Functional Variant in a Bitter-T aste Receptor (hTAS2R16)Influences Risk of Alcohol Dependence
Anthony L.Hinrichs,1,*Jen C.Wang,1,*Bernd Bufe,2Jennifer M.Kwon,1,†John Budde,1Rebecca Allen,1Sarah Berteln,1Whitney Evans,3Danielle Dick,1John Rice,1
Tatiana Foroud,4John Nurnberger,4Jay A.Tischfield,5Samuel Kuperman,6Raymond Crowe,6Victor Heslbrock,7Marc Schuckit,8Laura Almasy,9Bernice Porjesz,10Howard J.Edenberg,4Henri Begleiter,10Wolfgang Meyerhof,2Laura J.Bierut,1and Alison M.Goate 1
1
Washington University School of Medicine,St.Louis;2German Institute of Human Nutrition Potsdam-Rehbruecke,Nuthetal,Germany;3
Laboratory of Neurogenetics,National Institute of Aging,National Institute of Health,Bethesda;4Indiana University School of Medicine,Indianapolis;5Rutgers University,Piscataway,NJ;6University of Iowa School of Medicine,Iowa City;7University of Connecticut School of Medicine,Farmington;8University of California at San Diego School of Medicine,La Jolla;9Southwest Foundation,San Antonio;and 10State University of New Y ork Health Science Center at Brooklyn,Brooklyn
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A coding single-nucleotide polymorphism (cSNP),K172N,in hTAS2R16,a gene encoding a taste receptor for bitter b -glucopyranosides,shows significant association with alcohol dependence ().This gene is located on P p .00018chromosome 7q in a region reported elwhere to exhibit linkage with alcohol dependence.The SNP is located in the putative ligand-binding domain and is associated with an incread nsitivity to many bitter b -glucopyranosides in the prence of the N172allele.Individuals with the ancestral allele K172are at incread risk of alcohol dependence,regardless of ethnicity.However,this risk allele is uncommon in European Americans (minor-allele frequency [MAF]0.6%),whereas 45%of African Americans carry the allele (MAF 26%),which makes it a much more significant risk factor in the African American population.Received August 26,2005;accepted for publication October 11,2005;electronically published November 21,2005.
Address for correspondence and reprints:Dr.Alison M.Goate,Departments of Psychiatry,Neurology,and Genetics,Washington University School of Medicine,660S.Euclid Avneue,St.Louis,MO 63110.E-mail:******************.edu *The two authors contributed equally to this work.†
Prent affiliation:Departments of Neurology and Pediatrics,University of Rochester Medical Center,
Rochester,NY .
Am.J.Hum.Genet.2006;78:103–111.᭧2005by The American Society of Human Genetics.All rights rerved.0002-9297/2006/7801-0011$15.00
Alcohol dependence (MIM 103780)is one of the most common and costly health problems in the United States (Centers for Dia Control and Prevention 2004).It is a complex dia,with both genetic and environmen-tal contributions to the risk.Family,adoption,and twin studies provide convergent evidence of hereditary factors in alcoholism (Heath et al.1997).Heritable influences account for ∼40%–60%of the total variance in risk (Pick-ens et al.1991;Kendler et al.1994).The Collaborative Study of the Genetics of Alcoholism (COGA)was estab-lished to identify genes that modify susceptibility to al-coholism and related phenotypes.Genomewide linkage analys using COGA pedigrees have provided consis-tent evidence of an alcoholism-susceptibility locus on the long arm of chromosome 7in both the initial data t (Reich et al.1998)and the replication data t (Foroud et al.2000).Our recent studies have also shown linkage of an overlapping region of chromosome 7q with major depressive disorder (MIM 608516),composite pheno-types of alcohol dependence and/or depression,and elec-trophysiological measures derived from event-related os-cillations (Nurnberger et al.2001;Jones et al.2004;Wang et al.2004).Evidence of genetic linkage to al
cohol dependence has also been reported in two Native Amer-ican populations (Long et al.1998;Ehlers et al.2004)and in extended families from the Framingham Heart Study population (Ma et al.2003),although none of the studies showed linkage to chromosome 7q.
Elwhere,we have reported evidence of association between individual SNPs and specific haplotypes within the gene encoding the acetylcholine muscarinic receptor 2(CHRM2[MIM 118493])and alcohol dependence as well as major depressive syndrome (Wang et al.2004).Since this gene lies near the edge of the linkage peak,we suspected that additional alcoholism-susceptibility loci exist in this region of chromosome 7.A arch of the public databas revealed a cluster of bitter-taste recep-tors (TAS2Rs)in this region,which are potential candi-date genes.The TAS2R genes,with a size range of 876–1,014bp,have intronless coding regions,code for G protein–coupled receptors,and have recently been iden-tified in mice and in humans (Adler et al.2000;Matsu-nami et al.2000).A number of coding SNPs (cSNPs)have been identified in human bitter-taste–receptor genes (Ueda et al.2001;Kim et al.2003,2005;Soranzo et al.2005).Among the,three cSNPs in the hTAS2R38gene (MIM 607751and MIM 171200)and one cSNP in the hTAS2R16gene (MIM 604867)have been shown to al-
Figure1Location of the cluster of nine TAS2R genes on chromosome7q(not drawn to scale).(A color
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version of thisfigure is available in the online edition of the Journal.)
