Genome-Wide Association Study Implicates Atrial Natriuretic Peptide Rather Than B-Type Natriuretic Peptide in the Regulation of Blood Pressure in the General Population

1 The hea sec e es a ial na iu e ic pep ide (ANP) and B- ype na iu e ic pep ide (BNP) in o he ci cula ion in esponse o myoca dial s e ching. A ial ca diomyocy es mainly sec e e ANP, whe eas en icula ca diomyocy es p edominan ly p oduce BNP. Coded by he adjacen genes NPPA and NPPB in humans, he p oANP and p oBNP p o- ho mones a e clea ed o p oduce an inac i e N- e minal agmen and he ac i e ho mone. ANP and BNP educe ca - diac load ia inc eased na iu esis, aso elaxa ion, and o he physiological e ec s media ed by he na iu e ic pep ide ecep o A.1 Bo h he ac i e ho mones and he N- e minal agmen s may be used as bioma ke s o ca diac s ess. A pa icula ly aluable clinical applica ion is he use o low measu ed BNP o NT-p oBNP (N- e minal p oBNP) con- cen a ion o ule ou suspec ed hea ailu e.2 ANP and BNP a e, hus, egula o s o ca dio ascula unc ion and use ul clinical bioma ke s. See Edi o ial by A mando See Clinical Pe spec i e Na iu e ic pep ides a e a ac i e he apeu ic a ge s. O e exp ession o ei he NPPA o NPPB in mice leads o p onounced hypo ension.3,4 Dele ing NPPA in mice p edis- poses hem o hype ension, bu knocking ou NPPB ig- ge s ca diac ib osis ins ead o inducing hype ension.5–7 In con as o mice, he dele ion o NPPB in a hype ensi e a Backg ound—Ca diomyocy es sec e e a ial na iu e ic pep ide (ANP) and B- ype na iu e ic pep ide (BNP) in esponse o mechanical s e ching, making hem use ul clinical bioma ke s o ca diac s ess. Bo h human and animal s udies indica e a ole o ANP as a egula o o blood p essu e wi h con lic ing esul s o BNP. Me hods and Resul s—We used genome-wide associa ion analysis (n=6296) o s udy he e ec s o gene ic a ian s on ci cula ing na iu e ic pep ide concen a ions and compa ed he impac o na iu e ic pep ide–associa ed gene ic a ian s on blood p essu e (n=27 059). Eigh independen gene ic a ian s in 2 known (NPPA-NPPB and POC1B-GALNT4) and 1 no el locus (PPP3CC) associa ed wi h mid egional p oANP (MR-p oANP), BNP, amino e minal p oBNP (NT-p oBNP), o BNP:NT- p oBNP a io. The NPPA-NPPB locus con aining he adjacen genes encoding ANP and BNP ha bo ed 4 independen cis a ian s wi h e ec s speci ic o ei he mid egional p oANP o BNP and a a e missense single nucleo ide polymo phism in NT-p oBNP se iously al e ing i s measu emen . Va ian s nea he calcineu in ca aly ic subuni gamma gene PPP3CC and he polypep ide N-ace ylgalac osaminyl ans e ase 4 gene GALNT4 associa ed wi h BNP:NT-p oBNP a io bu no wi h BNP o mid egional p oANP, sugges ing e ec s on he pos - ansla ional egula ion o p oBNP. Ou o he 8 indi idual a ian s, only hose co ela ed wi h mid egional p oANP had a s a is ically signi ican albei weak impac on blood p essu e. The combined e ec o hese 3 single nucleo ide polymo phisms also associa ed wi h hype ension isk (P=8.2×10−4). Conclusions—Common gene ic di e ences a ec ing he ci cula ing concen a ion o ANP associa ed wi h blood p essu e, whe eas hose a ec ing BNP did no , highligh ing he blood p essu e–lowe ing e ec o ANP in he gene al popula ion. (Ci c Ca dio asc Gene . 2017;10:e001713. DOI: 10.1161/CIRCGENETICS.117.001713.) Key Wo ds: blood p essu e ◼ genes ◼ genome-wide associa ion s udy ◼ hype ension ◼ na iu e ic pep ide, b ain Ci c Ca dio asc Gene is a ailable a h p://ci cgene ics.ahajou nals.o g DOI: 10.1161/CIRCGENETICS.117.001713 Recei ed Janua y 23, 2017; accep ed Oc obe 3, 2017. *D s K is iansson and Pe ola con ibu ed equally o his wo k. The Da a Supplemen is a ailable a h p://ci cgene ics.ahajou nals.o g/lookup/suppl/doi:10.1161/CIRCGENETICS.117.001713/-/DC1. Co espondence o Pe u Salo, MSc, Ins i u e o Molecula Medicine Finland, Biomedicum 1, Haa maninka u 8 m. A331b, 00290 Helsinki, Finland. E-mail [email p o ec ed] © 2017 The Au ho s. Ci cula ion: Ca dio ascula Gene ics is published on behal o he Ame ican Hea Associa ion, Inc., by Wol e s Kluwe Heal h, Inc. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial-NoDe i s License, which pe mi s use, dis ibu ion, and ep oduc ion in any medium, p o ided ha he o iginal wo k is p ope ly ci ed, he use is noncomme cial, and no modi ica ions o adap a ions a e made. Genome-Wide Associa ion S udy Implica es A ial Na iu e ic Pep ide Ra he Than B-Type Na iu e ic Pep ide in he Regula ion o Blood P essu e in he Gene al Popula ion Pe u P. Salo, MSc; Aki S. Ha ulinna, PhD; Ta u Tukiainen, PhD; Olli Rai aka i, MD, PhD; Te ho Leh imäki, MD, PhD; Mika Kähönen, MD, PhD; Johannes Ke unen, PhD; Minna Männikkö, PhD; Johan G. E iksson, MD, PhD; An i Jula, MD, PhD; S e an Blankenbe g, MD, PhD; Tanja Zelle , PhD; Veikko Salomaa, MD, PhD; Ka i K is iansson, PhD*; Ma kus Pe ola, MD, PhD* O iginal A icle by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 2 Salo e al Na iu e ic Pep ide GWAS model has been epo ed o dec ease su i al and inc ease bo h sys olic and dias olic blood p essu e (BP).8 In humans, he e ec s o ANP o BNP in usions depend on baseline s a- us. In usions o ANP o BNP in pa ien s ha ing hea ailu e igge a ious hemodynamic changes, including a dec ease in a e ial p essu e, bu in heal hy males only induce na iu e- sis wi hou a ec ing a e ial p essu e.9–12 Bo h ANP and BNP ha e a BP-lowe ing e ec in hose ha ing essen ial hype en- sion, wi h BNP su p isingly showing a 2- o 3- old g ea e po ency han ANP despi e simila ecep o a ini y.13,14 A lack o associa ion o e en a pa adoxical nega i e associa ion o ANP wi h BP has been epo ed in obese men.15 How hea ailu e, hype ension, o obesi y may modi y ANP and BNP unc ion is incomple ely unde s ood. Recombinan BNP has also ailed o show a clea clinical bene i in ea ing acu e decompensa ed hea ailu e when used in addi ion o s an- da d ca e.9 A mo e de ailed unde s anding o ANP, BNP, and hei physiological ole may aid in success ully exploi ing hei po en ial. Gene ic s udies o ANP and BNP in humans a e o pa - icula in e es as a la ge pa o he li e a u e ega d knock- ou animal models and ela i ely high doses o in a enous in usions. Da a on a ia ion in hei concen a ion wi hin he no mal physiological ange a e mo e sca ce bu necessa y o unde s and he unc ion o hese pep ides unde nondiseased condi ions. The associa ion o human gene