Heredity and cardiometabolic risk

Original Article

Heredity and cardiometabolic risk naturallyoccurring polymorphisms in the human neuropeptideY2 receptor promoter disrupt multiple transcriptionalresponsemotifs

Zhiyun Weiabce Kuixing Zhangabc Gen Wenabc Karthika BalasubramanianabcPei-an B Shihabc Fangwen Raoabc Ryan S Frieseabc Jose P Miramontes-GonzalezabcGeert W Schmid-Schoenbeinabc Hyung-Suk Kimd Sushil K Mahataabc andDaniel T OrsquoConnorabc

C

Journal of Hypertension 2013 31123ndash133aDepartment of Medicine bDepartment of Pharmacology cDepartment of Bioengin-eering the Institute for Genomic Medicine University of California at San Diego theVA San Diego Healthcare System La Jolla California dDepartment of PathologyUniversity of North Carolina at Chapel Hill Chapel Hill USA and eBio-X InstitutesShanghai Jiao Tong University China

Correspondence to Daniel T OrsquoConnor MD Department of Medicine (0838) UCSDSchool of Medicine and VASDHS 9500 Gilman Drive La Jolla CA 92093-0838 USATel +1 858 5340661 fax +1 858 5340626 e-mail doconnorucsdedu

Received 7 June 2012 Revised 28 August 2012 Accepted 4 October 2012

J Hypertens 31123ndash133 2012 Wolters Kluwer Health | Lippincott Williams ampWilkins

Jo

Objectives The neuropeptide Y2 G-protein-coupledreceptor (NPY2R) relays signals from PYY or neuropeptideY toward satiety and control of body mass Targetedablation of the NPY2R locus in mice yields obesity andstudies of NPY2R promoter genetic variation in more than10 000 human participants indicate its involvement incontrol of obesity and BMI Here we searched for geneticvariation across the human NPY2R locus and probed itsfunctional effects especially in the proximal promoter

Methods and results Twin pair studies indicatedsubstantial heritability for multiple cardiometabolic traitsincluding BMI SBP DBP and PYY an endogenous agonistat NPY2R Systematic polymorphism discovery byresequencing across NPY2R uncovered 21 genetic variants10 of which were common [minor allele frequency (MAF)gt5] creating one to two linkage disequilibrium blocks inmultiple biogeographic ancestries In vivo NPY2Rhaplotypes were associated with both BMI (Pfrac14375E04)and PYY (Pfrac14401E06) Computational approachesrevealed that proximal promoter variants G-1606A C-599T and A-224G disrupt predicted IRF1 (AgtG) FOXI1(TgtC) and SNAI1 (AgtG) response elements Inneuroendocrine cells transfected with NPY2R promoterluciferase reporter plasmids all three variants and theirresulting haplotypes influenced transcription (G-1606APlt297E06 C-599T Plt117E06 A-224GPlt204E06) and transcription was differentiallyaugmented or impaired by coexpression of either thecognate full-length transcription factors or their specificsiRNAs at each site Endogenous expression of transcriptsfor NPY2R IRF1 and SNAI1 was documented inneuroendocrine cells and the NPY2R mRNA wasdifferentially expressed in two neuroendocrine tissues(adrenal gland brainstem) of a rodent model ofhypertension and the metabolic syndrome thespontaneously hypertensive rat

Conclusion We conclude that common genetic variationin the proximal NPY2R promoter influences transcriptionfactor binding so as to alter gene expression in

opyright copy Lippincott Williams amp Wilkins Unauthurnal of Hypertension

neuroendocrine cells and consequently cardiometabolictraits in humans These results unveil a novel control pointwhereby cis-acting genetic variation contributes to controlof complex cardiometabolic traits and point to newtranscriptional strategies for intervention into neuropeptideactions and their cardiometabolic consequences

Keywords autonomic genetics hypertension nervoussystem obesity

Abbreviations NPY neuropeptide Y NPY2Rneuropeptide Y2 receptor PYY peptide YY SNP singlenucleotide polymorphism

INTRODUCTION

The neuropeptide Y (NPY) receptor Y2 (NPY2ROMIM 162642 IUPHAR Y2) is a G-protein-coupledreceptor responding to hormones peptide YY (PYY)

[1] and NPY to control appetite and cardiovascular homeo-stasis There are five subtypes of NPY receptor identified inmammals four of which are functional in humans Sub-types Y1 and Y5 have known roles in the stimulation offeeding whereas Y2 and Y4 seem to have roles in appetiteinhibition NPY2R is widely expressed in tissues pertinentto cardiometabolic control including the arcuate nucleus amajor integrator of appetite control in the hypothalamus Inprevious studies NPY2R genetic variants were associated

orized reproduction of this article is prohibited

DOI101097HJH0b013e32835b053d

wwwjhypertensioncom 123

Wei et al

with obesity or BMI in several populations includingwhites [23] Asians [4] and Africans [5] Indeed studiesof NPY2R promoter genetic variation in more than 10 000individuals [236] indicate its involvement in control ofobesity or BMI (on-line Table 1 httplinkslwwcomHJHA209) NPY2R also cooperates with NPY in stress-induced obesity and the metabolic syndrome [7] NPY2Rgenetic variants associate with such human cardiometabolictraits as high-density lipoprotein cholesterol [8] SBP [3] type2 diabetes in men [9] and left ventricular hypertrophy [10]Hypothalamus-targeted NPY2R-knockout mice showed adecrease in body weight despite an increase in food intake[11] In the rat (httprgdmcwedu) the Npy2r genetic locusunderlies the confidence interval of a quantitative trait locus(QTL) for blood pressure (BP) BP QTL-90 (Bp90) [12] Suchdiverse evidence indicates that NPY2R plays an indispensa-ble role in the cardiometabolic syndrome

In these studies we first documented the role of heredityin cardiometabolic traits using twin pair variance com-ponents and then systematically searched for naturallyoccurring genetic variation across the human NPY2R locusBecause severalof thediscoveredcommonvariants occurredin a likely functional domain (thepromoter)weprobed theirmechanistic consequences beginning with bioinformaticmotif analysis and proceeding to transfected promoterluci-ferase reporter plasmids site-directed mutagenesis andcharacterization of trans-acting factors We developedevidence that variation in the NPY2R promoter especiallyat common variants G-1606A C-599T and A-224G disruptparticular motifs (IRF1 FOXI1 and SNAI1 elements respect-ively) creating differential cis-interactions and trans-interactions to alter transcriptional activity and ultimatelyBP body mass and associated risk traits in the population

PARTICIPANTS ANDMETHODS

Genomics

Systematic polymorphism discovery at the NPY2RlocusWe studied the NPY2R locus in nfrac14 80 participants (2nfrac14 160chromosomes) as described below under lsquoHuman partici-pantsrsquo Genomic DNA was prepared from leukocytes asdescribed previously [13] Public draft human genomesequences were obtained from the University of CaliforniaSanta Cruz Genome Bioinformatics website (httpgenomeucscedu) and used as a scaffold for primer designThe base position numbers were according to the NationalCenter for Biotechnology Information (NCBI) NPY2R sourceclone RefSeq genetranscript NM_0009102 Promoter pos-itions were numbered upstream of () the NPY2R exon-1start (cap) site PCR primers were designed by primer-3 [14](httpfrodowimiteduprimer3) to capture approxi-mately 2000bp of the proximal promoter between approxi-mately 500bp to approximately2000bp over each of the twoexons (including 50-UTR 30-UTR and exonintron borders)and regions highly conserved across species Target regionswere amplified and then dideoxy-sequenced using an ABI-3100 capillary sequencer (Applied Biosystems CarlsbadCaliforniaUSA) Polymorphism (typically as heterozygosity)was visualized on the Applied Biosystems (ABI) tracings

Copyright copy Lippincott Williams amp Wilkins Unauth124 wwwjhypertensioncom

using Codon Code Aligner (httpwwwcodoncodecomaligner)

Human participants

Resequencing the NPY2R locusHuman studies were approved by the University ofCalifornia San Diego (UCSD) Human Research ProtectionProgram Experiments were conducted with the under-standing and consent of each participant We studied theNPY2R locus in nfrac14 80 participants (2nfrac14 160 chromo-somes) from four diverse biogeographic ancestry groupssystematically sequenced for polymorphism discoveryacross the NPY2R locus white (European ancestry2nfrac14 46 chromosomes) black (sub-Saharan African ances-try 2nfrac14 50 chromosomes) Hispanic (Mexican American2nfrac14 32 chromosomes) and east Asian (2nfrac14 32 chromo-somes)

