 2011, 22 :1076-1086. 32.MattagajasinghI,KimCS,NaqviA,YamamoriT,HoffmanTA,JungSB, DeRiccoJ,KasunoK,IraniK: SIRT1promotesendothelium-dependent vascularrelaxationbyactivatingendothelialnitricoxidesynthase. Proc NatlAcadSciUSA 2007, 104 :14855-14860. 33.PotenteM,DimmelerS: EmergingrolesofSIRT1invascularendothelial homeostasis. CellCycle 2008, 7 :2117-2122.doi:10.1186/1479-5876-10-56 Citethisarticleas: Duarte etal .: EffectsofgeneticvariationinH3K79 methylationregulatorygenesonclinicalbloodpressureandblood pressureresponsetohydrochlorothiazide. JournalofTranslational Medicine 2012 10 :56. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Duarte etal JournalofTranslationalMedicine 2012, 10 :56 http://www.translational-medicine.com/content/10/1/56 Page9of9

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Supplementary Table 1. SNPs genotyped in SGK1 DOT1L SIRT1 and MLLT3 gene regions SGK1 rs2302061 rs3740051 rs10811350 rs2383138 rs7039016 rs9402571 rs8113174 rs10997866 rs10811352 rs2519454 rs7041793 rs7752937 rs8113528 rs12778366 rs10811358 rs2775265 rs7042746 rs1763527 rs7256735 rs752578 rs10811362 rs2780839 rs7046522 rs1057293 rs12459350 rs35671182 rs10811372 rs2780840 rs7047471 rs7755303 rs12462556 MLLT3 rs10811376 rs2780841 rs7874260 rs6911375 rs12611107 rs10964538 rs10964542 rs3764797 rs787490 9 rs3813344 rs12981806 rs4977421 rs10964545 rs3780826 rs10964539 rs2758152 rs12983678 rs10964597 rs10964547 rs3780829 rs16938091 rs2758151 rs1558118 rs6475454 rs10964552 rs3780830 rs3824576 rs2758150 rs886449 rs10121168 rs10964555 rs6475462 rs6475455 rs12663728 SIRT1 rs4977256 rs10964562 rs668703 rs10964600 rs1743966 rs34639502 rs7043548 rs10964566 rs7018627 rs10964605 DOT1L rs1966188 rs1016962 rs10964593 rs7020320 rs10964620 rs7251881 rs2234975 rs10738563 rs16938109 rs7020494 rs10964636 rs1003531 rs35671182 rs10757132 rs17177890 rs7020850 rs1111766 rs2074552 rs3758391 rs10811359 rs1929170 rs7023694 rs11999881 rs2240130 rs7069102 rs10757140 rs2073853 rs7025895 rs12003167 rs2269879 rs7096385 rs10811346 rs2188229 rs7029191 rs12004010 rs2269882 rs3 6107781 rs10811348 rs2188230 rs7029476 rs12004720 rs2286329 rs35706870 rs12685798 rs2301550 rs7036342 rs12005512

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MLLT3 rs12380857 rs1633699 rs3904577 rs623828 rs1411727 rs12006253 rs12554291 rs16925395 rs3934325 rs6475431 rs7866601 rs12335409 rs1268 2679 rs16938035 rs4246840 rs6475442 rs7866741 rs12337265 rs12683597 rs16938039 rs4448374 rs6475444 rs7868378 rs12342870 rs1291635 rs16938042 rs4977257 rs6475448 rs7870252 rs12344417 rs13289029 rs16938054 rs4977425 rs6475451 rs7873127 rs12346032 rs13299 141 rs16938062 rs4977427 rs734477 rs7874260 rs12350051 rs1360378 rs16938065 rs4977430 rs734772 rs7037941 rs12377027 rs1537069 rs3892039 rs4621895 rs7854241 rs12006034 rs12379192 rs1548375 rs3897271 rs5021140 rs7858673


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ui 1479-5876-10-56
ji 1479-5876
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dochead Research
bibl
title p Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide
aug
au id A1 ca yes snm Duartemi Dfnm Julioinsr iid I1 I2 email juliod@uic.edu
A2 ZinehIssamI3 Issam.Zineh@fda.hhs.gov
A3 BurkleyBenburkley@cop.ufl.edu
A4 GongYangong@cop.ufl.edu
A5 LangaeeYTaimourlangaee@cop.ufl.edu
A6 TurnerTStephenI4 sturner@mayo.edu
A7 ChapmanBArleneI5 Arlene_Chapman@emory.org
A8 BoerwinkleEricI6 Eric.Boerwinkle@uth.tmc.edu
A9 GumsGJohnjgums@UFL.EDU
A10 Cooper-DeHoffMRhondadehoff@cop.ufl.edu
A11 BeitelsheesLAmberI7 abeitels@medicine.umaryland.edu
A12 BaileyRKentI8 baileyk@mayo.edu
A13 FillingimBRogerI9 rfilling@ufl.edu
A14 KoneCBruceI10 I11 Bruce.C.Kone@uth.tmc.edu
A15 JohnsonAJuliejohnson@cop.ufl.edu
insg
ins Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, IL 60612, USA
Office of Clinical Pharmacology, Office of Translational Sciences CDER, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 55905, USA