ic a ia ion wi h ci cula ing ANP and BNP has been s udied o selec ed single nucleo ide polymo phisms (SNPs).16,17 Fou genome- wide associa ion s udies (GWAS) ha e s udied ci cula ing BNP o NT-p oBNP le els.18–21 The p io s udies ha e asso- cia ed a ian s nea NPPA-NPPB wi h p oANP, BNP, and NT-p oBNP, and he GWAS ha e associa ed ans loci nea LOXL2, SLC39A8, KLKB1, and GALNT4 wi h NT-p oBNP. No genome-wide s udies ha e been published on ANP. The p io s udies, hus, ei he did no ha e genome-wide co - e age o gene ic a ia ion o did no assay ANP, limi ing he in e p e a ion o hei esul s. We pe o med genome- wide associa ion es s o BNP, NT-p oBNP, and mid egional p oANP (MR-p oANP) and s udied he impac o he na i- u e ic pep ide–associa ed gene ic a ian s on BP. Because p oBNP is p ocessed pe iphe ally in o BNP and NT-p oBNP ha ha e di e en ci cula ing hal -li es, we also s udied he a io o BNP o NT-p oBNP concen a ions (BNP:NT- p oBNP a io) as a po en ial p oxy o he p ocessing and deg ada ion o BNP, p oBNP, and NT-p oBNP.1,22 Ma e ials and Me hods MR-p oANP, NT-p oBNP, and BNP we e measu ed in he GWAS disco e y (n=4932) and eplica ion samples (n=1373), o iginally e- c ui ed o he FINRISK 1997 s udy. The Na ional FINRISK S udy coho s a e collec ed e e y 5 yea s as ep esen a i e age- and sex- s a i ied samples o he popula ions o 5 geog aphical a eas o Finland, desc ibed in mo e de ail elsewhe e.23,24 We es ed he asso- cia ion o gene ic a ian s wi h na iu e ic pep ide ai s in he GWAS disco e y and eplica ion samples excluding pa icipan s who had p e alen diabe es melli us, hea ailu e, s oke, o co ona y hea disease. We hen s udied he BP associa ions o he gene ic a ian s de ec ed in he GWAS in an independen s udy popula ion, comp is- ing he FINRISK 1992 (n=4920), FINRISK 2002 (n=5,21), FINRISK 2007 (n=4996), he No he n Finland Bi h Coho 1966 (NFBC66, n=5363), he HBCS (Helsinki Bi h Coho S udy, n=1619), he YFS (Young Finns S udy, n=2443), and he Heal h2000 (n=1997) co- ho s.23–28 All s udy coho s we e popula ion-based samples o Finns, app o ed by hei espec i e ins i u ional e iew commi ees, and pa - icipan s ga e hei in o med consen . Na iu e ic Pep ide and BP Measu emen s Na iu e ic pep ide concen a ions we e measu ed in he MORGAM Bioma ke Labo a o y, Uni e si y o Mainz, Ge many, using he Abbo A chi ec i2000 BNP (BNP, UniP o acc. P16860, esidues 103–134), Roche Elecsys 2010 p oBNP (NT-p oBNP, acc. P16860 esidues 27–134), and B.R.A.H.M.S. MR-p oANP KRYPTOR (MR- p oANP, acc. P01160) assays, desc ibed in mo e de ail p e iously.29 The in e /in a-assay coe icien s o a ia ion we e 2.11%/4.28% (BNP), 2.58%/1.38% (NT-p oBNP), and 3.65%/2.33% (MR- p oANP). BP was measu ed om he pa icipan s’ igh a m, and hype ension was de ined as dias olic BP >90 mm Hg o sys olic BP >140 mm Hg o known use o an ihype ensi e medica ion. Geno yping and Impu a ion The GWAS disco e y sample and eplica ion samples we e geno yped using he Illumina HumanCo eExome beadchip a he Wellcome T us Sange Ins i u e (Camb idge, UK) and a he B oad Ins i u e o Ha a d and MIT (MA, USA), espec i ely. The da a we e p ephased and impu ed using he 1000 Genomes p ojec phase 1 and 3 haplo- ypes and a cus om haplo ype se o 2000 Finnish indi iduals. A e quali y con ol (Ha dy–Weinbe g equilib ium P alue <0.01, mino allele equency <1%, impu a ion quali y <0.9, geno yping success a e <95%) and emo al o a e SNPs (mino allele equency <1%), he disco e y phase GWAS da a se con ained a o al o 7 358 451 SNPs and 4932 samples. Coho s comp ising he BP s udy popula- ion we e geno yped on a ious genome-wide geno yping a ays and impu ed using he same me hods as used o he GWAS disco e y sample (Da a Supplemen ). All genomic coo dina es a e gi en using he GRCh37 human e e ence genome. Associa ion Tes s We used mul iple impu a ion o accoun o any missing alues o MR-p oANP (Nmissing=0), BNP (Nmissing =131), and NT-p oBNP (Nmissing=133) and andom-e ec s me a-analysis o combine esul s om he di e en coho s.30 We in e se-no mal ans o med he na- iu e ic pep ide measu emen s and used linea eg ession wi h an ad- di i e gene ic model adjus ed o geog aphical sampling egion, age2 sex, body mass index (BMI), cu en smoking (yes/no), sys olic BP, glome ula il a ion a e es ima ed using cys a in C and c ea inine as p oxies, and geno yping ba ch. We used leas absolu e sh inkage and selec ion ope a o eg ession implemen ed in he LLARMA package o ine-mapping he na iu e ic pep ide–associa ed loci o iden i y possible seconda y independen a ian s.31 The gene ic associa ion es s a e desc ibed in mo e de ail in he Da a Supplemen . We used linea eg ession implemen ed in he glm unc ion o R o s udy he associa ion o gene ic a ian s wi h sys olic and dias olic BP. We log- ans o med sys olic (bu no dias olic) BP and se he i s 2 genomic p incipal componen s, age, sex, BMI, cu en BP medica- ion use (yes/no, only o sys olic and dias olic BP), s udy yea , and geno yping ba ch as co a ia es ( he la e 2 only o he FINRISK samples). Fo hype ension, we used logis ic eg ession and he same co a ia es excluding BP medica ion. Pheno ypic Va iance Explained by SNPs Genome- Wide We used au osomal SNPs om he impu ed da a se o es ima e he ac ion o pheno ypic a iance explained by he SNPs genome-wide in he pa icipan s o he GWAS disco e y sample using PLINK 1.90 and GCTA 1.25.3.32,33 As a quali y con ol measu e, we de- i ed 4 genomic sco es co esponding o each o he 4 es ima es ( o MR-p oANP, BNP, NT-p oBNP, and BNP:NT-p oBNP a io) and es ed he associa ion o he genomic sco es wi h hei espec i e pheno ypes in he eplica ion sample (Da a Supplemen ). by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 3 Salo e al Na iu e ic Pep ide GWAS Coassocia ion Wi h Gene Exp ession We in es iga ed he coassocia ion o SNPs wi h bo h na iu e ic pep ides and gene exp ession in da a om 190 le en icula is- sue samples and 159 a ial appendage samples om he Geno ype- Tissue Exp ession (GTEx) conso ium ( elease V6, Oc obe 6, 2016).34 We used 3 me ics o con i m ha he same gene ic a i- an s co ela ed wi h bo h gene exp ession and na iu e ic pep ide concen a ions in a consis en way (Da a Supplemen ): we equi ed ha he mos s a is ically signi ican na iu e ic pep ide–associa ed SNPs (lead SNPs) associa ed wi h he genes’ exp ession le els and ha bo h he