UCSD twin pairsTwin recruitment included access to a population birthrecord-based twin registry [15] as well as by newspaperadvertisement as described [16] Description of the 362participants in the twin heritability and allelic associationstudies has been published [17] For human allelic andhaplotype association studies this twin group wasexpanded to 693 participants of European ancestry derivedfrom additional siblings from twinships and sibships aspreviously described [18]

Statistics and informatics

Linkage disequilibrium and haplotypesIn the resequenced participants patterns of linkage dis-equilibrium as well as haplotype frequencies were analyzedand visualized by the software Haploview (Broad InstituteMassachusetts USA) [19] Linkage disequilibrium blockswere derived by the confidence interval criterion andvisualized by r2 plot in Haploview from unphased diploidgenotypes of nfrac14 80 resequenced participants (2nfrac14 160chromosomes) from four diverse biogeographic ancestrygroups systematically sequenced across the NPY2R locusCommon variants (minor allele frequency gt5) were usedto establish linkage disequilibrium

In the twins and siblings haplotype-on-trait analyseswere conducted by regression in R (reporting effect sizeas b or slope per allele as well as its SEM) with Haploglmin Haplostats [20] (httpmayoresearchmayoeduschai-d_labsoftwarecfm) Trait-associated haplotype GTT waspresent on 111 of chromosomes analyzed

Bioinformatics computational prediction oftranscription factor-binding motifs overlying NPY2Rpromoter common variantsMultiple sequence alignments were performed by Clustal-W [21] (httpwwwebiacukToolsclustalw2) Potentialtranscription factor binding motifs were predicted from theJASPAR [22] (httpjaspargeneregnet) and ConSite [23](httpaspiiuibno8090cgi-binCONSITEconsite) data-bases

orized reproduction of this article is prohibitedVolume 31 Number 1 January 2013

NPY2R polymorphism

Heritability and pleiotropy (shared geneticdetermination or genetic covariance (rG)Estimates of heritability (h2) (h2frac14VGVP wherein VG isadditive genetic variance and VP is total phenotypicvariance) were obtained using the twin-pair variance-component methodology implemented in the SequentialOligogenic Linkage Analysis Routines (SOLAR) package[24] available at (httptxbiomedorgdepartmentsgenetics) This method maximizes the likelihood assuminga multivariate normal distribution of phenotypes intwin pairs (monozygotic versus dizygotic) with a meandependent on a particular set of explanatory covariatesThe null hypothesis (H0) of no heritability is tested bycomparing the full model which assumes genetic vari-ation (VG) and a reduced model which assumes nogenetic variation using a likelihood ratio test Heritabilityestimates were adjusted for age and sex because of theeffects of these covariates on several traits Pleiotropy(genetic covariance for two correlated heritable traitsie the cross-product of trait heritabilities) [25] wasestimated as the parameter rG in SOLAR [25] SOLARalso estimated the environmental covariance as para-meter rE

Functional studies of NPY2R genetic variation

Human phenotyping peptide YYHuman PYY (total) was measured using a Linco (MilliporeSt Charles Missouri USA) HRP-TMB ELISA kit (catalog EZHPYYT66K) EDTA-anticoagulated plasma was frozenand stored at 708C prior to assay this ELISA measures theenzyme by absorbance at 450 nm The assay sensitivity is14 pgml plasma with an intra-assay coefficient of varia-bility (CV) of 09ndash58 and interassay CV of 37ndash165The assay equivalently recognizes PYY1ndash36 and PYY3ndash36but does not cross-react (at up to 50 nmoll) with NPYghrelin gastric inhibitory polypeptide glucagon glucagon-like peptide-1 leptin insulin C-peptide amylin or adipo-nectin PYY distribution in human individuals was tested bythe one-sample two-tailed nonparametric KolmogorovndashSmirnov test in SPSS (IBM Corporation New York USA)untransformed PYY deviated from normality (Pfrac14 0002)whereas log[10]-transformed PYY did not display suchdeviation (Pfrac14 0291) Estimates of heritability (by variancecomponents in SOLAR see above) did not differ whenperformed on untransformed versus log[10]-transformedPYY data (see RESULTS)

Human single nucleotide polymorphism genotypingand marker-on-trait associationSingle nucleotide polymorphism (SNP) genotypes atrs6851222 (Promoter G-1606A) rs6857715 (PromoterC-599T) and rs1047214 (Exon-2 TC Ile195Ile) werechosen to span the NPY2R locus and typed by the TaqManmethod on an ABI-7900HT Fast Real-Time PCR Systemwith labeled probes synthesized at Applied BiosystemsEach SNP was in Hardy Weinberg equilibrium (allPgt 005) Haplotypes were derived from diploid genotypedata and haplotype-on-trait analyses were conducted byregression [20] or by Generalized Estimating Equations

Copyright copy Lippincott Williams amp Wilkins UnauthJournal of Hypertension

with analyses adjusted for age sex and biogeographicancestry

NPY2R promoter haplotypeluciferase reporterdesign and constructionHuman NPY2R promoter fragments corresponding toNPY2R-2323thorn130bp in NPY2R (NCBI NPY2R sourceclones RefSeq genetranscript NM_0009102) were PCR-amplified from genomic DNA (after resequencing) andcloned into the polylinker (between KpnI and BglII sites)of the promoterless firefly luciferase reporter plasmid pGL3-Basic (Promega Madison Wisconsin USA) as described[13] Site-directed mutagenesis (QuikChange StratageneSanta Clara California USA) created the required variantat position 1606 599 and 224 (Supplemental DigitalContent Fig S1 httplinkslwwcomHJHA209) Super-coiled plasmids were purified using NucleoBond Xtra Maxikits (74041410 Machery-Nagel Bethlehem PennsylvaniaUSA) prior to transfection and verified by sequencingPromoter positions are numbered upstream () of the tran-scriptional start (cap) site

Luciferase reporter assays of NPY2R promotervariantsPC12 rat pheochromocytoma cells were transfected(at 60ndash80 confluence 1 day after 1 4 splitting in 24-wellplate) with 500 ng of supercoiled promoterfirefly luciferasereporter plasmid per well by the liposome method (Trans-fectin Bio-Rad Hercules California USA) The firefly luci-ferase activity in cell lysates was measured 24 h aftertransfection using the luciferase assay system (Promega)and the results were expressed as the ratio of firefly activitytotal protein in the lysate as described [13] Each experi-ment included at least three replicates Results wereexpressed as mean SEM Statistical significance wascalculated using Studentrsquos t-test or ANOVA and significancewas established at the P value less than 005 level Inspec-tion of the NCBI Gene Expression Omnibus (GEO) data-base (httpwwwncbinlmnihgovgeo) indicates thattranscripts for NPY2R are abundantly expressed in theadrenal gland (GEO dataset GDS3556 and GDS2374) aswell as PC12 chromaffin cells (GDS2555)

Exogenouscotransfected transcription factorsEukaryotic expression plasmids containing cDNAs encod-ing transcription factors IRF1 (rat clone ID 7099391) FOXI1(human clone ID 5185923) and SNAI1 (human clone ID4537122) were from Open Biosystems (HuntsvilleAlabama USA) cDNAs were obtained in eitherpExpress-1 or cytomegalovirus promoter (pCMV)-SPORT6plasmids and subcloned if needed into a eukaryotic pCMVexpression vector (pcDNA-31) One hundred nanogramsof each transcription factor expression plasmid or 100 ngpcDNA-31 empty vector (control) was cotransfected intoPC12 cells along with 500 ng of NPY2R promoterluciferasereporter wild-type versus variants After 24 h cells werelysed and luciferase activities were assayed as describedabove and normalized by total protein Response of theNPY2R promoter to exogenous transcription factor wasrevealed by comparison of the normalized luciferase

orized reproduction of this article is prohibitedwwwjhypertensioncom 125

Wei et al

activity between the transcription factor-transfected groupand the mock-transfected (empty vector pcDNA 31)group

Exogenouscotransfected siRNAsSilencer select predesigned siRNAs targeting IRF1 (rat siRNAID s127967) FOXI1 (rat siRNA ID s220491) or SNAI1 (ratsiRNA ID s137986)were from Ambion (AppliedBiosystems)Silencer select negative control 1 siRNA (part number4390843) was used as the negative control Six nanomolesper litre final concentration of each transcription factorsiRNA or negative control siRNA was cotransfected intoPC12 cells along with 500ng of NPY2R promoterluciferasereporter wild-type versus variant After 24h cells were lysedand luciferase activitieswere assayed as described above andnormalized by total protein Response of the NPY2R pro-moter to exogenous siRNAs was revealed by comparison ofthe normalized luciferase activity between the transcriptionfactor siRNA-transfected group and the mock-transfected(negative control siRNA) group