Renal Division, Department of Medicine, Emory University, Atlanta, GA 30322, USA
Human Genetics Center and Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA
Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland, Baltimore, MD 21201, USA
Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN 55905, USA
Department of Community Dentistry and Behavioral Science, University of Florida, Gainesville, FL 32610, USA
Division of Nephrology, Hypertension & Renal Transplantation, University of Florida, Gainesville, FL 32610, USA
Division of Renal Diseases and Hypertension, University of Texas Health Science Center, Houston, TX 77030, USA
source Journal of Translational Medicine
section Clinical translationissn 1479-5876
pubdate 2012
volume 10
issue 1
fpage 56
url http://www.translational-medicine.com/content/10/1/56
xrefbib pubidlist pubid idtype doi 10.1186/1479-5876-10-56pmpid 22440088
history rec date day 14month 11year 2011acc 2232012pub 2232012cpyrt 2012collab Duarte et al; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg kwd PharmacogenomicsPharmacogeneticshydrochlorothiazidehypertensionblood pressureit DOT1LSIRT1MLLT3SGK1histone methylation
abs
sec st Abstract
Background
Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure.
Methods
We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant.
Results
In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed.
Conclusions
Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.
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Background
Hydrochlorothiazide (HCTZ) is one of the most commonly prescribed antihypertensive drug in the US, with approximately 118 million prescriptions dispensed in 2010, either alone or combined with another antihypertensive abbrgrp abbr bid B1 1B2 2. HCTZ and other thiazide diuretics are recommended by current hypertension treatment guidelines in the United States as first-line treatment for most patients with uncomplicated essential hypertension, and are strongly recommended for all patients requiring two or more antihypertensives for blood pressure control B3 3.
Patient response to thiazides varies widely, with differential responses between and within races B4 4. Because of this, clinicians have difficulty predicting which patients will achieve good blood pressure response with thiazide treatment. Pharmacogenetic studies can not only help explain this variability in drug response, but can also provide further information on the mechanistic basis of thiazides.
Thiazides achieve their initial diuretic action by preventing renal sodium reabsorption via inhibition of the Nasup +/Cl- cotransporter (NCC) in the distal convoluted tubule B5 5B6 6B7 7. However, the mechanism by which thiazides chronically lower blood pressure remains poorly understood. Also involved in sodium reabsorption is the distally-located epithelial sodium channel (ENaC). Although ENaC contributes to the reabsorption of approximately 5% of total filtered sodium load, it provides a fine-tuning mechanism for sodium, body fluid volume, and, ultimately, blood pressure homeostasis B8 8. Because ENaC is distal to NCC in the nephron, inhibition of NCC, such as occurs with thiazide therapy, results in altered ion concentrations in the tubular lumen, particularly increased sodium concentrations at ENaC-expressed regions. Consequently, the clinical effect of variations in ENaC expression could be magnified in thiazide-treated patients. In fact, evidence already exists showing association between variation in NEDD4L, a gene involved in ENaC regulation, and blood pressure response to diuretics B9 9. In addition, pharmacogenetic research has previously implicated ENaC in thiazide response, as polymorphisms in SCNN1G (which encodes the ENaCγ subunit) have been associated with HCTZ response B10 10B11 11. In addition, ENaC is expressed in the vascular smooth muscle and may also play some role in regulating vascular resistance B12 12.