associa ion P alues and he e ec es ima es (βs) we e co ela ed ac oss he SNPs in he na iu e ic pep ide–associa ed e- gions. Because P alues depend on allele equencies, we used bo h Spea man ank ( o P alues) and Pea son p oduc momen ( o βs) co ela ion coe icien s as measu es o he co ela ion be ween he na iu e ic pep ide and gene exp ession associa ions and de i ed he P alues empi ically. Resul s Baseline Cha ac e is ics The baseline cha ac e is ics o he GWAS disco e y sam- ple, he eplica ion sample, and he BP s udy popula ion a e desc ibed in Table I in he Da a Supplemen . The s a a we e b oadly simila , and he main di e ence was ha pa icipan s wi h p e alen ca dio ascula disease we e no excluded om he BP s udy popula ion. GWAS and Va iance Explained by All SNPs To quan i y he o al amoun o gene ic signal p esen in he da a, we i s es ima ed he p opo ion o a iance in he na i- u e ic pep ide ai s join ly explained by all SNPs genome- wide. The poin es ima es we e 13.9% o MR-p oANP, 13.5% o BNP, 23.0% o NT-p oBNP, and 17.9% o BNP:NT- p oBNP a io, bu he coa se p ecision o he es ima es p e- en s anking he 4 pheno ypes in any pa icula o de in e ms o a iance explained (Table II in he Da a Supplemen ). The magni ude o he 4 es ima es none heless indica es ha he SNPs oge he explained a mode a e p opo ion o he pheno- ypic a iance. Ha ing es ima ed he p opo ion o a iance explained by all SNPs genome-wide, we es ed he SNPs indi idually o associa ion wi h he pheno ypes. Va ian s in 4 loci nea NPPA- NPPB, PPP3CC, GALNT4, and NCOR12 me he p especi ied h eshold o genome-wide signi icance P<5×10−8 o associa- ion (Figu e 1; Table 1; Figu e I in he Da a Supplemen ; Table III in he Da a Supplemen ). We selec ed he SNP wi h he smalles P alue (lead SNP) a each locus o eplica ion. Only he associa ion o s701041 wi h MR-p oANP nea NCOR12 did no eplica e (P=0.94). Associa ions nea NPPA-NPPB and GALNT4 ha e been epo ed p e iously, whe eas he associa- ion o s7000551 wi h BNP:NT-p oBNP a io on ch omosome 8 nea PPP3CC is a no el inding.16–20 Fine-mapping he loci using leas absolu e sh inkage and selec ion ope a o eg ession iden i ied independen seconda y signals nea NPPA-NPPB and GALNT4. Th ee independen SNPs nea NPPA associa ed wi h MR-p oANP le els, whe eas 2 independen SNPs nea GALNT4 associa ed wi h BNP:NT-p oBNP a io. P e iously de ec ed associa ions eplica ed success ully in he p esen da a in e ms o he di ec ion o associa ion (Table IV in he Da a Supplemen ). O hese, all bu 1 o he cis associa ions nea NPPA-NPPB also eached s a is ical signi icance. Two o he 3 p e iously published ans asso- cia ions, s13107325 in SLC39A8 and s3733402 in KLKB1, associa ed wi h BNP:NT-p oBNP a io in he me a-analy- sis o he disco e y and eplica ion samples ( s13107325 P=2.19×10−9; s3733402 P=0.00277) and he me a-analysis P alue o s13107325 wi h NT-p oBNP (P=0.00496) was also nominally signi ican . The hi d, s6557662 in LOXL2, did no each s a is ical signi icance. None o he ans loci associa ed wi h BNP o MR-p oANP. Mos common a ian s a e hough o a ec pheno ypes by al e ing gene exp ession.35,36 We, hus, s udied da a om 190 le en icula issue samples and 159 a ial appendage samples om he GTEx conso ium o iden i y coassocia ion o SNPs wi h bo h na iu e ic pep ide ai s and gene exp es- sion.34 The esul s o hese es s, oge he wi h hose o he ine-mapping es s wi h leas absolu e sh inkage and selec ion ope a o eg ession, a e p esen ed in de ail below o he loci mee ing genome-wide signi icance in he p esen s udy. NPPA-NPPB on Ch omosome 1 SNPs associa ing wi h he na iu e ic pep ide on ch omosome 1 we e loca ed nea he NPPA and NPPB genes (Figu e 2; Figu e II in he Da a Supplemen ). P e iously, associa ions in his locus ha e been epo ed using a GWAS s a egy o NT-p oBNP and a candida e SNP app oach o ANP.16–20 To ex end he p e iously epo ed esul s, we ocus he e on he ex ensi e panel o SNPs and he mo e de ailed pheno yping, which we e no a ailable in he p io s udies. The NPPA-NPPB locus con ained 3 ini ial associa ion signals o BNP, NT-p oBNP, and BNP:NT-p oBNP a io, depending on which o he pheno ypes was es ed (Table 1; Table III in he Da a Supplemen ). Rs198379, si ua ed 2055 base pai s downs eam om he las exon o NPPB, associ- a ed wi h BNP (P=4.42×10−52). Fo NT-p oBNP and BNP:NT- p oBNP a io, s61761991 was he mos s a is ically signi ican SNP (P=8.76×10−68 and P=4.81×10−103, espec i ely), and leas absolu e sh inkage and selec ion ope a o eg ession de ec ed s12406089 as a seconda y signal o NT-p oBNP (P=8.31×10−48). Howe e , nei he o hese 2 SNPs associ- a ed wi h BNP when s198379 was included in he model. The NPPA-NPPB locus, he e o e, ha bo ed only 1 a i- an , s198379, independen ly associa ed wi h bo h BNP and NT-p oBNP, wi h e e y C allele inc easing BNP concen a ion by ≈4.5 pg/mL and NT-p oBNP concen a ion by 9.6 pg/mL. Th ee SNPs associa ed independen ly wi h MR-p oANP a he NPPA-NPPB locus (Table 1). The mos s a is ically signi ican was s3753584 (P=3.85×10−13), bu he e ec sizes o he 3 SNPs we e b oadly simila . Each allele o he SNPs co ela ed wi h a 2.5 o 5.0 pmol/L di e ence in MR-p oANP concen a ion. The SNPs a e ound ≈40 kb downs eam om NPPA wi hin an a ea bound by egula o y p o eins in human ca diomyocy es (ENCODE: Encyclopedia o DNA Elemen s, h ps://www.encodep ojec .o g, expe imen ENCSR000ENJ).37 Because obesi y dis u bs he associa ion o MR-p oANP wi h BP, we s udied he e ec o body mass on he SNP associa ions by in oducing body mass*SNP in e - ac ion e ms o he eg ession models.15 The in e ac ion e ms we e s a is ically nonsigni ican (P>0.05) o bo h BMI as a con inuous a iable and obesi y (BMI>30) as a ca ego ical by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 4 Salo e al Na iu e ic Pep ide GWAS a iable. Fu he mo e, because NPPA and NPPB a e sepa- a ed by <10 kb, any a ian in his egion migh a ec ei he bo h genes o only 1 o he 2. We explo ed his by i ing models con aining all o he p e iously men ioned SNPs o he NPPA-NPPB locus and ound ha SNPs associa ed wi h MR-p oANP did no associa e wi h BNP o NT-p oBNP and ice e sa (Table V in he Da a Supplemen ), indica - ing ha hei e ec s we e speci ic o ei he MR-p oANP o NT-p oBNP (and BNP). Gene exp ession p o iling in human ca diac issue sam- ples con i med ha he associa ions o SNPs wi h BNP o