Quantification of endogenous transcripts by real-time PCR NPY2R itself and transcripts for factorswhose binding motifs are disrupted by NPY2Rpromoter variants (IRF1 FOXI1 SNAI1)Total RNA was extracted from cells (neuroendocrine PC12)or organs under each experimental state using an ABI 6700automated nucleic acid workstation and quantitative real-time PCR (RT-PCR) was performed on mRNAcDNA withthe ABI-7700 TaqMan platform using fluorescent reporter-tagged oligonucleotide primers and normalization of datato b-actin expression in the same sample Threshold cycle(Ct) is determined for both the specific target mRNAcDNAas well as b-actin and the difference in Ct (for target mRNAversus b-actin mRNA) is normalized to the average for thatstate (eg control versus experimental) by the DDCt

method [26]

Copyright copy Lippincott Williams amp Wilkins Unauth

0

02

04

Her

itab

ility

(h

2 = V

GV

P)

06

08

1

(a) (bHeritability

BMI (kgm2)

h2 = 086+ndash002

P lt 00001

h2 = 046+ndash006

P lt 00001

h2 = 052+ndash006

P lt 00001

h2 = 051+ndash006

P lt 00001

SBP (mmHg) DBP (mmHg)

Trait

PYY (pgml)

FIGURE 1 Heredity pleiotropy and human cardiometabolic traits (a) Heritability (h2) twgenetic variance (ie h2frac14VGVP) h2 ( SEM with significance for h2) is displayed for BMshared genetic determination or genetic covariance (rG) for BMI with other cardiometabpair studies above are illustrated graphically as mean SEM for each covariance with P

126 wwwjhypertensioncom

Experimental animals spontaneously hypertensiverat and WistarndashKyoto ratAnimal studies were performed with age-matched adult(12ndash17 weeks) male spontaneously hypertensive rat (SHR)and WistarndashKyoto (WKY) rat strains from Charles RiverLaboratories (Wilmington Massachusetts USA) Features ofthe Charles River colonies including BP monitoringare given at (httpwwwcrivercomEN-USPRODSERVBYTYPERESMODOVERRESMODPagesSHRRataspx)Isoflurane was used for terminal anesthesia of SHR andWKY rats Adrenal glands and brainstem were isolated fromeach rat (nfrac14 9 per group) immediately frozen in liquidnitrogen and then stored at 808C prior to RNA extractionand RT-PCR Rats were studied according to a protocolapproved by the Animal Subjects Committee of the Uni-versity of California at San Diego and research was con-ducted in accordance with institutional guidelines

RESULTS

Heredity pleiotropy and cardiometabolic traitsin humansTwin pair variance component analyses indicate thatmultiple cardiometabolic traits display substantial andsignificant (Pfrac14 00001) heritability (h2) (Fig 1a) includ-ing BMI (h2frac14 86 2) SBP (h2frac14 46 6) DBP(h2frac14 52 6) and circulating PYY (h2frac14 51 6) theprincipal endogenous ligand for the NPY2R Heritabilityestimates for BMI SBP and DBP were consistent withpreviously reported values [17] Using the twin methodwe also investigated genetic pleiotropy (shared geneticdetermination or genetic covariance) between BMI andother cardiometabolic traits (Fig 1b) BMI displayed sig-nificant genetic covariance with SBP (Pfrac14 931E05) DBP(Pfrac14 774E04) and PYY (Pfrac14 30E02) by contrastenvironmental covariance (or shared environmental deter-mination rE) was not significant for these same traits


ndash03ndash03

ndash02

ndash02

ndash01

ndash01

00

0

01

01

Rho_G (genetic covariance)

Rh

o_E

(en

viro

nm

enta

l co

vari

ance

)

02

02

03

03

04

04

05

)

05

SBPRho_G P = 931E-05Rho_E P = 076

DBPRho_G P = 774E-04Rho_E P = 021PYY

Rho_G P = 30E-02Rho_E P = 072

Genetic covariance with BMI

Y = X(line of

identity)

in pair variance components h2 is the fraction of trait variance accounted for byI SBP DBP and circulating plasma peptide YY (PYY) concentration (b) Pleiotropyolic traits Genetic (rG) and environmental (rE) covariance estimates from the twin-value for its significance

Volume 31 Number 1 January 2013

Promoter Human NPY2R (85 kbp)

Humanmousehomology

RefSeqNM_0009102Amplicons

Polyadenylationsignal (AAUAAA)Common SNPs

ndash1606ndash1324ndash1192ndash810

ndash224

+5895

+6242

ndash737ndash627ndash599

FIGURE 2 NPY2R resequencing strategy and identified variants Sequences conserved between mouse and human NPY2R were visualized with VISTA (httpgenomelblgovvistaindexshtml) Locations of common (minor allele frequency5) single nucleotide polymorphisms (SNPs) are indicated Positions are numbered upstream () ordownstream (thorn) of the CAP (transcription initiation) site Solid blocks open reading frame (ORF) hatched blocks UTRs Bi-directional horizontal arrows resequencingamplicons with sequencing direction(s) indicated by arrowheads

NPY2R polymorphism

Polymorphism discovery across NPY2RLocated on chromosome 4q31 NPY2R spans two exons(one coding) with one intervening sequence (intron) Weresequenced approximately 1800 bp of proximal promotereach of exon-1 and exon-2 [down to the first polyadeny-lation site (bold) 50-TACTAAATAAAACAAT-30] and adja-cent intronexon borders (Fig 2) in 2nfrac14 160 chromosomesderived from four biogeographic ancestry groups (Table 1)We identified 21 variants (18 SNPs 3InsDel) in theseindividuals Of these variants 10 are common [minor allele


TABLE 1 NPY2R polymorphism discovery in nfrac1480 individuals (ie 2

NPY2RSNPs Alleles

SNPposition

RefSNPnumber

Aminochang

1 AG 1637 promoter rs57869523 none

2 GA 1606 promoter rs6851222 none

3 TC 1449 promoter rs10212938 none

4 T 1324 promoter rs36032070 none



7 AGAG 807 promoter rs34874489 none

8 AT 737 promoter rs12507396 none


10 CT 599 promoter rs6857715 none

11 CA 314 promoter NA none

12 CG 265 promoter NA none



15 GA 220 promoter NA none

16 GC 186 promoter NA none

17 CT thorn85 exon-1 (50-UTR) NA none

18 CT thorn324 exon-1 (50-UTR) rs72972775 none

19 TC thorn5469 exon-2 rs2342674 L-53-L

20 TC thorn5895 exon-2 rs1047214 I-195-I


RefSNP reference SNP SNP single nucleotide polymorphism Positions and allele frequencies fois represented by population Ethnicity-specific frequencies are given if overallglobal MAF is gt5and those downstream from the cap site are positive (thorn) A RefSNP number from NCBI is given(lower case) alleles The high-frequency G-1606A C-599T and A-224G promoter variants whicnumbers Italics three variants chosen for inclusion in haplotypes to span the NPY2R locus for cC-599T) and rs1047214 (Exon-2 TC Ile195Ile)

Journal of Hypertension

frequency (MAF) gt5] including two in the open readingframe within coding exon-2 (both synonymous) Tthorn 5895C(Ile195Ile) and Tthorn 6242C (Ile312Ile) whereas the rest arelocated in the proximal promoter

Biogeographic ancestry and NPY2R linkagedisequilibriumNPY2R common allele frequencies did not differ across thefour biogeographic ancestry groups (Table 1) To visualizepatterns of marker-on-marker association pair-wise


nfrac14160 chromosomes) from four biogeographic ancestries

acide

Minor allele frequency

Whitenfrac1423

Blacknfrac1425

Hispanicnfrac1416

Asiannfrac1416

Allnfrac1480

ndash ndash ndash ndash 0018

025 016 025 047 0270


025 022 025 047 0290

011 000 012 019 0090

045 012 038 025 0287


012 008 022 020 0140

029 065 041 047 0470

029 036 041 047 0470




024 020 033 053 0310






048 012 034 007 0250

050 033 043 013 0360

r each common (global frequency 5) and rare (global frequency lt5) polymorphism Polymorphisms in the promoter region (upstream from the cap site) are numbered ()if available in the public database SNPs are represented as major (upper case) and minor

h we focused on during molecular biology experiments are marked with bold RefSNPlinical marker-on-trait associations rs6851222 (Promoter G-1606A) rs6857715 (Promoter


White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24

GTT on trait (regression)PYY P = 401Endash06BMI P = 375Endash04

NPY2R haplotype GTTCoordinate effects on PYY and BMI

BM

I (kg

m2 )