An epigenetic pathway was recently discovered that regulates ENaCα expression in the kidney by methylation of histone protein H3 at lysine 79 (H3K79) B13 13B14 14B15 15. At the center of this pathway is a complex including the methyltransferase Disruptor of telomeric silencing 1 (Dot1) and DNA-binding protein ALL1 fused gene from chromosome 9 (Af9) 15. Af9 (in humans, encoded by MLLT3) binds to the ENaCα promoter and localizes Dot1 for di- and tri-methylation at H3 Lys79, which is associated with ENaCα gene repression B16 16. This repression can be prevented by Serum/glucocorticoid-induced kinase (encoded by SGK1), which disrupts the assembly of the Af9/Dot1 complex 14. Evidence indicates that the deacetylase Sirtuin-1 (encoded by SIRT1) can also form a complex with Dot1 to decrease ENaCα expression. However, the mechanism for this interaction is still unclear B17 17.
We hypothesized that genetic variation in this epigenetic regulatory pathway plays a role in the antihypertensive effects of thiazides, through its regulation of ENaC. Secondarily, we hypothesized that variation in this pathway affects human blood pressure homeostasis. To test the first hypothesis, we evaluated whether polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 affect clinical blood pressure response to HCTZ in well-defined clinical cohorts. To test the second, we assessed associations of these polymorphisms with untreated clinical blood pressures as an exploratory analysis.
Methods
Participants
Study participants arose from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR; clinicaltrials.gov #NCT00246519) and the Genetic Epidemiology of Responses to Antihypertensives (GERA; clinicaltrials.gov #NCT00005520). Both studies were approved by the institutional review boards at each center where they were conducted, and all subjects provided informed, written consent before being screened for enrollment.
PEAR was a multi-center clinical trial examining the role of genetic variability on blood pressure response to HCTZ and/or atenolol B18 18. Men and women of any race between the ages of 17 and 65 with essential hypertension (clinic diastolic blood pressure ≥ 90 mmHg, ≤ 110 mmHg) were recruited to participate. After a four-week antihypertensive washout, included participants were randomized to receive either HCTZ 12.5 mg daily or atenolol 50 mg daily, with most receiving dose escalations to 25 mg and 100 mg, respectively for blood pressure greater than 120/70 mmHg. To assure a four-week washout sufficiently erased blood pressure effects of any previous antihypertensive treatments, we confirmed that post-washout blood pressure levels in previously-treated participants were nearly identical to those who had never received antihypertensive medication. After nine weeks, blood pressure response was assessed and for blood pressure greater than 120/70 mmHg, the other study drug was added with another dose titration and response assessment after six to nine weeks. Participants were not given sodium restrictions, but were counseled to maintain consistent dietary intakes. The primary response phenotype was home blood pressure, which participants were required to take in triplicate upon rising and before retiring at least five of seven days prior to their blood pressure assessment visit. The coefficients of variance were approximately 7% for both systolic and diastolic measurements. The first 297 self-reported Caucasians and African-Americans to complete HCTZ monotherapy (PEAR HCTZ) are included in this report. In these participants randomized to HCTZ, greater than 90% received dose increases to 25 mg. For Caucasian and African-Americans randomized to atenolol (PEAR ATEN), 374 participants were included for untreated blood pressure analyses.
GERA was a two-center clinical trial designed to determine whether polymorphisms in renin-angiotensin-aldosterone system genes were predictive of the blood pressure response to HCTZ B19 19. Briefly, participants were self-reported non-Hispanic Caucasians and African-Americans between the ages of 30 and 59 who had blood pressure greater than 140/90 mmHg or a previous diagnosis of essential hypertension and current antihypertensive prescription. Previously treated hypertensives had all antihypertensive drugs discontinued for four weeks, then were assessed for blood pressure at baseline. If diastolic blood pressure remained between 90 and 110 mmHg, participants were included in the study and treated with HCTZ 25 mg daily for four weeks. Participants were counseled to stabilize sodium intake at about 1 mmol/kg/day beginning at washout and continuing throughout the study period. All blood pressure measurements were made in a clinic setting.