ter receptor functions or taste nsitivity to bitter com-pounds,which suggests that genetic variation of the TAS2Rs may correlate with susceptibility to diet-related dia(Tepper1998;Kim et al.2003;Wooding et al. 2004;Bufe et al.2005;Soranzo et al.2005).Further-more,variation in the hTAS2R38gene has been associ-ated with drinking behavior but not alcohol dependence (Duffy et al.2004a,2004b).Whereas the hTAS2R38and other members of the cluster are located telomeric to the CHRM2gene(fig.1),the hTAS2R16gene is located between the CHRM2gene and our linkage peak.In this study,we genotyped the entire COGA linkage sam-ple with four SNPs within andflanking the hTAS2R16 gene,including two nonsynonymous cSNPs(K172N and R222H),and examined the association between the variations and alcohol dependence.
Material and Methods
Study Subjects and Asssment
Linkage sample.—Alcohol-dependent probands,defined by DSM-IIIR alcohol dependence(American Psychiatric Associa-tion1987)and Feighner-criteria for definite alcoholism(Feigh-ner et al.1972),were systematically recruited from alcohol-treatment units,and their biological relatives were invited to part
icipate in the study.All subjects were assd using the Semi-Structured Asssment for the Genetics of Alcoholism (Bucholz et al.1994;Heslbrock et al.1999),a mi-struc-tured interview designed as a polydiagnostic instrument that generates Feighner,DSM-IIIR,DSM-IV(American Psychiatric Association1994),and ICD-10(World Health Organization 1993)diagnos of alcohol dependence.The diagnos are esntially nested,with DSM-IIIR and Feighner definite alco-holism defining the broadest diagnosis,and ICD-10,the narrow-est definition of dependence(Culverhou et al.2005).In-formed connt was obtained from all subjects.A total of262 families—including2,310individuals,with an average of4.6 alcohol-dependent individuals per pedigree—were lected for genetic-linkage studies.Among the pedigrees,298individ-uals from35pedigrees are African American,and8pedigrees are of mixed ancestry(by lf-report).
Additional trios.—The COGA sample contains additional pedigrees with cell lines that were not informative for linkage and had therefore not been lected for the linkage sample. From the,we identified85trios consisting of a DSM-IV–defined alcohol-dependent individual and two parents.This sample of“additional trios,”includingfive African American trios,was typed for SNP rs846664.
Identity-by-Descent(IBD)Sharing
Nonparametric multipoint linkage analysis of independent ()affected sibling pairs was conducted using ASPEX, nϪ1
which allows large sibships to be included in analys.Linkage analys were performed using the SIBPHASE option,which infers allele sharing if there is ambiguity between identity by state and IBD,by u of marker frequencies in the sample.To avoid bias due to ethnic stratification,maximum-likelihood allele-frequency estimates were obtained,from the USERM13 subroutine of MENDEL(Boehnke1991),parately for Afri-can American and European American pedigrees.Maximum-likelihood estimates of sharing are displayed infigure2.
清脆的反义词Association Analysis
Transmit(Clayton1999),an extension of the transmission/ diquilibrium test(Spielman and Ewens1996)ud to test for association in extended pedigrees to allow for missing parental genotypes,was ud to test each SNP individually for evidence of linkage and association.The three cloly correlated alco-hol-dependence phenotypes—DSM-IIIR and Feighner definite alcoholism,DSM-IV alcohol dependence,and ICD-10alco-hol dependence—were tested to examine the consistency of re-
Figure2Affected-sibling-pair sharing on chromosome7.(A color version of thisfigure is available in the online edition of the Journal.) Sharing computed,by ASPEX sib_pha,using all parents,with large pedigrees down-weighted to.The solid line reprents all pedigrees;
nϪ1
the dotted line reprents pedigrees in which no individual has a copy of the rare polymorphism;the dashed line reprents pedigrees in which at least one individual has a copy of the rare polymorphism.
sults.For the additional trios,association wasfirst tested,using Transmit,in this sample alone and was then computed again when combined with the linkage sample.