MR-p oANP concen a ion and wi h NPPB and NPPA gene Figu e 1. Genome-wide associa ion s udy P alues. P alues o he genome-wide associa ion es s and hei genomic loca ions. Y axis cu a Y=18, he peak on ch omosome 1 ex ends o Y=80. Table 1. Associa ion o Gene ic Va ian s Wi h Na iu e ic Pep ides in he Genome-Wide Signi ican Loci T ai SNP Ch omosome Posi ion Alleles* (MAF) Impu a ion Quali y† Genes (Dis ance‡, Loca ion) Model PGWAS PREPLICATION β (SE; 95% CI) PCOMBINED BNP s198379 1 11915467 /C (0.365) 0.989 NPPB (3.5 kb, 3′) GWAS 6.85×10−41 7.99×10−13 0.249 (0.0164; 0.217 o 0.282) 4.42×10−52 BNP:NT- p oBNP s61761991 1 11918444 c/T (0.029) 0.996 NPPB (0.5 kb, coding exon) GWAS 7.17×10−79 5.71×10−26 1.114 (0.0517; 1.013 o 1.215) 4.81×10−103 s7000551 8 22276251 a/G (0.369) 0.994 SLC39A14 (38.6 kb, in onic) PPP3CC (22.5 kb, 5′) GWAS 2.16×10−8 0.0248 0.109 (0.0181; 0.073 o 0.144) 2.00×10−9 s11105298 12 89876143 /C (0.211) 0.992 POC1B (59 kb, in onic) GALNT4 (43.2 kb, 3′)GWAS 3.06×10−18 4.11×10−6 0.21 (0.0213; 0.169 o 0.252) 6.77×10−23 s11105298 12 89876143 /C (0.211) 0.992 POC1B (59 kb, in onic) GALNT4 (43.2 kb, 3′)Condi ional-1 3.67×10−20 2.01×10−6 0.189 (0.02185; 0.189 o 0.275) 2.96×10−26 s61378614 12 89903654 a/C (0.16) 0.994 POC1B (87 kb, in onic) GALNT4 (15.7 kb, 3′)Condi ional-1 1.70×10−10 0.0453 0.101 (0.03242; 0.101 o 0.228) 4.13×10−7 MR- p oANP s3753584 1 11864586 /C (0.149) 1 MTHFR (3 kb, in onic) NPPA (43.5 kb, 3′)GWAS 4.63×10−38 3.48×10−7 0.275 (0.038; 0.201 o 0.35) 4.19×10−13 s4845875 1 11824133 A/c (0.355) 0.944 C1o 167 (11 kb, in onic) NPPA (84 kb, 3′) Condi ional-2 3.53×10−7 0.0031 −0.156 (0.0198; −0.156 o −0.079) 3.37×10−9 s6540997 1 11827355 A/g (0.274) 0.995 C1o 167 (8 kb, in onic) NPPA (80.8 kb, 3′) Condi ional-2 9.03×10−10 0.0326 0.074 (0.0195; 0.074 o 0.171) 7.13×10−7 s3753584 1 11864586 /C (0.149) 1 MTHFR (3 kb, in onic) NPPA (43.5 kb, 3′)Condi ional-2 1.85×10−20 1.80×10−4 0.162 (0.023; 0.162 o 0.282) 3.85×10−13 s701041 12 124999344 G/c (0.106) 0.928 NCOR2 (126 kb, in onic) GWAS 1.23×10−8 0.9381 −0.088 (0.0782; −0.241 o 0.066) 0.2624 NT- p oBNP s61761991 1 11918444 c/T (0.029) 0.996 NPPB (0.5 kb, coding exon) GWAS 1.72×10−51 5.33×10−18 −0.766 (0.044; −0.853 o −0.68) 8.76×10−68 s61761991 1 11918444 c/T (0.029) 0.996 NPPB (0.5 kb, coding exon) Condi ional-3 3.85×10−43 1.26×10−15 −0.782 (0.0425; −0.782 o −0.616) 1.41×10−60 s12406089 1 11921181 c/G (0.291) 0.995 NPPB (2.2 kb, 5′) Condi ional-3 2.45×10−33 3.54×10−14 0.201 (0.0172; 0.201 o 0.264) 8.31×10−48 s10858906 12 89934474 c/T (0.21) 0.996 GALNT4 (15.2 kb, 5′) GWAS 1.08×10−12 0.0388 −0.12 (0.0338; −0.186 o −0.054) 3.91×10−4 Associa ion es ed wi h an addi i e gene ic model using single SNP (GWAS) o condi ional models which included all SNPs o each model simul aneously. All models adjus ed o geog aphical sampling egion, age, age2, sex, cu en smoking s a us (yes o no), sys olic blood p essu e, es ima ed glome ula il a ion a e, and geno yping ba ch. Genomic posi ions gi en ela i e o he GRCh37 e e ence genome build. BNP indica es B- ype na iu e ic pep ide; MAF, mino allele equency; MR-p oANP, mid egional p oa ial na iu e ic pep ide; and NT-p oBNP, amino e minal p o-B- ype na iu e ic pep ide. *Alleles gi en as ( e e ence allele)/(e ec allele) wi h mino alleles in lowe case le e s. †IMPUTE in o me ic. Rs4845875 was di ec ly geno yped wi h missing geno ypes impu ed. ‡Median dis ance o he ansc ip ion s a si es o he candida e gene(s). by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 5 Salo e al Na iu e ic Pep ide GWAS exp ession we e posi i ely co ela ed. In le en icula is- sue samples, SNPs associa ed wi h ci cula ing MR-p oANP concen a ion also associa ed wi h NPPA exp ession le el (Figu e III in he Da a Supplemen ; Spea man ank co ela ion o P alues, P=0.014), and SNPs associa ed wi h BNP con- cen a ion also associa ed wi h NPPB exp ession (P=0.004). Fu he mo e, he e ec es ima es o ci cula ing MR-p oANP concen a ion and NPPA exp ession in he le en icle co - ela ed (Pea son =0.611; P=0.024) as did hose o BNP and NPPB ( =0.735; P=0.001). A somewha a enua ed end was also p esen in he a ial appendage samples, whe e he co e- la ions be ween he e ec es ima es we e s a is ically signi i- can (MR-p oANP e sus NPPA =0.508; P=0.021 and BNP e sus NPPB =0.481; P=0.033), bu he co ela ions be ween associa ion P alues we e no . In addi ion o NPPA and NPPB, he co ela ions we e also signi ican o EXOSC10 and ENSG00000272482 (wi h MR-p oANP) and EXOSC10 and MTHFR (wi h BNP). The egula o y e ec s unde lying he MR-p oANP and BNP associa ions nea NPPA-NPPB may, he e o e, be s onge in he le en icle compa ed wi h he a ium and also selec i ely a ec he exp ession o o he nea by genes. PPP3CC and GALNT4 on Ch omosomes 8 and 12 Rs7000551 on ch omosome 8 nea PPP3CC associa ed wi h BNP:NT-p oBNP a io (P=2.27×10−9). This co ela ion was d i en by an e ec on he NT-p oBNP concen a ion as s7000551 associa ed wi h NT-p oBNP (P=3.72×10−5) bu no wi h BNP (P=0.87) in he disco e y GWAS sample. Howe e , only he associa ion o s7000551 wi h BNP:NT-p oBNP a io me genome-wide signi icance and eplica ed. The gen- o ype-speci ic mean BNP:NT-p oBNP a ios o s7000551 (AA=0.386; AG=0.412; GG=0.463) sugges an addi i e o mul iplica i e gene ic e ec wi h each G-allele aising he a io by ≈0.04 U o 10%. The associa ion peak on ch omosome 8 ex ends om he 3′ end o SLC39A14 in o he p omo e egion and 5′ end o PPP3CC, wi h s7000551 i sel loca ed in an in on o SLC39A14 (Figu e 3). When we s udied he coassocia ion o SNPs wi h BNP:NT-p oBNP a io and gene exp ession, PPP3CC and 2 an isense RNA genes ENSG00000245025 and ENSG00000248738 ma ched he p especi ied c i e ia. SNPs associa ed wi h inc eased BNP:NT-p oBNP a io also associa ed wi h educed exp ession o PPP3CC in bo h a ial and le en icula issue samples (a ial appendage, Pea son =−0.70; P=0.006 and le en icle, =−0.81; P=0.021; Figu e III in he Da a Supplemen ). The coassocia ion wi h he 2 RNA genes was signi ican only in he le en icula issue samples. Bo h he physical loca ion nea he p omo e o PPP3CC and he coassocia ion wi h i s exp ession, he e- o e, sugges ha he BNP:NT-p oBNP a io–associa ed SNPs ag a egula o y a ian ha al e s he exp ession o PPP3CC in he hea . SNPs nea POC1B and GALNT4 on ch omosome 12 associa ed wi h NT-p oBNP and BNP:NT-p