110 120 130

Black2n = 50

FIGURE 3 Haplotype analyses at the NPY2R locus (a) Linkage disequilibrium (LD) blocks across NPY2R in several biogeographic ancestry groups derived by confidenceintervals in Haploview Numerical values shown in diamonds are r2100 r2 color scheme r2frac140 white 0ltr2lt1 shades of grey r2frac141 black Common single nucleotidepolymorphisms (SNPs) MAF 5 Rectangles exons Diagonal shading noncoding (UTR) Solid shading coding (open reading frame) (b) Polymorphism at NPY2Rinfluences human cardiometabolic traits haplotype effects across the locus lsquotaggedrsquo by three SNPs (see Fig 1 rs6851222 (Promoter G-1606A) rs6857715 (Promoter C-599T) and rs1047214 (Exon-2TC Ile195Ile) chosen to span the NPY2R locus Each SNP was in HardyndashWeinberg equilibrium (all Pgt005) Both BMI and circulating peptideYY (PYY) were significantly influenced by the GTT haplotype and the effects displayed evidence of joint determination (genetic pleiotropy)

Wei et al

linkage disequilibrium correlations among the eight com-mon (MAF gt5) SNPs were quantified by the confidenceinterval method across the NPY2R locus In each biogeo-graphic ancestry group twoblocks of linkagedisequilibriumwere maintained with one in the promoter region (Fig 3a)

Neuropeptide Y2 receptor haplotype effects ontraitsWe lsquotaggedrsquo the human NPY2R gene with three SNPsspanning the locus (Fig 3b) haplotype GTT (foundon 111 of chromosomes) was associated significantlywith both BMI (Pfrac14 375E04) and PYY secretion(Pfrac14 401E06) and the principal effect accrued to GTThomozygotes (with two copies of that haplotype per


01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY

Differential expression of NPY2Rbetween WKY and SHR rats

SHR

P-value

Brain stem0027

FIGURE 4 Transcript (mRNA) expression for the NPY2R system in tissues in vivo as weDDCt method for normalization and condition comparisons (a) Experimental (genetic) hytissues (adrenal gland and brainstem) WKY WistarndashKyoto rat as a normotensive controeach group (b) Cultured neuroendocrine cells presence of transcripts for NPY2R as wellcontrol (human elastin gene) was measured to define the threshold of expression Genesasterisk () Nfrac144 samples in each group


diploid genome) the GTT effect size (or slope) was positivefor BMI (193 048 kgm2 per copy) although negative forPYY (263 565 pgml per copy) Perhaps these pleio-tropic effects of haplotype GTT involve increased responseto PYY with consequent fall in this anorexigenic hormoneand ultimately an increase in BMI

Endogenous NPY2R mRNA expression in adisease model in rodents spontaneouslyhypertensive ratWistarndashKyoto ratNPY2R mRNA expression was increased significantly in twokey neuroendocrine tissues of the SHR (Fig 4a) both theadrenal gland (by 26-fold Pfrac14 0002) and the brainstem(by 15-fold Pfrac14 0027)


01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6

Expression of NPY2R IRF1 FOXI1 and SNAI1in rat PC12 cells

ll as in cultured neuroendocrine cells Results were obtained by RT-PCR using thepertension differential expression of NPY2R in SHR and WKY neuroendocrine

l SHR spontaneously hypertensive rat as a polygenic hypertension model Nfrac149 inas transcription factors IRF1 FOXI1 and SNAI1 in rat PC12 cells The negativewith significantly higher expression than negative control are marked by an


NPY2R polymorphism

Genetic variation in the proximal human NPY2Rpromoter consensus motifs

Core promoter nonpolymorphic motifsMotifs identified did not include a consensus TATA box nearthe transcriptional start site the closest partial TATA (ieTA-rich) match on the (thorn) strand was 50-(113bp)-AAAcTT-(108bp)-30 whereas the nearest potentialCAAT box was on the () strand at 50 (420bp)-CCAAT(424bp)-30 There was no proximal cAMP responseelement The 13GC-rich (consecutive GC 6bp) regionswere noted in the proximal promoter as were 4 E-boxes(CANNTG) One of the GC-rich domains constituted aconsensus match for a B recognition element [27] on thevery proximal (thorn) strand at 50-(49bp) GGGCGCC(43bp)-30 The closest potential initiator (Inr) elements[28] (consensus 50-YYAthorn1NWYY-30) were located at 50-(244bp)CCAGTCC(238bp)-30 (thorn strand) and 50-(thorn151bp)TTACACT(thorn145bp)-30 ( strand) None of thesecore elements were polymorphic across 2nfrac14 160 humanchromosomes

PolymorphismsWe identified 16 polymorphisms in the promoter (Table 1)eight of which were common (MAFgt5) Of note the veryproximal lsquocorersquo promoter (186thorn85 bp) was devoidof common variation At promoter variants G-1606AC-599T and A-224G we identified motifs likely to bedisrupted by the sequence change (see below)

NPY2R promoter haplotypes affect gene expressionConstructed from three common SNPs (G-1606A C-599Tand A-224G) that were predicted to be functional (seebelow) eight haplotypes were created by site-directed


NPY2R haplotypes influenceluciferase reporter expression

One-way ANOVA P lt 1124 104

3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14

FIGURE 5 NPY2R haplotypes influence luciferase reporter expression (a) Eight haplotypehave significantly different effects on expression of the reporter (one-way ANOVA Pfrac141haplotype in our resequencing sample is shown at the bottom Results for nfrac148 groupsusing two-way ANOVA and found to all have significant effects on reporter expression


mutagenesis from the most common promoter haplotype(alleles G-1606 C-599 and A-224 554 of chromosomes inour sample) NPY2R promoterluciferase reporters with var-ious haplotypes had significantly different expression activi-ties (one-way ANOVA Pfrac14 112E23 Fig 5a) We used two-way ANOVA to probe individual SNP effects on gene expres-sion each individual SNP as well as their binary and ternaryinteractions displayed significant influences on reporterexpression (Pfrac14 500E06 Fig 5b)

Neuropeptide Y2 receptor G-1606Apolymorphism role of an IRF1 activator-binding site

Sequence conservationalignmentG-1606A is located in a region highly conserved acrosssequenced primates (Fig 4a) with the G allele ancestral inthe human lineage as judged by the chimp sequence(Fig 6a) In this conserved local region there is a partialconsensus match for an IRF1 site (VAAARYGAAASY1606in bold) with an improved match for the A allele (1012 bpmatch) over the G allele (912 bp match) (Fig 4a)

Exogenous IRF1 transcription factor increasedNPY2R promoter-driven reporter expression AgtGalleleDuring NPY2R promoterluciferase reporter transfectionexpression into chromaffin cells (cotransfection with emptyvector pcDNA 31 Fig 6b) the A allele displayed greaterexpression than the G allele (AgtG) Cotransfectionexpres-sion of the IRF1 transcription factor increased reporterexpression and amplified the difference in expressionbetween the two alleles (Fig 4b Pfrac14 0001)


Endash23(b)

Two-way ANOVA

Promoter SNP P value

297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64

s constructed by the combination of three single nucleotide polymorphisms (SNPs)12E23) The minor allele for each SNP is shown in bold Frequency of eachare shown (b) Single SNPs and their binary and ternary interactions were examined


IRF1 motif VAAARYGAAASY Match ScoreHuman minor allele TTAAGTG AACT 1012 859Human major allele TTAAGTGGAACT 912 435Chimp TTAAGTGGAACT 912 -Rhesus TTCAGTGGAACT 812 -Orangutan TTAAGTGGAACT 912 -Marmoset GTAAGTGGAACT 1012 -Conserved uarr

G-1606A(rs6851222)

Bold Motif matchItalics Position of variant Conserved across primates

IUPAC symbolsV ACGR GA Y TCS GC

0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA

G-1606AIRF1Interaction

P = 877Endash09P = 426Endash05P = 0001

12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G

Neg ctrlsiRNArs6851222

IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA


P = 233Endash14P = 175Endash09P = 872Endash06

12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)

FIGURE 6 NPY2R promoter variant G-1606A role of IRF1 (a) Consensus motif match for IRF1 (interferon regulatory factor-1) at G-1606A across primate species(b) Exogenous IRF1 cDNA enhanced reporter expression driven by NPY2R promoter significantly more on the 1606A allele (c) Exogenous IRF1 siRNA impaired theactivator function of IRF1 on reporter expression driven by NPY2R promoter significantly more on the 1606A allele Nfrac146 in each group