Two normotensive samples were also used in an attempt to replicate untreated blood pressure associations found in GERA and PEAR African-Americans. The first sample drawn from was the Ethnic Pain Sensitivity trial, a single center study designed to examine ethnic differences in pain sensitivity B20 20. 206 healthy men and women between the ages of 18-53, representing multiple ethnic groups, were enrolled at the University of Florida. Mean resting systolic and diastolic blood pressure, mean arterial pressure, and mean resting heart rate were measured in each participant. For replication purposes, 88 available African-American participants were included in analyses. The second sample was a hypertension database from the University of Florida that enrolled 730 participants, both hypertensive and normotensive. Normotensive participants (systolic blood below 140 mm Hg and diastolic blood pressure below 90 mm Hg) were never diagnosed with high blood pressure and had no parents, siblings, or children with high blood pressure diagnosed before age 65. For replication purposes, 121 available African-American normotensives were included in analyses.
Selection of polymorphisms and determination of genotypes
A tagSNP approach was used to assure maximum coverage of common SNPs in each candidate gene region. A tagSNP selection tool using the Multipop-TagSelect algorithm B21 21 provided online by the Genome Variation Server http://gvs.gs.washington.edu/GVS/ was queried for the genetic regions of DOT1L, MLLT3, SIRT1, and SGK1 in the HapMap YRI (African Ancestry) and CEPH (European Ancestry) populations. SNPs with a minor allele frequency less than 5% were excluded. The Genome Variation Server provided a comprehensive list of 144 SNPs meeting these criteria which tagged all four gene regions.
To more precisely investigate the strongest candidate SNPs, putative functional SNPs (pfSNPs) were added to the lists generated by the Genome Variation Server. These were computed in silico by two separate programs, Pupasuite B22 22 and FastSNP B23 23. From this combined list of pfSNPs, those with a minor allele frequency greater than 0.05 for either African or European ancestry were added to the already established tagSNP list. The resulting final list contained 180 SNPs to genotype (Additional file supplr sid S1 1; Table S1).
suppl
Additional file 1
text b Supplementary Table 1. SNPs genotyped in SGK1, DOT1L, SIRT1, and MLLT3 gene regions.
file name 1479-5876-10-56-S1.DOC
Click here for file
Genotypes in HCTZ-treated GERA and PEAR participants were determined using a custom GoldenGate Assay for the BeadXpress Reader System (Illumina Inc., San Diego, CA). Genotyping was carried out according to the manufacturer's protocol. Raw data conversion and quality control were completed in GenomeStudio software (Illumina Inc., San Diego, CA). Samples were excluded if their genotype call rate was below 90%. Individual SNPs were excluded from analysis if they were monomorphic in our cohorts, their call frequencies were below 75%, or their GenTrain scores were less than 0.3.
For untreated blood pressure replication analyses, genotypes were determined using Taqman SNP Genotyping Assays and the Taqman 7900HT Real Time PCR System (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.
Statistical methods
Associations between genotype and blood pressure responses to HCTZ were tested by linear regression after adjustment for covariates, including gender, age, and untreated blood pressure. Associations between untreated systolic blood pressure and diastolic blood pressure were tested in the same manner as described for HCTZ response, except covariate adjustments included only gender and age. Statistical analyses were completed in JMP Genomics 4 and SAS 9.2 (SAS Institute, Cary, NC).
Because of the large number of SNPs tested, adjustments for multiple comparisons were necessary to avoid false positives. A Bonferroni correction, assuming 180 independent tests, would require a P ≤ 0.00028 for significance (0.05/180 = 0.00028). However, the SNPs analyzed are strong biological candidates, and do not represent independent tests due to linkage disequilibrium. Additionally, replication of findings is essential in genetic association studies. In our analyses, SNPs were considered significant if they associated with HCTZ blood pressure response in either GERA or PEAR with P ≤ 0.01, replicated with P ≤ 0.05 in the other study group, and had matching directions of effect. With this replication strategy, the overall P-value threshold for significance is 0.01 × 0.05 × 0.5 (for matching direction of effect) = 0.00025, which therefore meets the Bonferroni criterion, as similarly justified previously B24 24.