oBNP a io. Rs11105298 and s61378614, loca ed in di e en in ons o he POC1B gene (Figu e 3), independen ly associa ed wi h he a io (P=1.52×10−26 and P=3.98×10−9, espec i ely). The genes’ exp ession on ch omosome 12 did no show a clea coassocia ion wi h BNP:NT-p oBNP a io because none o hem was signi ican o all 3 p ede ined c i e ia. Associa ion Wi h BP Ha ing iden i ied he se o SNPs associa ed wi h he na i- u e ic pep ide ai s in he genome-wide signi ican loci, we nex s udied hei co ela ion wi h sys olic BP, dias olic BP, and hype ension in an independen sample. We i ed all SNPs simul aneously in each locus, excluding s61761991 and s12406089 on ch omosome 1, which did no independen ly Figu e 2. SNPs associa ed wi h na iu e ic pep ides on ch o- mosome 1 nea NPPA and NPPB. Linkage disequilib ium on ch omosome 1 nea NPPA and NPPB. R2 and D′ calcula ed wi h Haplo iew 4.2 om 22 374 un ela ed Finnish samples. Genes a e depic ed as anno a ed in GENCODE 19, po en ial egula- o y egions iden i ied by digi al genomic oo p in ing in human ca diac myocy es (ENCODE: Encyclopedia o DNA Elemen s, expe imen numbe ENCSR000ENJ) indica ed by he black g aph. SNPs independen ly associa ed wi h mid egional p oa ial na iu e ic pep ide (MR-p oANP) colo ed wi h blue, SNPs inde- penden ly associa ed wi h B- ype na iu e ic pep ide (BNP) o NT-p oBNP (N- e minal p o-B- ype na iu e ic pep ide) colo ed in pink. SNPs associa ed wi h MR-p oANP o NT-p oBNP in p e i- ous s udies colo ed in black. by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 6 Salo e al Na iu e ic Pep ide GWAS associa e wi h BNP. A e geno yping quali y con ol, he s udy sample con ained 27 059 pa icipan s wi h bo h BP mea- su emen s and SNP geno ypes a ailable. The 3 SNPs associa ed wi h MR-p oANP also associa ed weakly wi h BP (Table 2; Figu e IV in he Da a Supplemen ). The poin es ima es o he MR-p oANP inc easing alleles’ e ec s we e ≈0.25 mm Hg (dias olic BP) and 0.50 mm Hg (sys olic BP). Only 1 o hese SNPs was independen ly asso- cia ed wi h hype ension as a bina y end poin ( s3753584; P=6.8×10−4). To assess he combined e ec o he gene ic di e ences in MR-p oANP concen a ion on BP, we o med an allele-coun ing sco e o he 3 SNPs. The sco e explained 2.36% o he a iance in MR-p oANP concen a ion and a uni inc ease in he sco e associa ed wi h a 9% dec ease in he odds a io o hype ension (odds a io=0.91; SE=0.0283; P=8.2×10−4). In con as o MR-p oANP, none o he SNPs co ela ed wi h BNP, NT-p oBNP, o BNP:NT-p oBNP a io associ- a ed wi h BP. Rs198379, associa ed wi h NPPB exp ession and ci cula ing BNP le els, did no associa e wi h sys olic o dias olic BP when adjus ed o he nea by MR-p oANP– co ela ed SNPs. SNPs nea PPP3CC and GALNT4, co e- la ed wi h NT-p oBNP and BNP:NT-p oBNP a io, simila ly did no associa e wi h BP o hype ension. Discussion We pe o med a GWAS o ci cula ing MR-p oANP, BNP, and NT-p oBNP concen a ion and BNP:NT-p oBNP con- cen a ion a io in 4932 samples wi h eplica ion in 1373 samples. We hen s udied he e ec o he na iu e ic pep ide– associa ed loci on sys olic BP, dias olic BP, and hype en- sion in 27 059 addi ional samples. We de ec ed a no el locus o BNP:NT-p oBNP a io on ch omosome 8 nea PPP3CC and ine-mapped 2 published loci on ch omosomes 1 and 12 o hei associa ion wi h ANP and BNP and BP. The en i e genome-wide SNP da a explained om 14% o 23% o he a ia ion in he na iu e ic pep ide ai s in ou popula ion- based sample. These es ima es a e simila o hose, o exam- ple, BMI (14%) o sys olic BP (24%) published elsewhe e, showing ha he na iu e ic pep ide ai s conside ed he e ha e an addi i e gene ic componen compa able o adi ional ca dio ascula isk ac o s.38 The p esen s udy is he i s o assess he NPPA-NPPB locus wi h a dense SNP panel simul aneously o MR-p oANP, BNP, and NT-p oBNP, ex ending he esul s o p e ious in es- iga ions.16–21 We iden i ied 3 s a is ically independen cis a i- an s associa ed wi h MR-p oANP, and 1 a ian associa ed wi h BNP and NT-p oBNP. Analysis o gene exp ession da a sug- ges s ha he p o ein-le el cis associa ions s em om e ec s on NPPA and NPPB gene exp ession, a ec ing bo h a ial and en- icula issue. Fu he mo e, e en i he 2 genes a e sepa a ed by <10 000 bp, hei ansc ip ional egula ion is decoupled o he ex en ha he ANP-associa ed SNPs had no obse able e ec on BNP and ice e sa. Each o hese SNPs, howe e , co ela es wi h bo h MR-p oANP and BNP concen a ions, i he analysis is no adjus ed o he o he SNPs. This is c ucial o he in e p e a ion o esul s om Mendelian andomiza ion s udies using SNPs in his locus as ins umen s, such as hose pe o med in ela ion wi h ype 2 diabe es melli us.39 SNPs on ch omosome 8 nea SLC39A14 and PPP3CC associa e wi h BNP:NT-p oBNP a io. SLC39A14 belongs o he same la ge amily o solu e ca ie p o eins as SLC39A8 in he p e iously de ec ed NT-p oBNP associa ed locus on ch omosome 4, bu i is di icul o assess whe he his is only coinciden al.19,40 SNPs associa ed wi h inc eased BNP:NT-p oBNP a io co ela ed wi h dec eased exp ession Figu e 3. B- ype na iu e ic pep ide (BNP):NT-p oBNP (N- e minal p o-B- ype na iu e ic pep ide) associa ed SNPs on ch omosomes 8 and 12. Associa ion o SNPs wi h BNP:NT-p oBNP a io on ch omosomes 8 and 12 a e me a-analyzing he esul s om he genome- wide associa ion s udy (GWAS) and eplica ion samples wi h lead SNPs om he GWAS indica ed wi h pu ple diamonds. by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 7 Salo e al Na iu e ic Pep ide GWAS o PPP3CC in bo h le en icula and a ial issue samples, whe eas no such co ela ion was p esen o SLC39A14. PPP3CC codes o 1 o he 3 al e na i e ca aly ic subuni s o calcineu in, a phospha ase wi h a wide ange o unc ions including he egula ion o ca diac hype ophic signaling.41 O iginally cha ac e ized as a es is-speci ic calcineu in sub- uni , PPP3CC has been la e de ec ed in mul iple issues.34 Because o i s cen al ole in spe ma ogenesis, d ugs inhibi - ing PPP3CC ha e been sugges ed as a po en ial male con- acep i e.42 Ou esul s indica e ha sys emic inhibi ion o calcineu in con aining he subuni coded by PPP3CC may ha e unin ended ca dio ascula side e ec s. Two independen SNPs nea POC1B and GALNT4 asso- cia ed wi h NT-p oBNP le els and BNP:NT-p oBNP a io in ou s udy. An associa ion o a SNP wi h NT-p oBNP in his locus has been p e iously