Wei et al

Exogenous IRF1 siRNA decreased NPY2R promoter-driven reporter expression AgtG alleleDuring NPY2R promoterluciferase reporter cotransfectionwith negative control siRNA into chromaffin cells (Fig 6c)the A allele once again displayed greater expression thanthe G allele (AgtG) Cotransfection of IRF1 siRNA decreasedreporter expression and attenuated the difference ofexpression between the two alleles (Fig 6C Pfrac14 872E06)

Neuropeptide Y2 receptor C-599Tpolymorphism role of an activator FOXI1binding site

Sequence conservationalignmentC-599T is located in a region highly conserved acrosssequenced primates (Fig 7a) with the T allele ancestralin the human lineage as judged by the chimp sequence(Fig 7a) In this conserved local region there is a totalconsensus match for a FOXI1 site (TRTTTRKWD 599 inbold) with an improved match for the T allele (99 bpmatch) over the C allele (89 bp match) (Fig 7a)


FOXI1 motif TRTTTRKWD Match ScoreHuman minor allele TGTTTGGAG 99 886Human major allele CGTTTGGAG 89 432Chimp TGTTTGGAG 99 -Rhesus TGTTTGGAG 99 -Orangutan TGTCTGGAG 89 -Marmoset GGTTTGGAG 89 -Conserved

uarrC-599T

(rs6857715)


IUPAC symbolsR GAK TGW TAD TGA

0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)

FIGURE 7 NPY2R promoter variant C-599T role of FOXI1 (a) Consensus motif match focDNA enhanced reporter expression driven by NPY2R promoter significantly more on thFOXI1 on reporter expression driven by NPY2R promoter significantly more on the 599


Exogenous FOXI1 transcription factor increase inNPY2R promoter-driven reporter expression TgtCalleleDuring NPY2R promoterluciferase reporter transfec-tion into chromaffin cells (cotransfection with emptyvector pcDNA 31 Fig 7b) the T allele displayed greaterexpression than the C allele (TgtC) Cotransfectionexpression of FOXI1 transcription factor increasedreporter expression and amplified the difference ofexpression between the two alleles (Fig 7bPfrac14 557E06)

Exogenous FOXI1 siRNA decrease in NPY2Rpromoter-driven reporter expressionTgtC alleleDuring NPY2R promoterluciferase reporter cotransfectionwith negative control siRNA into chromaffin cells (Fig 7c)the T allele displayed greater expression than the C allele(TgtC) Cotransfection of FOXI1 siRNA decreased reporterexpression and attenuated the difference of expressionbetween two alleles (Fig 7c Pfrac14 0010)


NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA

C-599TFOXI1Interaction

P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)

r FOXI1 (Forkhead Box I-1) at C-599T across primate species (b) Exogenous FOXI1e 599T allele (c) Exogenous FOXI1 siRNA impaired the activator function ofT allele Nfrac146 in each group


NPY2R polymorphism

Neuropeptide Y2 receptor A-224Gpolymorphism role of a SNAI1 repressorbinding site

Sequence conservationalignmentA-224G is located in a region highly conserved acrosssequenced primates (Fig 8a) with the A allele ancestralin the human lineage as judged by the chimp sequence(Fig 8a) In this conserved local region there is a partialconsensus match for an SNAI1 site (CAGGTG 224 inbold) with an improved match for the A allele (56 bpmatch) over the G allele (46 bp match) (Fig 8a)

Exogenous SNAI1 transcription factor decrease inNPY2R promoter-driven reporter expression AgtGalleleDuring NPY2R promoterluciferase reporter transfectioninto chromaffin cells (cotransfection with empty vectorpcDNA 31 Fig 8b) the G allele displayed greater expres-sion than the A allele (GgtA) Cotransfection of the SNAI1transcription factor decreased reporter expression andamplified the difference of expression between the twoalleles (Fig 8b Pfrac14 0034)

Exogenous SNAI1 siRNA increase in NPY2Rpromoter-driven reporter expression AgtG alleleDuring NPY2R promoterluciferase reporter cotransfectionwith negative control siRNA into chromaffin cells (Fig 8c)the G allele displayed greater expression than the A allele(GgtA) Cotransfection of SNAI1 siRNA increased reporterexpression and attenuated the difference of expressionbetween two alleles (Fig 8c Pfrac14 0019)

Endogenous mRNA expression in neuroendocrinecells NPY2R and transcription factors whosebinding is disrupted by NPY2R promoter commongenetic variation (IRF1 FOXI1 SNAI1)We used PC12 (rat pheochromocytoma) cells as anexperimental system to test the effects of potentiallyallele-specific transcription factors but are the receptorand these transcription factors endogenously expressed


SNAI 1 motif CAGGTG Match ScoreHuman minor allele CAGGAG 56 6028Human major allele CGGGAG 46 1346Chimp CAGGAG 56 -Rhesus CAGGAG 56 -Orangutan CAGGAG 56 -Marmoset CAGGAG 56 -Conserved

uarrA- 224G

rs2234759


0A

pcDNAcDNArs2234759 G

5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)

FIGURE 8 NPY2R promoter variant A-224G role of SNAI1 (a) Consensus motif match focDNA inhibited reporter expression driven by NPY2R promoter significantly more on theon reporter expression driven by NPY2R promoter significantly more on the A-224 allele


in this model system (Fig 4b) NPY2R itself as well as thetranscription factors IRF1 and SNAI1 displayed substan-tial expression in PC12 cells whereas FOXI1 expressionwas undetectable

DISCUSSION

OverviewNPY2R represents a central control point for the PYYNPYregulatory pathway In this study we explored whether andhow common genetic variations in the NPY2R promoteraffect gene expression We present evidence from severalapproaches (genomic bioinformatic transfection trans-activation and siRNA inhibition) in which we found thatpromoter variants G-1606A C-599T and A-224G conferredfunctional changes onto NPY2R expression and thatparticular transcription factors were implicated We thuspresent evidence of previously unexpected cis-variation inthe regulation of NPY2R expression

Cardiometabolic traits and NPY2R geneticvariationWe found that multiple cardiometabolic traits are highlyheritable and also display shared genetic determination(Fig 1) Associations between NPY2R SNPs and obesity arewidely investigated in multiple populations with substan-tial agreement that significant marker-on-trait effects occur[29] We too could replicate such effects in that a haplotypeacross the NPY2R locus influenced both BMI and PYY(Fig 3b) Thus in this report we describe a potential geneticcontributor to dysregulation of body mass genetic variationat the NPY2R locus (Figs 2 and 3)

Neuropeptide Y2 receptor promoter variantsG-1606A C-599T and A-224GWe focused on three promoter polymorphisms that are notonly common (high MAF) but also predicted to influencetranscription factor binding by bioinformatic analyses Onthe basis of this strategy the G-1606A C-599T and A-224Gwere advanced to further investigation Frequencies of theirpromoter haplotypes are shown in Fig 5


rat SNAI1A G

I1 cDNA

A-224GSNAI1Interaction


0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104

SNAI1 siRNAA-224GSNAI1Interaction

P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)

r SNAI1 (Snail homolog 1) at A-224G across primate species (b) Exogenous SNAI1A-224 allele (c) Exogenous SNAI1 siRNA impaired the repressor function of IRF1 Nfrac146 in each group


Wei et al

Of note for the physiological significance of these resultswe detected abundant transcripts in neuroendocrine PC12cells (Fig 4b) for NPY2R itself IRF1 and SNAI1 In additionquery of the NCBI GEO database (httpwwwncbinlmnihgovgeo) indicates that transcripts for NPY2R IRF1(binding G-1606A) FOXI1 (binding C-599T) and SNAI1(binding A-224G) are expressed endogenously in PC12chromaffin cells by inspection of the following GEO tran-script datasets GDS3436 [30] GDS1038ndash1039 [31] andGDS2555 [32]

Results in context with the literatureCommon genetic variation in the NPY2R promoter [236]has been associated with obesity or BMI traits in studies ofover 10 000 individuals (on-line Table 1 httplinkslwwcomHJHA209) in one case [6] the effect size (asCramerrsquos phi) suggested that NPY2R promoter geneticvariation might account for up to approximately 93 oftrait variance in the population Among the three promotervariants evaluated in depth in our studies C-599T(rs6857715) was implicated in one of these associationstudies C-599T was associated with both adult and child-hood obesity in a French sample [6] and this variant alsohad an effect on high-density lipoprotein cholesterol [8]C-599T was a component of functional promoter haplo-types on gene expression (Figs 5 and 7) as well as the BMIPYY-associated GTT haplotype in our population (Fig 3b)