As a quality control procedure, Hardy-Weinberg equilibrium was tested via χ2 analysis separately by race and study group. Those SNPs with Hardy-Weinberg equilibrium P-values less than a Bonferroni-corrected 0.00028 were flagged and analyzed under suspicion of genotyping error.
Results
Study cohorts
Baseline characteristics were similar among both study cohorts (Table tblr tid T1 1). Both normotensive replication cohorts varied from the study cohorts in racial make-up and untreated blood pressure (by design), and also varied slightly from all other groups in age and BMI. In PEAR, HCTZ treatment decreased blood pressure by approximately 12/7 mmHg in African Americans and 8/4 mmHg in Caucasians. In GERA, HCTZ treatment decreased blood pressure by approximately 18/9 mmHg in African Americans and 11/6 mmHg in Caucasians.
tbl Table 1caption Baseline demographics of GERA and PEAR clinical cohortstblbdy cols 6
r
c
left
GERA
PEAR
HCTZ
PEAR
ATEN
HTNDB AA Normotensives
EPS AA Population
cspan
hr
N
583
297
374
121
88
Age (y)
48.2 ± 6.7
50.3 ± 8.9
48.6 ± 9.2
45.1 ± 7.1
22.7 ± 5.1
Sex (% female)
47.2
51.2
57.7
52.9
51.1
Race (%)
Caucasian
50.3
57.1
58.9
0
0
African-American
49.7
42.9
41.1
100
100
BMI (kg/m2)
31.3 ± 6.0
30.8 ± 5.6
31.0 ± 6.2
30.1 ± 6.8
25.2 ± 4.3
Mean Clinic BP
Systolic (mmHg)
146.0 ± 14.4
152.6 ± 13.0
151.4 ± 12.4
118.3 ± 9.1
122.5 ± 8.9
Diastolic (mmHg)
96.1 ± 5.4
98.7 ± 6.1
98.3 ± 6.2
76.7 ± 6.3
68.4 ± 5.9
Mean Home BP
Systolic (mmHg)
N/A
146.7 ± 11.2
145.2 ± 9.8
N/A
N/A
Diastolic (mmHg)
N/A
94.3 ± 6.2
93.5 ± 6.0
N/A
N/A
tblfn
Values are listed as mean ± SD
GERA Genetic Epidemiology of Responses to Antihypertensives trial, PEAR HCTZ Randomized to HCTZ in the Pharmacogenomic Evaluation of Antihypertensive Responses trial, PEAR ATEN Randomized to atenolol in the Pharmacogenomic Evaluation of Antihypertensive Responses trial, EPS AA population African Americans from the Ethic Pain Sensitivity trial, HTNDB AA Normotensives Normotensive African Americans from a University of Florida hypertension database, BP blood pressure, N/A Not Available.
Out of a total 180 SNPs genotyped with the GoldenGate assay, six SNPs were excluded for having a SNP call frequency less than 75% or a GenTrain score less than 0.3. An additional two SNPs failed these quality controls in GERA only, so were analyzed exclusively in PEAR. In GERA, 19 participant samples were excluded from analysis because of low genotype call rates (< 90%). In PEAR, one participant sample was excluded from analyses because of low call rates. Taqman genotyping of rs12350051 was completed in replication samples with approximately 7% duplication, revealing 98% concordance. Both rs2269879 and rs12350051 were in Hardy-Weinberg equilibrium in all groups analyzed.