epo ed in whi es.19 Analysis o gene exp ession in ca diac issue ailed o highligh any o he nea by genes bu , as p e iously no ed, GALNT4 is an a ac i e candida e.19 I codes o an aminoacyl an e ase ha ini ia es O-linked glycosyla ion, and p oBNP is known o be O-glycosyla ed.43,44 Acco ding o da a p esen ed he e, he associa ion nea GALNT4 is speci ic o NT-p oBNP, suppo - ing he hypo hesis ha p oBNP may be a a ge o GALNT4. Because BNP and NT-p oBNP a e p oduced as a single polypep ide, de ia ions in hei ci cula ing concen a ion a io should e lec hei di e en ial sec e ion o emo al, he p o- cessing o p oBNP, o ac o s dis u bing he de ec ion o he pep ides. The la e is p obably he case wi h s61761991, loca ed wi hin he egion o he NT-p oBNP p oho mone (NP_002512.1:p.A g72His) used as he an igen o p epa e he assay’s p ima y an ibody.45,46 The a ian , which e ec i ely blocked he signal o he NT-p oBNP assay, is a e o absen in o he popula ions bu signi ican ly en iched in Finns, whe e he equency o he T allele is ≈3%.47 One in 20 Finns will, he e o e, ha e a measu ed concen a ion o NT-p oBNP, which is ≈50% lowe han he co esponding C- e minal BNP alue, po en ially causing alse ule-ou o suspec ed hea ail- u e. The associa ions o SNPs nea GALNT4 wi h BNP:NT- p oBNP a io may also ela e o he de ec ion o NT-p oBNP a he han changes in i s concen a ion, i hey a e indeed linked o he possible glycosyla ion o p oBNP by GALNT4. How PPP3CC may a ec BNP:NT-p oBNP a io is unclea . We adjus ed he analysis o he es ima ed glome ula il a ion a e, bu con ounding by kidney unc ion canno be uled ou . Expe imen al da a has poin ed o ei he simila o di e - en ca dio ascula e ec s o ANP and BNP, depending on he expe imen al se ing.6–8,48 The esul s o his s udy a e in line wi h some o he p e ious s udies ha iden i ied ANP a he han BNP as an impo an egula o o BP. Gene ically de e mined inc eases in ANP concen a ion dec eased sys- olic and dias olic BP, bu a smalle gene ic dec ease in BNP did no . Obesi y did no modi y he associa ions o SNPs wi h MR-p oANP, showing ha he ansc ip ional egula- ion o NPPA is a leas pa ially una ec ed by he epo ed ANP-dec easing e ec o high body mass.15,49 Acco ding o da a p esen ed he e, ea lie gene ic associa ions o he NPPA- NPPB locus wi h BP we e d i en by ANP-associa ed a ian s and should no be aken as e idence o any BP lowe ing e ec o BNP.16 We conclude ha he e a e in e es ing di e ences be ween ANP and BNP in humans ha a e ye o be ully elucida ed and ha gene ics p o ides unique insigh s in o he e ec s o li elong al e a ions o hese ho mones. Table 2. Independen E ec s o Gene ic Va ian s on Na iu e ic Pep ides and Blood P essu e SNP Ch Posi ion Alleles* Candida e Genes GWAS and Replica ion (n=6296) Blood P essu e S udy Popula ion (n=27 059) BNP, pg/mL NT-p oBNP, pg/mL BNP:NT- p oBNP Ra io MR-p oANP, pmol/L Dias olic BP, mm Hg Sys olic BP, mm Hg Hype ension (OR) s4845875 1 11824133 A/c NPPA 0.84, ns. 5.00, ns. 0.01, ns. −2.40, P=2.1×10−8 0.40, P=0.0033 0.63, ns. 1.00, ns. s6540997 1 11827355 A/g NPPA 0.11, ns. −2.20, ns. −0.01, ns. 3.10, P=5.8×10−7 −0.25, ns. −0.47, P=0.029 0.93, ns. s3753584 1 11864586 /C NPPA 1.50, ns. 8.70, ns. −0.00, ns. 5.00, P=1.2×10−6 −0.38, P=0.022 −0.36, ns. 0.88, P=6.8×10−4 s198379 1 11915467 /C NPPB 4.50, P=1.2×10−20 9.60, P=7.2×10−19 −0.00, ns. 0.33, ns. −0.02, ns. −0.29, ns. 1.00, ns. s7000551 8 22276251 a/G SLC39A14 and PPP3CC −0.34, ns. −3.80, ns. 0.03, P=5.4×10−9 −0.11, ns. 0.16, ns. −0.07, ns. 1.00, ns. s11105298 12 89876143 /C GALNT4 0.66, ns. −7.40, P=0.0067 0.06, P=3×10−26 1.30, ns. −0.01, ns. −0.04, ns. 1.00, ns. s61378614 12 89903654 a/C GALNT4 0.71, ns. −4.40, ns. 0.04, P=4.1×10−7 1.00, ns. 0.23, ns. 0.38, ns. 0.99, ns. Independen e ec s o SNPs om eg ession models whe e, pe each locus, all SNPs we e simul aneously included. E ec s es ima ed using un ans o med ai alues, P alues de i ed using un ans o med (dias olic BP), in e se-no mal ans o med (na iu e ic pep ides), o log- ans o med (sys olic BP) alues. Fo na iu e ic pep ide ai s, he models we e adjus ed o geog aphical sampling egion, age, age2, sex, cu en smoking s a us (yes/no), sys olic BP, es ima ed glome ula il a ion a e, and geno yping ba ch. Fo BP ai s, he models we e adjus ed o he i s 2 genomic p incipal componen s, age, sex, BMI, cu en BP medica ion use (yes/no, only o sys olic and dias olic BP), coho yea , and geno yping ba ch ( he la e 2 only o he FINRISK samples). BP indica es blood p essu e; BNP, B- ype na iu e ic pep ide; MR-p oANP, mid egional p oa ial na iu e ic pep ide; NT-p oBNP, amino e minal p o-B- ype na iu e ic pep ide; and SNP, . *Alleles gi en as ( e e ence allele)/(e ec allele) wi h mino alleles in lowe case le e s. by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 8 Salo e al Na iu e ic Pep ide GWAS Appendix F om he Na ional Ins i u e o Heal h and Wel a e, Helsinki, Finland (P.P.S., A.S.H., J.K., J.G.E., A.J., V.S., K.K., M.P.); Ins i- u e o Molecula Medicine Finland, Helsinki (P.P.S., A.S.H., T.T., K.K., M.P.); Diabe es and Obesi y Resea ch P og am (K.K., M.P.) and Depa men o Gene al P ac ice and P ima y Heal h Ca e, Helsinki Uni e si y Hospi al (J.G.E.), Uni e si y o Helsinki, Finland; The Resea ch Cen e o Applied and P e- en i e Ca dio ascula Medicine (O.R.) and Depa men o Clinical Physiology, Tu ku Uni e si y Hospi al (O.R.), Uni e - si y o Tu ku, Finland; Depa men o Clinical Chemis y, Fim- lab Labo a o ies and Finnish Ca dio ascula Resea ch Cen e Tampe e, Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Finland (T.L.); Depa men o Clinical Physiology, Tampe e Uni e si y Hospi al, Finland (M.K.); Depa men o Clinical Physiology, Uni e si y o Tampe e School o Medi- cine, Finland (M.K.); Ins i u e o Compu a ional Medicine, Cen e o Li e Cou se Heal h Resea ch, Facul y o Medi- cine (J.K.), Biocen e Oulu (J.K.), and Cen e o Li e Cou se Heal h Resea ch, Facul y o Medicine (M.M.), Uni e si y o Oulu, Finland; Folkhälsan Resea ch Cen e , Helsinki, Finland (J.G.E.); Depa men o Gene al and In e en ional Ca diology, Uni e si y Hea Cen e Hambu g, Ge many (S.B., T.Z.); Ge - man Cen e o Ca dio ascula esea ch, pa ne si e Hambu g/ Lübeck/Kiel, Hambu g, Ge many (S.B., T.Z.); and Es onian Genome Cen e , Uni e si y o Ta u, Es onia (M.P.). Sou ces o Funding This s udy was suppo ed by Aa ne Koskelo Founda ion; he Academy o Finland g an s 269517, 250207, 269517, 283045, 297338, 286284 (D Leh imäki), 134309(Eye), 126925, 121584, 124282, 129378(Sal e), 117787(Gendi), and 41071(Skidi); Biomedicum Helsinki Founda ion; he Compe i i e S a e Resea ch Financing o he Expe Responsibili y a ea o Tampe e, Tu ku; Kuopio Uni e si y Hospi al (g an X51001); he Diabe es Resea ch Founda ion o he Finnish Diabe es Associa ion; Emil Aal onen Founda ion; he EU FP7 g an s 313010 (BBMRI-LPC), 305280 (MIMOmics), and HZ2020 633589 (Ageing wi h Elegans); Finnish Cul u al Founda ion; Finnish Founda ion o Ca dio ascula Resea ch; Ida Mon in Founda ion; In eg a i e Li e Science Doc o al P og am o he Uni e si y o Helsinki; Juho Vainio Founda ion; Paa o Nu mi Founda ion; Signe and Ane Gyllenbe g Founda ion; he Social Insu ance Ins i u ion o Finland, Tampe e Tube culosis Founda ion; and Y jö Jahnsson Founda ion. Disclosu es None. Re e ences 1. 