Furthermore each of the three transcription factors(Figs 6ndash8) whose binding is altered by NPY2R promotervariants is already implicated in cardiometabolic function Ameta-analysis of genome-wide association studies revealedthe influence of IRF1 on circulating C-reactive protein levelwhich is strongly associated with cardiovascular disease [33]IRF1 also plays a key role in development of insulitis anddiabetes in a mouse model [34] FOXI1 may be necessary forexpression of at least four subunits and proper assembly ofthe vacuolar Hthorn-ATPase complex [35] whose activity has animpact on hypertension [36] SNAI1 transcriptionally controlscardiovascular progenitor cell formation through epicardialepithelial-mesenchymal transition [37] and such function isregulated by glucose metabolism [38]

Limitations of this studyA number of issues remain unexplored by our studiesFor effects in very large sample sizes (gt10 000 participantson-line Table 1 httplinkslwwcomHJHA209) we relyon the findings of other groups [236] that NPY2R promoterpolymorphism influences obesity especially for C-599T [6]although we did find evidence for such effects in our ownpopulation (Fig 3b) Second the cis-interactionstrans-interactions that we observed in transfected cells(Figs 6ndash8) are novel and thus not yet established in vivoalthough we did find evidence of differential expression ofNPY2R in neuroendocrine tissues of the SHR (Fig 4a) aswell as endogenous expression of the pertinent transcriptsin neuroendocrine cells (Fig 4b)

Conclusions and perspectivesWe conclude that cardiometabolic traits are highly heritablethat NPY genetic variation influences such traits (including


BMI and PYY) and that within the NPY2R promotercommon polymorphisms are associated with alterations intranscriptional efficiency The functional effects of polymor-phism seem to arise from differential actions of specifictranscription factors at the NPY2R promoter IRF1 function-ing as an activator disrupted by G-1606A bi-allelic variationFOXI1 acting as an activator disrupted by C-599T and SNAI1acting as a repressor disrupted by A-224G The results raisethe potential for novel alterations in cis-interactions forcontrol of PYY responses thus augmenting our understand-ing of molecular events underlying interindividual variationin energy balance and the genetic predisposition towardobesity a potent risk factor for cardiovascular disease

ACKNOWLEDGEMENTSSources of funding are National Institutes of Health[HL58120 1UL1RR031980 (UCSD Clinical and TranslationalResearch Institute) MD000220 (UCSD ComprehensiveResearch Center in Health Disparities CRCHD)] Depart-ment of Veterans Affairs

Conflicts of interestThe authors have no conflicts of interest to declare

REFERENCES1 Batterham RL Cowley MA Small CJ Herzog H Cohen MA Dakin CL

et al Gut hormone PYY(3-36) physiologically inhibits food intakeNature 2002 418650ndash654

2 Torekov SS Larsen LH Andersen G Albrechtsen A Glumer C Borch-Johnsen K et al Variants in the 50 region of the neuropeptide Yreceptor Y2 gene (NPY2R) are associated with obesity in 5971 whitesubjects Diabetologia 2006 492653ndash2658

3 Lavebratt C Alpman A Persson B Arner P Hoffstedt J Commonneuropeptide Y2 receptor gene variant is protective against obesityamong Swedish men Int J Obes (Lond) 2006 30453ndash459

4 Zhang J Wang HJ Ma J Association between obesity and the poly-morphism of neuropeptide Y2 receptor gene in children and adoles-cents Zhonghua Liu Xing Bing Xue Za Zhi 2009 30695ndash698

5 Friedlander Y Li G Fornage M Williams OD Lewis CE Schreiner Pet al Candidate molecular pathway genes related to appetite regulatoryneural network adipocyte homeostasis and obesity results from theCARDIA Study Ann Hum Genet 2010 74387ndash398

6 Siddiq A Gueorguiev M Samson C Hercberg S Heude B Levy-Marchal C et al Single nucleotide polymorphisms in the neuropeptideY2 receptor (NPY2R) gene and association with severe obesity inFrench white subjects Diabetologia 2007 50574ndash584

7 Kuo LE Kitlinska JB Tilan JU Li L Baker SB Johnson MD et alNeuropeptide Y acts directly in the periphery on fat tissue and mediatesstress-induced obesity and metabolic syndrome Nat Med 200713803ndash811

8 Takiguchi E Fukano C Kimura Y Tanaka M Tanida K Kaji HVariation in the 50-flanking region of the neuropeptide Y2 receptorgene and metabolic parameters Metabolism 2010 591591ndash1596

9 Campbell CD Lyon HN Nemesh J Drake JA Tuomi T Gaudet D et alAssociation studies of BMI and type 2 diabetes in the neuropeptide ypathway a possible role for NPY2R as a candidate gene for type 2diabetes in men Diabetes 2007 561460ndash1467

10 Arnett DK Devereux RB Rao DC Li N Tang W Kraemer R et al Novelgenetic variants contributing to left ventricular hypertrophy the Hyper-GEN study J Hypertens 2009 271585ndash1593

11 Sainsbury A Schwarzer C Couzens M Fetissov S Furtinger S JenkinsA et al Important role of hypothalamic Y2 receptors in body weightregulation revealed in conditional knockout mice Proc Natl Acad SciU S A 2002 998938ndash8943

12 Garrett MR Rapp JP Multiple blood pressure QTL on rat chromosome2 defined by congenic Dahl rats Mamm Genome 2002 1341ndash44


NPY2R polymorphism

13 Wen G Mahata SK Cadman P Mahata M Ghosh S Mahapatra NR et alBoth rare and common polymorphisms contribute functional variationat CHGA a regulator of catecholamine physiology Am J Hum Genet2004 74197ndash207

14 Rozen S Skaletsky H Primer3 on the WWW for general users and forbiologist programmers Method Mol Biol 2000 132365ndash386

15 Cockburn M Hamilton A Zadnick J Cozen W Mack TM The occur-rence of chronic disease and other conditions in a large population-based cohort of native Californian twins Twin Res 2002 5460ndash467

16 Zhang L Rao F Wessel J Kennedy BP Rana BK Taupenot L et alFunctional allelic heterogeneity and pleiotropy of a repeat polymor-phism in tyrosine hydroxylase prediction of catecholamines andresponse to stress in twins Physiol Genomics 2004 19277ndash291

17 Wessel J Moratorio G Rao F Mahata M Zhang L Greene W et alC-reactive protein an lsquointermediate phenotypersquo for inflammationhuman twin studies reveal heritability association with blood pressureand the metabolic syndrome and the influence of common poly-morphism at catecholaminergicbeta-adrenergic pathway loci J Hyper-tens 2007 25329ndash343

18 Shih PA Wang L Chiron S Wen G Nievergelt C Mahata M et alPeptide YY (PYY) gene polymorphisms in the 3rsquo-untranslated andproximal promoter regions regulate cellular gene expression and PYYsecretion and metabolic syndrome traits in vivo J Clin EndocrinolMetab 2009 944557ndash4566

19 Barrett JC Fry B Maller J Daly MJ Haploview analysis and visual-ization of LD and haplotype maps Bioinformatics 2005 21263ndash265

20 Schaid DJ Rowland CM Tines DE Jacobson RM Poland GA Scoretests for association between traits and haplotypes when linkage phaseis ambiguous Am J Hum Genet 2002 70425ndash434

21 Thompson JD Higgins DG Gibson TJ CLUSTAL W improving thesensitivity of progressive multiple sequence alignment throughsequence weighting position-specific gap penalties and weight matrixchoice Nucleic Acids Res 1994 224673ndash4680

22 Wasserman WW Sandelin A Applied bioinformatics for the identifi-cation of regulatory elements Nat Rev Genet 2004 5276ndash287

23 Sandelin A Wasserman WW Lenhard B ConSite Web-based predic-tion of regulatory elements using cross-species comparison NucleicAcids Res 2004 32(suppl 2)W249ndashW252

24 Almasy L Blangero J Multipoint quantitative-trait linkage analysis ingeneral pedigrees Am J Hum Genet 1998 621198ndash1211

25 Falconer DS Mackay TFC Introduction to quantitative genetics 4thed Harlow Essex UK Longman 1996

26 Livak KJ Schmittgen TD Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) MethodMethods 2001 25402ndash408


27 Lagrange T Kapanidis AN Tang H Reinberg D Ebright RH New corepromoter element in RNA polymerase II-dependent transcriptionsequence-specific DNA binding by transcription factor IIB GenesDev 1998 1234ndash44

28 Javahery R Khachi A Lo K Zenzie-Gregory B Smale ST DNAsequence requirements for transcriptional initiator activity in mamma-lian cells Mol Cell Biol 1994 14116ndash127