Association of candidate gene variation with hydrochlorothiazide response
A Manhattan plot of -log P-values for genetic associations with blood pressure response in GERA and PEAR HCTZ (Figure figr fid F1 1) indicate no SNP associations were replicated according to predetermined criteria for significance. The DOT1L SNP rs2269879 came the closest to our criteria for a significant association. The variant T allele at rs2269879 was associated with a 5.5 mmHg greater mean systolic (P = 0.0002) and a 3.5 mmHg greater mean diastolic (P = 0.0016) response to HCTZ in PEAR HCTZ Caucasians (Figure F2 2), while no association was found in PEAR HCTZ African-Americans. Blood pressure response associations in GERA Caucasians possessed the same direction of effect as those in PEAR HCTZ (Figure 2), but were not statistically significant (systolic P = 0.73, diastolic P = 0.29). Because GERA P-values did not meet our predetermined criteria for significance, this SNP was not considered a replicated association.
fig Figure 1Association of candidate SNPs with blood pressure response to HCTZ in GERA and PEAR HCTZ cohorts
Association of candidate SNPs with blood pressure response to HCTZ in GERA and PEAR HCTZ cohorts. The lower line represents P = 0.05, while the higher line represents P = 0.01. B African American, W Caucasian, BL Untreated, SBP systolic blood pressure response, DBP diastolic blood pressure response.
graphic 1479-5876-10-56-1 hint_layout double
Figure 2Blood pressure response to HCTZ by rs2269879 genotype in GERA and PEAR in Caucasian samples
Blood pressure response to HCTZ by rs2269879 genotype in GERA and PEAR in Caucasian samples. Adjusted for age, gender, and untreated blood pressure. Error bars indicate standard error. SBP Systolic blood pressure; DBP diastolic blood pressure; ‡ P ≤ 0.001; † P ≤ 0.01.
1479-5876-10-56-2 single
Association of candidate gene variation with untreated blood pressure
In a pre-defined secondary analysis, we observed associations between baseline, untreated blood pressure and rs12350051 (in MLLT3) genotype in both PEAR HCTZ (systolic P = 0.005, diastolic P = 0.049) and GERA African-Americans (systolic P = 0.001, diastolic P = 0.010) at the pre-defined thresholds for association and replication (Figure F3 3). African-Americans with the variant C allele at rs12350051 had mean untreated systolic blood pressures 6.4 mmHg higher in PEAR HCTZ and 6.9 mmHg higher in GERA than those with the T/T genotype (Figure F4 4). Mean untreated diastolic blood pressures were 2.8 mmHg higher in PEAR HCTZ and 1.8 mmHg higher in GERA (Figure 4). No association was observed in Caucasians.
Figure 3Association of candidate SNPs with untreated blood pressure in GERA and PEAR HCTZ cohorts
Association of candidate SNPs with untreated blood pressure in GERA and PEAR HCTZ cohorts. The lower line represents P = 0.05, while the higher line represents P = 0.01. B African American, W Caucasian, BL Untreated, SBP systolic blood pressure, DBP diastolic blood pressure.
1479-5876-10-56-3
Figure 4Untreated blood pressure by rs12350051 genotype in GERA and PEAR HCTZ African-American samples
Untreated blood pressure by rs12350051 genotype in GERA and PEAR HCTZ African-American samples. Adjusted for age and gender. Error bars indicate standard error. SBP Systolic blood pressure; DBP diastolic blood pressure; † P (trend) ≤ 0.01; P (trend) ≤ 0.05.
1479-5876-10-56-4
Since this discovered association was with untreated blood pressure, baseline data could be used from African-Americans randomized to atenolol in PEAR ATEN as a further replication. While the untreated blood pressures of PEAR ATEN African-Americans followed a similar trend to GERA and PEAR HCTZ African-Americans, the means between genotype groups differed by less than 1 mmHg for both systolic and diastolic blood pressures and the difference was not statistically significant (Figure F5 5). Further association testing in the African-American normotensive replication samples revealed no associations with untreated blood pressure (data not shown).
Figure 5Untreated blood pressure by rs12350051 genotype in PEAR ATEN African-American sample
Untreated blood pressure by rs12350051 genotype in PEAR ATEN African-American sample. Adjusted for age and gender. Error bars indicate standard error. SBP Systolic blood pressure; DBP diastolic blood pressure.