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CLINICAL PERSPECTIVE A ial na iu e ic pep ide and B- ype na iu e ic pep ide a e unique ho mones sec e ed by ca diomyocy es, o en used in he diagnos ics o hea ailu e. They bind o he same ecep o , bu unexpec ed di e ences in hei e ec s ha e been epo ed in bo h human and animal models. We used genome-wide associa ion analysis o s udy gene ic a ia ion a ec ing hei ci cula ing concen a ion, iden i ying 8 a ian s nea he genes NPPA, NPPB, PPP3CC, and GALNT4. Subsequen ly, we in es iga ed he co ela ion be ween he na iu e ic pep ide–associa ed gene ic a ian s and blood p essu e. Gene ic a ian s lowe ing he concen a ion o mid egional p oa ial na iu e ic pep ide associa ed wi h highe blood p essu e, bu we did no obse e a simila blood p essu e co ela ion wi h gene ic a ian s a ec ing B- ype na iu e ic pep ide o NT-p oBNP (N- e minal p o-B- ype na iu e ic pep ide). The e ec sizes o he mid egional p oa ial na iu e ic pep ide co ela ed gene ic a ian s on blood p essu e we e small, om 0.25 o 0.50 mm Hg pe allele. Thei combined e ec , howe e , associ- a ed wi h a 9% di e ence in he odds a io o hype ension, con ibu ing signi ican ly o he bu den o high blood p essu e in he gene al popula ion. by gues on Janua y 8, 2018h p://ci cgene ics.ahajou nals.o g/Downloaded om 6 he mean RMSD o he e ained SNPs agains he ange o possible cu o alues ( om 0 o 2 by inc emen s o 0.001). Fo alues g ea e han 0.075, he mean RMSD inc eased sha ply in a non-linea manne indica ing he inclusion o SNPs wi h pa icula ly high popula ion di e ences in LD. Fo alues smalle han 0.075, he ela ionship was app oxima ely linea . Pheno ypic a iance explained genome-wide We used he uni o mly impu ed da ase o es ima e he ac ion o pheno ypic a iance explained by he SNPs bu modi ied he impu a ion quali y and HWE es P- alue h esholds. Because impu a ion quali y is posi i ely co ela ed wi h MAF, we included SNPs wi h IMPUTE INFO me ic > 0.3 in o de o a oid unnecessa ily penalizing a e SNPs. Due o he inc ease in he size o he da ase as compa ed o he disco e y phase GWAS, we used a nume ically smalle HWE P- alue limi by excluding SNPs wi h P < 0.005 o a es on HWE. We es ima ed he gene ic ela ionship ma ix (GRM) o he samples using PLINK 1.90.[10] Nex , we used he GRM o es ima e he pheno ypic a iance explained by he geno ypes wi h he GCTA 1.25.3 p og am in he pa icipan s o he GWAS disco e y sample, se ing aside 863 samples o which duplica e geno ypes om o he geno yping a ays we e a ailable o be used in quali y con ol.[11] In o de o assess he eliabili y o he es ima es o pheno ypic a iance explained, we de i ed ou genomic sco es co esponding o each o he ou es ima es and s udied he associa ion o he genomic sco es wi h hei espec i e pheno ypes in he eplica ion sample as ollows: Fo each pheno ype, we de i ed he con ibu ion o he indi idual SNPs o he o al gene ic e ec using GCTA and used hese as weigh s o es ima e he o al gene ic e ec o genomic sco e o each o he s udy pa icipan s o he ou ai s. The genomic sco es we e echnically obus as he co ela ion be ween he 863 geno yping eplica es was high (Pea son's p oduc -momen co ela ion > 0.99) o all 7 pheno ypes. We hen es ed he associa ion o he genomic sco es o MR-p oANP, NT-p oBNP, BNP, and BNP:NT-p oBNP a io wi h said ai s in he eplica ion sample using linea eg ession. We con i med a s a is ically signi ican associa ion (P < 0.05) wi h NT-p oBNP, BNP, and BNP:NT-p oBNP a io bu no wi h MR-p oANP (Supplemen al Table 2). 8 Supplemen al Tables Supplemen al Table 1. Sample cha ac e is ics GWAS Sample Replica ion Sample Blood P essu e S udy Sample N4,932 1,373 27,059 Age (yea s) 46.18 (21.39) 48.67 (20.5) 42.41 (25.42) Females (n/%) 2,592 (52.55%) 711 (51.78%) 14,377 (53.13%) Body Mass Index (kg/m2) 25.73 (5.473) 25.97 (5.116) 25.69 (5.714) Dias olic Blood P essu e (mm Hg) 82 (15) 82 (14) 80 (16) Sys olic Blood P essu e (mm Hg) 132 (26) 134 (26) 130 (25) Hype ension (n/%) 2,090 (42.38%) 605 (44.06%) 10,404 (39.3%) Smoking (n/%) 1,230 (24.94%) 300 (21.85%) 3,893 (24.9%) P e alen Hea Failu e (n/%) 0 (0%) 0 (0%) na. Inciden Hea Failu e (n/%) 289 (5.86%) 84 (6.118%) na. NT-p oBNP (pg/ml) 39.26 (56.49) 46.75 (57.2) na. MR-p oANP (pmol/L) 41.3 (25.2) 43.4 (25.4) na. BNP (pg/ml) 12.9 (17.6) 14.9 (19.6) na. Quan i a i e a iables: median (in e qua ile ange) Quali a i e a iables: numbe (p opo ion) BNP: B- ype na iu e ic pep ide, MR-p oANP: mid- egional p oa ial na iu e ic pep ide, NT-p oBNP: amino e minal p o-B- ype na iu e ic pep ide 9 Supplemen al Table 2. T ai a iance explained by he genome-wide geno ype da a T ai Va iance Explained (SE ) Genomic Sco e Associa ion* MR-p oANP 0.139 (0.071) Be a=1.60, P=0.31 BNP 0.135 (0.070) Be a=2.70, P=0.0049 NT-p oBNP 0.230 (0.072) Be a=9.40, P=0.015 BNP:NT-p oBNP 0.179 (0.071) Be a=0.03, P=0.0055 P opo ion o pheno ypic a iance explained by au osomal SNPs in he GWAS disco e y sample and he associa ion o he co esponding genomic sco es wi h he ai s in he eplica ion sample. BNP: B- ype na iu e ic pep ide, MR-p oANP: mid- egional p oa ial na iu e ic pep ide, NT-p oBNP: amino e minal p o-B- ype na iu e ic pep ide, SE: s anda d e o * Be a coe icien s gi en as dimensionless uni s (BNP:NT-p oBNP a io), pg/ml (BNP and NT-p oBNP), o pmol/L (MR-p oANP) pe one s anda d de ia ion di e ence in he genomic sco e 10 Supplemen al Table 3. Associa