29 Naveilhan P Hassani H Canals JM Ekstrand AJ Larefalk A ChhajlaniV et al Normal feeding behavior body weight and leptin responserequire the neuropeptide Y Y2 receptor Nat Med 1999 51188ndash1193

30 Yamada M Shida Y Takahashi K Tanioka T Nakano Y Tobe T Prg1is regulated by the basic helix-loop-helix transcription factor Math2J Neurochem 2008 1062375ndash2384

31 Impey S McCorkle SR Cha-Molstad H Dwyer JM Yochum GS BossJM et al Defining the CREB regulon a genome-wide analysis oftranscription factor regulatory regions Cell 2004 1191041ndash1054

32 Lattanzi W Bernardini C Gangitano C Michetti F Hypoxia-like tran-scriptional activation in TMT-induced degeneration microarrayexpression analysis on PC12 cells J Neurochem 2007 1001688ndash1702

33 Dehghan A Dupuis J Barbalic M Bis JC Eiriksdottir G Lu C et alMeta-analysis of genome-wide association studies in gt80 000 subjectsidentifies multiple loci for C-reactive protein levels Circulation 2011123731ndash738

34 Nakazawa T Satoh J Takahashi K Sakata Y Ikehata F Takizawa Yet al Complete suppression of insulitis and diabetes in NOD micelacking interferon regulatory factor-1 J Autoimmun 2001 17119ndash125

35 Vidarsson H Westergren R Heglind M Blomqvist SR Breton S Ener-back S The forkhead transcription factor Foxi1 is a master regulator ofvacuolar H-ATPase proton pump subunits in the inner ear kidney andepididymis PLoS ONE 2009 4e4471

36 Wei Z Biswas N Wang L Courel M Zhang K Soler-Jover A et al ACommon Genetic Variant in the 3rsquo-UTR of Vacuolar Hthorn-ATPaseATP6V0A1 Creates a Micro-RNA Motif to Alter Chromogranin A(CHGA) Processing and Hypertension Risk Circ Cardiovasc Genet2011 4381ndash389

37 Martınez-Estrada OM Lettice LA Essafi A Guadix JA Slight J VelecelaV et al Wt1 is required for cardiovascular progenitor cell formationthrough transcriptional control of Snail and E-cadherin Nat Genet2010 4289ndash93

38 Park SY Kim HS Kim NH Ji S Cha SY Kang JG et al Snail1 isstabilized by O-GlcNAc modification in hyperglycaemic conditionEMBO J 2010 293787ndash3796

Reviewersrsquo Summary Evaluations

Reviewer 1Neuropeptide Y receptors are activated by neuropeptide Ypeptide YY and pancreatic polypeptide Subtypes Y1 andY5 are involved in stimulation of feeding while Y2 and Y4appear to be involved in satiety By extension there isinterest in this pathway being involved in metabolic traitsPeptide YY is related to pancreatic peptide and is releasedpostprandially primarily from the ileum and the colon andhas a role in appetite suppression This study shows thatpeptide YY levels have a high heritability of 51 and showthat 3 promoter polymorphisms in the NPY2R influencetranscriptional activity using luciferase reporter constructswith IRF1 and SNAI1 as putative transcription factors Acausal relation between these polymorpisms or peptide YYand cardiometabolic traits is not established and future

studies should validate this finding and well poweredassociation and functional studies

Reviewer 2Satiety and obesity are interdependently subject to gene xenvironment interactions Significant genetic componentconfirmed in twinrsquos studies actually illustrates a superiorhereditary determination for obesity than for hypertensionWhile neuropeptide Y pathway has been associated in largestudies with obesity this paper provides novel evidence offunctional relevance of polymorphisms within the pro-moter region of NPY2R in cis- as well as trans- modesThe fact that these genomic variances are present in aquarter of several populations is teaching us that theirimpact should be included in future preventive strategiesof satiety obesity and hypertension control


Wei et al

with obesity or BMI in several populations includingwhites [23] Asians [4] and Africans [5] Indeed studiesof NPY2R promoter genetic variation in more than 10 000individuals [236] indicate its involvement in control ofobesity or BMI (on-line Table 1 httplinkslwwcomHJHA209) NPY2R also cooperates with NPY in stress-induced obesity and the metabolic syndrome [7] NPY2Rgenetic variants associate with such human cardiometabolictraits as high-density lipoprotein cholesterol [8] SBP [3] type2 diabetes in men [9] and left ventricular hypertrophy [10]Hypothalamus-targeted NPY2R-knockout mice showed adecrease in body weight despite an increase in food intake[11] In the rat (httprgdmcwedu) the Npy2r genetic locusunderlies the confidence interval of a quantitative trait locus(QTL) for blood pressure (BP) BP QTL-90 (Bp90) [12] Suchdiverse evidence indicates that NPY2R plays an indispensa-ble role in the cardiometabolic syndrome

In these studies we first documented the role of heredityin cardiometabolic traits using twin pair variance com-ponents and then systematically searched for naturallyoccurring genetic variation across the human NPY2R locusBecause severalof thediscoveredcommonvariants occurredin a likely functional domain (thepromoter)weprobed theirmechanistic consequences beginning with bioinformaticmotif analysis and proceeding to transfected promoterluci-ferase reporter plasmids site-directed mutagenesis andcharacterization of trans-acting factors We developedevidence that variation in the NPY2R promoter especiallyat common variants G-1606A C-599T and A-224G disruptparticular motifs (IRF1 FOXI1 and SNAI1 elements respect-ively) creating differential cis-interactions and trans-interactions to alter transcriptional activity and ultimatelyBP body mass and associated risk traits in the population

PARTICIPANTS ANDMETHODS

Genomics

Systematic polymorphism discovery at the NPY2RlocusWe studied the NPY2R locus in nfrac14 80 participants (2nfrac14 160chromosomes) as described below under lsquoHuman partici-pantsrsquo Genomic DNA was prepared from leukocytes asdescribed previously [13] Public draft human genomesequences were obtained from the University of CaliforniaSanta Cruz Genome Bioinformatics website (httpgenomeucscedu) and used as a scaffold for primer designThe base position numbers were according to the NationalCenter for Biotechnology Information (NCBI) NPY2R sourceclone RefSeq genetranscript NM_0009102 Promoter pos-itions were numbered upstream of () the NPY2R exon-1start (cap) site PCR primers were designed by primer-3 [14](httpfrodowimiteduprimer3) to capture approxi-mately 2000bp of the proximal promoter between approxi-mately 500bp to approximately2000bp over each of the twoexons (including 50-UTR 30-UTR and exonintron borders)and regions highly conserved across species Target regionswere amplified and then dideoxy-sequenced using an ABI-3100 capillary sequencer (Applied Biosystems CarlsbadCaliforniaUSA) Polymorphism (typically as heterozygosity)was visualized on the Applied Biosystems (ABI) tracings


using Codon Code Aligner (httpwwwcodoncodecomaligner)

Human participants

Resequencing the NPY2R locusHuman studies were approved by the University ofCalifornia San Diego (UCSD) Human Research ProtectionProgram Experiments were conducted with the under-standing and consent of each participant We studied theNPY2R locus in nfrac14 80 participants (2nfrac14 160 chromo-somes) from four diverse biogeographic ancestry groupssystematically sequenced for polymorphism discoveryacross the NPY2R locus white (European ancestry2nfrac14 46 chromosomes) black (sub-Saharan African ances-try 2nfrac14 50 chromosomes) Hispanic (Mexican American2nfrac14 32 chromosomes) and east Asian (2nfrac14 32 chromo-somes)

UCSD twin pairsTwin recruitment included access to a population birthrecord-based twin registry [15] as well as by newspaperadvertisement as described [16] Description of the 362participants in the twin heritability and allelic associationstudies has been published [17] For human allelic andhaplotype association studies this twin group wasexpanded to 693 participants of European ancestry derivedfrom additional siblings from twinships and sibships aspreviously described [18]

Statistics and informatics

Linkage disequilibrium and haplotypesIn the resequenced participants patterns of linkage dis-equilibrium as well as haplotype frequencies were analyzedand visualized by the software Haploview (Broad InstituteMassachusetts USA) [19] Linkage disequilibrium blockswere derived by the confidence interval criterion andvisualized by r2 plot in Haploview from unphased diploidgenotypes of nfrac14 80 resequenced participants (2nfrac14 160chromosomes) from four diverse biogeographic ancestrygroups systematically sequenced across the NPY2R locusCommon variants (minor allele frequency gt5) were usedto establish linkage disequilibrium