1479-5876-10-56-5
Discussion
We investigated associations in a candidate pathway for HCTZ blood pressure response, which controls expression of ENaC. Our study showed no association between common variation in DOT1L, MLLT3, SIRT1, or SGK1 and blood pressure responses to HCTZ that met our pre-defined criteria for significance. In an exploratory analysis, we also evaluated the role of SNPs in this pathway on untreated blood pressure and identified rs12350051 in MLLT3 as being associated with baseline blood pressure in both GERA and PEAR African-Americans. However, in another PEAR and two normotensive cohorts, this association did not replicate.
The strongest pharmacogenetic association with HCTZ response was with rs2269879 in DOT1L, and was only observed in PEAR Caucasians. Results with systolic and diastolic response in GERA were directionally consistent, but nonsignificant. Located in intron 7, rs2269879 was chosen for genotyping as a tagSNP. Upon review in the HapMap CEPH population, the SNP was found to be in perfect linkage with rs8113528 (r2 = 1.0), in intron 3. FastSNP showed the variant A allele at rs8113528 creates a possible binding site for p300, transcriptional co-activator that functions as a histone acetyltransferase. Additionally, UCSC Genome Browser http://genome.ucsc.edu/ indicates rs8113528 exists in an area surrounded by moderate histone acetylation.
Because the association only met the significance threshold in PEAR, and did not replicate in GERA, we cannot rule out that this is a chance finding. One reason for a lack of replication in GERA may be that the effect of this SNP can only be detected using home blood pressure. PEAR was the only study with the home blood pressure phenotype available. We decided a priori to use it as the response phenotype in PEAR because home blood pressure is a more accurate phenotype, as home blood pressure predicts cardiovascular risk better than office blood pressure B25 25B26 26. In addition, we previously found ambulatory blood pressure measurement, another potentially better predictor of cardiovascular risk, correlated with home blood pressure more than with office blood pressure in a subset of PEAR participants B27 27. PEAR home blood pressure entries were averages of multiple measurements spanning at least five days, thus they likely give a better estimate of participants' actual blood pressures. Home blood pressure is also a more precise phenotype, as evidenced by the smaller standard deviations in home systolic blood pressure measurements we observed in PEAR compared with office measurements (Table 1 systolic: P < 0.001, diastolic: P = 0.689). Office measurements, the only blood pressure phenotypes available in GERA, may not possess high enough fidelity to detect this association with rs2269879. Supporting this theory, we observed similar, but much weaker associations with office blood pressure response in PEAR. Perhaps if another large hypertensive cohort, prospectively treated with HCTZ, becomes obtainable for analysis of home blood pressure responses, the association we found could be tested again for replication.
The lack of association we found with HCTZ response suggests that genotyping polymorphisms in this pathway would likely not help predict patient response to thiazide diuretics. The likelihood that common SNP associations were missed with DOT1L, MLLT3, SIRT1, SGK1 and blood pressure associations is low. TagSNPs within 5000 bases of each candidate gene were selected to try to detect any possible cis-regulatory regions. Great effort was spent on identifying pfSNPs in silico for each candidate gene, which were not required to be in the pre-defined gene region for tagSNP development. However, only SNPs with a minor allele frequency of 0.05 were considered for genotyping, so our study cannot rule out very rare SNPs in the candidate genes with large effect sizes affecting blood pressure response. Additionally, our data do not rule out whether or not this pathway plays any role thiazide response. If HCTZ did have some small effect on H3K79 methylation, redundancy in ENaCα regulation B28 28 could conceivably overcome the changes in H3K79 methylation and leave behind no measurable change in patient blood pressure response.
Little is known about the effect of this histone H3K79 methylation pathway on blood pressure regulation in humans, so exploratory analyses testing associations in untreated blood pressure phenotypes could also provide valuable information. The SNP that associated and replicated with untreated blood pressure was rs12350051 in MLLT3. It was chosen as a tagSNP, and is located in intron 2, with no linkage to any known functional SNPs. In silico, rs12350051 was not observed in any known miRNA sequences, transcription factor binding sites, exonic splice sites, splice enhancer, or silencer sequences. Because the same blood pressure association was not seen in Caucasians, one possibility could be that this SNP is in high linkage disequilibrium with an undiscovered functional polymorphism in African-Americans.