ion o genome-wide signi ican gene ic a ian s wi h na iu e ic pep ide ai s T ai SNP* Ch Posi ion Alleles** PGWAS PREPLICATION Be a (95% CI) PCOMBINED BNP s3753584 1 11864586 T/C 8.49 × 10-17 1.33 × 10-4 0.2047 (0.1609 o 0.2486) 5.71 × 10-20 s198379 1 11915467 T/C 6.85 × 10-41 7.99 × 10-13 0.2494 (0.2172 o 0.2816) 4.42 × 10-52 s61761991 1 11918444 C/T 0.0079 0.0904 -0.1465 (-0.2378 o -0.0552) 0.0017 s7000551 8 22276251 A/G 0.3909 0.4384 -0.0046 (-0.0414 o 0.0322) 0.8069 s11105298 12 89876143 T/C 0.3759 0.2768 0.0032 (-0.056 o 0.0623) 0.9168 s10858906 12 89934474 C/T 0.1767 0.3064 -0.0022 (-0.0699 o 0.0654) 0.9482 s701041 12 124999344 G/C 0.0047 0.084 -0.0011 (-0.1767 o 0.1745) 0.9905 BNP:NT-p oBNP s3753584 1 11864586 T/C 0.061 0.3392 -0.0523 (-0.1011 o -0.0036) 0.0354 s198379 1 11915467 T/C 0.0709 0.7244 -0.0321 (-0.0679 o 0.0037) 0.0786 s61761991 1 11918444 C/T 7.17 × 10-79 5.71 × 10-26 1.1138 (1.0125 o 1.215) 4.81 × 10-103 s7000551 8 22276251 A/G 2.16 × 10-8 0.0248 0.1085 (0.0731 o 0.144) 2.00 × 10-9 s11105298 12 89876143 T/C 3.06 × 10-18 4.11 × 10-6 0.2103 (0.1685 o 0.2521) 6.77 × 10-23 s10858906 12 89934474 C/T 2.06 × 10-17 5.87 × 10-7 0.2105 (0.1686 o 0.2524) 7.27 × 10-23 s701041 12 124999344 G/C 0.3886 0.5158 -0.0313 (-0.0886 o 0.0261) 0.2858 MR-p oANP s3753584 1 11864586 T/C 4.63 × 10-38 3.48 × 10-7 0.2752 (0.2008 o 0.3495) 4.19 × 10-13 s198379 1 11915467 T/C 3.97 × 10-7 0.0016 0.0914 (0.0614 o 0.1214) 2.46 × 10-9 s61761991 1 11918444 C/T 1.97 × 10-4 0.3494 -0.1562 (-0.253 o -0.0595) 0.0016 s7000551 8 22276251 A/G 0.6066 0.6892 -0.004 (-0.0337 o 0.0258) 0.7944 s11105298 12 89876143 T/C 0.0369 0.0109 0.0217 (-0.1111 o 0.1545) 0.7489 s10858906 12 89934474 C/T 0.0457 0.0225 0.0179 (-0.1046 o 0.1404) 0.7746 s701041 12 124999344 G/C 1.23 × 10-8 0.9381 -0.0876 (-0.2408 o 0.0656) 0.2624 NT-p oBNP s3753584 1 11864586 T/C 1.29 × 10-21 5.65 × 10-6 0.2235 (0.182 o 0.2649) 4.23 × 10-26 s198379 1 11915467 T/C 4.36 × 10-47 5.76 × 10-16 0.2565 (0.2261 o 0.287) 2.30 × 10-61 s61761991 1 11918444 C/T 1.72 × 10-51 5.33 × 10-18 -0.7663 (-0.8526 o -0.68) 8.76 × 10-68 s7000551 8 22276251 A/G 3.72 × 10-5 0.8706 -0.0442 (-0.1086 o 0.0201) 0.1781 s11105298 12 89876143 T/C 2.54 × 10-11 0.0929 -0.107 (-0.1796 o -0.0344) 0.0039 s10858906 12 89934474 C/T 1.08 × 10-12 0.0388 -0.1199 (-0.1862 o -0.0536) 3.91 × 10-4 s701041 12 124999344 G/C 0.0139 0.0366 0.0135 (-0.1602 o 0.1871) 0.8792 Associa ion o SNPs wi h na iu e ic pep ides in he GWAS. Associa ion es ed wi h an addi i e gene ic model adjus ed o geog aphical sampling egion, age, age2, sex, cu en smoking s a us (yes o no), 11 sys olic blood p essu e, es ima ed glome ula il a ion a e, and geno yping ba ch. Genomic posi ions gi en ela i e o he GRCh37 e e ence genome build. BNP: B- ype na iu e ic pep ide, MR-p oANP: mid- egional p oa ial na iu e ic pep ide, NT-p oBNP: amino e minal p o-B- ype na iu e ic pep ide * Genome-wide signi ican lead SNPs o each ai and locus a e ma ked wi h bold unde lined ex ** Alleles gi en as [ e e ence allele]/[e ec allele] 12 13 Supplemen al Table 4. Replica ion o p e iously published associa ions. NT-p oBNP MR-p oANP BNP BNP:NT-p oBNP Snp Ch Pos Alleles* Be a P AP** Be a P AP** Be a P AP** Be a P AP** s1023252 111899033 G/T 0.2277 4.71 × 10-43 T[12] 0.0834 3.33 × 10-7 0.1755 1.31 × 10-23 -0.1112 1.13 × 10-8 s198358 1 11904076 T/C 0.1724 6.71 × 10-20 0.2008 4.60 × 10-27 C[13] 0.1945 1.94 × 10-22 C[13] 0.0156 0.4823 s5063 111907648 C/T 0.2532 1.30 × 10-9 -0.055 0.1813 C[14] 0.1904 0.0041 -0.1637 0.1528 s198389 1 11919271 A/G 0.2535 1.03 × 10-60 G[15] 0.0905 2.79 × 10-9 0.2462 2.48 × 10-51 -0.0328 0.0705 s35207557 111917620 T/TA 0.2502 2.98 × 10-58 0.0919 2.20 × 10-9 0.2448 5.32 × 10-50 TA[16] -0.0284 0.1585 s5068 111905974 A/G 0.2213 4.26 × 10-6 0.3298 7.68 × 10-30 G[13] 0.1782 4.27 × 10-11 G[13] -0.1265 0.0097 s549596 111916095 T/C 0.2474 4.69 × 10-58 0.0932 8.76 × 10-10 0.2417 8.42 × 10-50 C[16] -0.027 0.1366 s632793 1 11910677 A/G 0.254 3.80 × 10-59 0.0938 1.30 × 10-9 G[13] 0.2448 1.81 × 10-49 G[13] -0.034 0.0647 s13107325 4103188709 C/T 0.1824 0.005 T[17] -0.0498 0.4356 -0.0658 0.3373 -0.4549 2.19 × 10-9 s3733402 4 187158034 G/A 0.0053 0.9054 0.0136 0.7592 -0.033 0.3429 G[18] -0.0529 0.0028 s6557662 823230898 A/G -0.0061 0.7792 A[16] -0.0189 0.6837 -0.0251 0.3838 -0.0217 0.4283 s11105306 12 89897388 C/T -0.1159 2.77 × 10-5 C[17] 0.0213 0.7416 -0.0002 0.9935 0.2084 9.55 × 10-22 Associa ion o p e iously published SNPs wi h na iu e ic pep ide ai s in he me a-analysis o he GWAS and eplica ion samples. All models adjus ed o geog aphical sampling egion, age, age2, sex, cu en smoking s a us (yes o no), sys olic blood p essu e, es ima ed glome ula il a ion a e, and geno yping ba ch. BNP: B- ype na iu e ic pep ide, MAF: mino allele equency, MR-p oANP: mid- egional p oa ial na iu e ic pep ide, NT-p oBNP: amino e minal p o-B- ype na iu e ic pep ide * Alleles gi en as [ e e ence allele]/[e ec allele] ** AP: P e iously published ai inc easing-allele 14 Supplemen al Table 5. Mul i-SNP models on ch omosome 1 T ai SNP Be a SE P BNP s198379_C 0.2385 0.03445 4.42 × 10-12 BNP s3753584_C 0.03386 0.02994 0.2581 BNP s6540997_G 0.01056 0.02655 0.6909 BNP s4845875_C 0.01701 0.02356 0.4703 BNP s61761991_T -0.03811 0.0527 0.4696 BNP s12406089_G 0.03313 0.05278 0.5302 MR-p oANP s198379_C -0.03905 0.03207 0.2234 MR-p oANP s3753584_C 0.1937 0.05636 5.88 × 10-4 MR-p oANP s6540997_G 0.1249 0.02474 4.47 × 10-7 MR-p oANP s4845875_C -0.1264 0.02197 8.60 × 10-9 MR-p oANP s61761991_T 0.01139 0.04926 0.8171 MR-p oANP s12406089_G 0.06408 0.05276 0.2246 NT-p oBNP s198379_C 0.1347 0.05406 0.0127 NT-p oBNP s3753584_C 0.00616 0.02794 0.8255 NT-p oBNP s6540997_G 0.01158 0.04709 0.8057 NT-p oBNP s4845875_C -0.01402 0.02191 0.5223 NT-p oBNP s61761991_T -0.6503 0.04891 2.42 × 10-40 NT-p oBNP s12406089_G 0.1222 0.05417 0.0241 BNP:NT-p oBNP s198379_C 0.1083 0.04947 0.0286 BNP:NT-p oBNP s3753584_C 0.02899 0.04006 0.4694 BNP:NT-p oBNP s6540997_G -0.02637 0.0508 0.6037 BNP:NT-p oBNP s4845875_C 0.04523 0.02601 0.0821 BNP:NT-p oBNP s61761991_T 1.085 0.05833 3.33 × 10-77 BNP:NT-p oBNP s12406089_G -0.1126 0.03479 0.0012 Mul i a ia e models o SNP-na iu e ic pep ide associa ion wi h all iden i ied SNPs on ch omosome included simul aneously. 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