In the twins and siblings haplotype-on-trait analyseswere conducted by regression in R (reporting effect sizeas b or slope per allele as well as its SEM) with Haploglmin Haplostats [20] (httpmayoresearchmayoeduschai-d_labsoftwarecfm) Trait-associated haplotype GTT waspresent on 111 of chromosomes analyzed

Bioinformatics computational prediction oftranscription factor-binding motifs overlying NPY2Rpromoter common variantsMultiple sequence alignments were performed by Clustal-W [21] (httpwwwebiacukToolsclustalw2) Potentialtranscription factor binding motifs were predicted from theJASPAR [22] (httpjaspargeneregnet) and ConSite [23](httpaspiiuibno8090cgi-binCONSITEconsite) data-bases


NPY2R polymorphism











Wei et al




method [26]


0

02

04

Her

itab

ility

(h

2 = V

GV

P)

06

08

1

(a) (bHeritability

BMI (kgm2)

h2 = 086+ndash002

P lt 00001

h2 = 046+ndash006

P lt 00001

h2 = 052+ndash006

P lt 00001

h2 = 051+ndash006

P lt 00001


Trait

PYY (pgml)




RESULTS



ndash03ndash03

ndash02

ndash02

ndash01

ndash01

00

0

01

01


Rh

o_E

(en

viro

nm

enta

l co

vari

ance

)

02

02

03

03

04

04

05

)

05



Rho_G P = 30E-02Rho_E P = 072


Y = X(line of

identity)




Humanmousehomology




ndash224

+5895

+6242



NPY2R polymorphism




NPY2RSNPs Alleles

SNPposition

RefSNPnumber

Aminochang




























acide


Whitenfrac1423

Blacknfrac1425

Hispanicnfrac1416

Asiannfrac1416

Allnfrac1480


025 016 025 047 0270


025 022 025 047 0290

011 000 012 019 0090

045 012 038 025 0287


012 008 022 020 0140

029 065 041 047 0470

029 036 041 047 0470




024 020 033 053 0310






048 012 034 007 0250

050 033 043 013 0360




White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24



BM

I (kg

m2 )

110 120 130

Black2n = 50


Wei et al




01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY


SHR

P-value

Brain stem0027






01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6





NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































NPY2R polymorphism











Wei et al




method [26]


0

02

04

Her

itab

ility

(h

2 = V

GV

P)

06

08

1

(a) (bHeritability

BMI (kgm2)

h2 = 086+ndash002

P lt 00001

h2 = 046+ndash006

P lt 00001

h2 = 052+ndash006

P lt 00001

h2 = 051+ndash006

P lt 00001


Trait

PYY (pgml)




RESULTS



ndash03ndash03

ndash02

ndash02

ndash01

ndash01

00

0

01

01


Rh

o_E

(en

viro

nm

enta

l co

vari

ance

)

02

02

03

03

04

04

05

)

05



Rho_G P = 30E-02Rho_E P = 072


Y = X(line of

identity)




Humanmousehomology




ndash224

+5895

+6242



NPY2R polymorphism




NPY2RSNPs Alleles

SNPposition

RefSNPnumber

Aminochang




























acide


Whitenfrac1423

Blacknfrac1425

Hispanicnfrac1416

Asiannfrac1416

Allnfrac1480


025 016 025 047 0270


025 022 025 047 0290

011 000 012 019 0090

045 012 038 025 0287


012 008 022 020 0140

029 065 041 047 0470

029 036 041 047 0470




024 020 033 053 0310






048 012 034 007 0250

050 033 043 013 0360




White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24



BM

I (kg

m2 )

110 120 130

Black2n = 50


Wei et al




01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY


SHR

P-value

Brain stem0027






01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6





NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































Wei et al




method [26]


0

02

04

Her

itab

ility

(h

2 = V

GV

P)

06

08

1

(a) (bHeritability

BMI (kgm2)

h2 = 086+ndash002

P lt 00001

h2 = 046+ndash006

P lt 00001

h2 = 052+ndash006

P lt 00001

h2 = 051+ndash006

P lt 00001


Trait

PYY (pgml)




RESULTS



ndash03ndash03

ndash02

ndash02

ndash01

ndash01

00

0

01

01


Rh

o_E

(en

viro

nm

enta

l co

vari

ance

)

02

02

03

03

04

04

05

)

05



Rho_G P = 30E-02Rho_E P = 072


Y = X(line of

identity)




Humanmousehomology




ndash224

+5895

+6242



NPY2R polymorphism




NPY2RSNPs Alleles

SNPposition

RefSNPnumber

Aminochang




























acide


Whitenfrac1423

Blacknfrac1425

Hispanicnfrac1416

Asiannfrac1416

Allnfrac1480


025 016 025 047 0270


025 022 025 047 0290

011 000 012 019 0090

045 012 038 025 0287


012 008 022 020 0140

029 065 041 047 0470

029 036 041 047 0470




024 020 033 053 0310






048 012 034 007 0250

050 033 043 013 0360




White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24



BM

I (kg

m2 )

110 120 130

Black2n = 50


Wei et al




01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY


SHR

P-value

Brain stem0027






01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6





NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism


































Humanmousehomology




ndash224

+5895

+6242



NPY2R polymorphism




NPY2RSNPs Alleles

SNPposition

RefSNPnumber

Aminochang




























acide


Whitenfrac1423

Blacknfrac1425

Hispanicnfrac1416

Asiannfrac1416

Allnfrac1480


025 016 025 047 0270


025 022 025 047 0290

011 000 012 019 0090

045 012 038 025 0287


012 008 022 020 0140

029 065 041 047 0470

029 036 041 047 0470




024 020 033 053 0310






048 012 034 007 0250

050 033 043 013 0360




White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24



BM

I (kg

m2 )

110 120 130

Black2n = 50


Wei et al




01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY


SHR

P-value

Brain stem0027






01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6





NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































White2n = 46

(a) (b)

Hispanic2n = 32

Asian2n = 32

6025

26

27

28

29

30

31

32

70 80 90 100

Plasma PYY (pgml)

GTT1-copyn = 315

GTT0-copiesn = 354

GTT2-copies

n = 24



BM

I (kg

m2 )

110 120 130

Black2n = 50


Wei et al




01

001

Rel

ativ

e ex

pre

ssio

n to

bet

a-ac

tin

0001

(a)

Adrenal grand0002

WKY


SHR

P-value

Brain stem0027






01

(b)

001

0001

NPY2R

Rel

ativ

e ex

pre

ssio

n to

bet

s-ac

tin

IRF1

FOXI1 SNAI1

Negative ctrl

00001

10ndash5

10ndash6





NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































NPY2R polymorphism








3 104

2 104

1 104

0

(a)

Haplotype

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

G-1606A

C-599T

A-224G

Frequency

1

G

C

A

554

2

AC

A

00

3

G

C

G160

4

AC

G00

5

G

T

A

34

6

AT

A

174

7

G

T

G14








Endash23(b)

Two-way ANOVA


297Endash06

204Endash06

275Endash16

446Endash06

353Endash09

443Endash16

117Endash06

G-1606A

G-1606A C-599T

G-1606A C-599T A-224G

G-1606A A-224G

C-599T A-224G

C-599T

A-224G

8

AT

G64




G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism


































G-1606A(rs6851222)



0G

pcDNAcDNArs6851222

IRF1GA A

2000

4000

6000

8000

1 104

IRF1 cDNA



12 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

0G


IRF1GA A

2 104

4 104

6 104

8 104

1 105

IRF1 siRNA



12 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b) (c)


Wei et al






uarrC-599T

(rs6857715)



0C

pcDcDNArs6857715

5000

1 104

15 104

2 104

F

C-599TFOXI1Interact

25 104

1

1

2

2 3 4 5 6 7 8 9 10 11 12

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)






NA rat FOXI1CT T

OXI1 cDNA

ion


0C


rat FOXI1CT T

2 104

4 104

6 104

8 104

FOXI1 siRNA


P = 212Endash09P = 0005P = 0010

1 105

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































NPY2R polymorphism








uarrA- 224G

rs2234759


0A


5000

1 104

15 104

2 104

SNA

25 1041

1

2

2 3 4 5 6

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(a)

(b)




DISCUSSION





rat SNAI1A G

I1 cDNA



0A


rat FOXI1AG G

1 104

2 104

3 104

5 104

6 104

4 104


P = 330Endash07P = 0941P = 0019

7 104

Lu

cife

rase

rep

ort

er a

ctiv

ity

(no

rmal

ized

to to

tal p

rote

in c

on

cen

trat

ion

)

(c)



Wei et al
























NPY2R polymorphism

































Wei et al
























NPY2R polymorphism

































NPY2R polymorphism

































Documents

Heredity and cardiometabolic risk