One would expect using a patient population with a wide range of blood pressures to be the best method to detect genetic associations with untreated blood pressure. So the fact that untreated blood pressure associations were seen in PEAR and GERA is somewhat surprising, as these studies enrolled hypertensives spanning a relatively small blood pressure range. This was one of the reasons we attempted to replicate these findings in normotensive blood pressure ranges not represented in PEAR and GERA. However, the normotensive groups also had a narrow blood pressure range. The fact that no replication was observed in normotensives could be because of this narrow blood pressure range, the fact that they were younger, the differences in study protocols, leading to differences in blood pressure measurement precision, or the sample sizes were too small and lacked the power to detect the effect we observed in the larger hypertensive cohorts. Another possibility is that perhaps the effect of this SNP is easier to detect or only exerts an effect with higher blood pressures.
Our findings are not the first to detect associations between this H3K79 methylation pathway and blood pressure regulation. Dot1 conditional knockout mice were shown to exhibit salt sensitive hypertension B29 29. Conversely, mice null for Af17, which has been shown to compete with Af9 for Dot1 binding at the ENaCa promoter, B30 30 exhibit renal salt wasting and hypotension with diminished renal H3K79 methylation and renal ENaCα gene expression B31 31. Also, Sirtuin-1 deacetylation of endothelial nitric oxide synthase B32 32 has been suggested as a possible mechanism for the blood pressure reduction seen during caloric restriction, a well-known inducer of Sirtuin-1 B33 33.
Conclusions
In conclusion, associations likely do not exist between common variation in MLLT3, SIRT1, or SGK1 and blood pressure responses to HCTZ in hypertensives. One SNP in DOT1L (rs2269879) could play a role in HCTZ response, but requires further investigation to replicate the association found in PEAR. Additionally, rs12350051 in MLLT3 was associated with untreated blood pressure in African-American hypertensive individuals. Because this was an exploratory analysis, and the association was not replicated in smaller normotensive samples, questions remain as to whether this polymorphism is involved in the blood pressure regulation of normotensives, and the mechanism by which rs12350051 exerts an effect on blood pressure. Further study in clinical populations with broader blood pressure ranges would help answer these questions.
Abbreviations
ATEN: atenolol; HCTZ: hydrochlorothiazide; H3K79: histone H3 lysine 79; NCC: sodium chloride cotransporter; ENaC: epithelial sodium channel; ENaCα: epithelial sodium channel α subunit; SNP: single nucleotide polymorphism; pfSNP: putative functional SNP; GERA: Genetic Epidemiology of Responses to Antihypertensives; PEAR: Pharmacogenomic Evaluation of Antihypertensive Responses.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JDD participated in the study design, participated in genotyping, performed statistical analyses and drafted the manuscript. IZ participated in the design of the study and helped to draft the manuscript. BB participated in sample processing and genotyping and helped to draft the manuscript. KRB assisted with statistical analysis, STT and ALB participated in study coordination and helped to draft the manuscript. YG, TYL, ABC, EB, JGG, RMC, RBF participated in study coordination. BCK conceived the study, participated in its design, and helped to draft the manuscript. JAJ conceived the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
GERA: This work was supported by NIH grants HL74735, HL53335, and the Mayo Foundation.
PEAR: This work is supported by a grant from the National Institutes of Health (Bethesda, MD), grant U01 GM074492, funded as part of the Pharmacogenetics Research Network. This work is also supported by the following grants from the NIH National Center for Research Resources: grant M01 RR00082 and UL1 RR029890 to the University of Florida, grants UL1 RR025008 and M01 RR00039 to Emory University, and UL1 RR024150 to Mayo Clinic.
This research was also supported by NIH grants R01 DK075065 (B.C.K.), R01 HL064691 (J.A.J.), R01 NS42754 (R.L.F.), K23 HL091120 (A.L.B.), and T32 DK007518 (J.D.D.). While Dr. Zineh is an employee of the FDA, no official FDA endorsement of this manuscript is intended nor should be inferred.
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