SNP Assays
The dbSNP databa was ud to identify SNPs within and flanking the hTAS2R16gene.Both pyroquencing(Biotage Pyroquencing)and mass spectrometry(Sequenom)methods were ud for SNP genotyping.For pyroquencing,PCR prim-ers were lected using the MacVector6.5.3program(Accel-rys)to yield200–500-bp genomic fragments containing the SNP.Standard procedures were followed to generate PCR prod-ucts.Sequencing primers were designed using the Pyroquenc-ing Primer Design program.For mass spectrometry,PCR prim-ers,termination mixes,and multiplexing capabilities were de-termined with Sequenom Spectro Designer software v2.00.17. Standard PCR procedures were ud to amplify PCR products. All unincorporated nucleotides were deactivated with shrimp alkaline phosphata.A primer-extension r
eaction was then performed with the mass-extension primer and the appropriate termination mix.The primer-extension products were then cleaned with resin and were spotted onto a silicon SpectroChip. The chip was scanned by mass spectrometry(Bruker),and the resulting genotype spectra were analyzed with the Sequenom SpectroTYPER software.Sequence Analysis to Identify Additional Variants
饱的意思The entire coding region of hTAS2R16was quenced in both directions in DNA from14people,including one Eu-ropean American and six African Americans homozygous for the minor allele of rs846664and ven African Americans heterozygous for the same SNP.Publicly available quence databas were ud to lect PCR primers,to amplify the coding exon plus at least60bp offlanking intronic quence. The PCR product was purified using a QIAquick PCR puri-fication kit(Qiagen)to remove excess primers.Purified PCR product was quenced using the BigDye Terminator Cycle Se-quencing method and then was electrophored on an ABI3100 automated DNA quencer(Applied Biosystems[ABI]).Elec-tropherograms were analyzed using ABI DNA Sequencing An-alysis Software,version3.4.前无古人后无来者什么意思
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Heterologous Expression
Generation of the hTAS2R16haplotypes and functional an-alysis in HEK293cells were performed as described elwhere (Bufe et al.2002;Soranzo et al.2005).
Results
The entire COGA linkage sample was genotyped with four SNPs,including two nonsynonymous cSNPs,K172N (rs846664)and R222H(rs860170).Since the three
Figure3Predicted topology of hTAS2R16.E p extracellular domain;TM p transmembrane domain;I p intracellular domain.
a trend of association was also obrved between the synonymous cSNP rs1204014and DSM-IV alcohol de-pendence.The significance of this association incread ()when the test for association was restricted P p.003
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to the subt of African American families.Neither the noncoding SNP rs978739nor the nonsynonymous cSNP rs860170showed any association with alcohol depen-dence in our sample.Haplotype analys using the two nonsynonymous cSNPs(rs846664and rs860170)and the two significantly associated SNPs(rs846664and rs1204014)were less significant than the single SNP as-sociation results for rs846664(data not shown).
To further explore the role of SNP rs846664,we strat-ified the linkage sample into tho families containing the minor allele K172and tho without and performed affected-sibling-pair linkage analysis with ASPEX.The families,including62nuclear pedigrees,with the minor allele exhibited IBD sharing of61.0%at the linkage peak (D7S1799)on chromosome7for DSM-IIIR and Feigh-ner definite alcoholism,whereas families,including344 nuclear pedigrees,without the K172allele exhibited IBD sh
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aring of55.7%(overall sharing is56.5%)(fig.2). Thus,it appears that,although only15%of the nuclear families have one or more individuals carrying the minor allele,tho families contribute disproportionately to the linkage signal on chromosome7.The procedure of com-paring IBD sharing in pedigrees with and without a pu-tative risk allele is conrvative and may,in fact,under-estimate the effect of the allele(Li et al.2004).
To extend our results with rs846664,we genotyped this SNP in an independent sample:85trios(consisting of a DSM-IV alcohol-dependent individual and two par-ents)(table2).With u of Transmit,the independent trios showed an overtransmission of the K172allele, with a trend of association with DSM-IV alcohol depen-dence.When the data from the85trios were combined with that of the linkage sample,we obrved a P value of.00018for DSM-IV dependence and substantial over-transmission of the K172allele(79obrved/62expected transmission).Strong association was also detected with the correlated alcohol-dependence diagnos—DSM-IIIR and Feighner definite alcoholism()and
P p.002
ICD-10dependence().Sequencing of the cod-
P p.002
ing region of the hTAS2R16gene in individuals ho-mozygous and heterozygous for the minor allele(K172) confirmed that there were two nonsynonymous coding changes in the gene—the lysine r asparagine mutation at codon172(rs846664)and the arginine r histidine muta-tion at codon222(rs860170)—and a synonymous cSNP at codon282(rs1204014)(fig.3).No additional SNPs were obrved.Given the LD pattern in both European Americans and Africans derived from the International HapMap Project and the fact that the neighboring genes (CADPS2and SLC13A1)are each1100kb from the
Figure 4
Pairwi LD between markers flanking three SNPs in TAS2R16(500kb)from Yoruba in population from Ibadan,Nigeria.version of this figure is available in the online edition of the Journal.)
Table 3
Average EC 50Values of hTAS2R16Variants for Four Bitter-Taste Agonists
A GONIST
EC 50V ALUES (mM)
FOR
H APLOTYPE
P V ALUE FOR
t T EST
N172ϩR222
N172ϩH222
K172ϩH222NR/KH NH/KH NR/NH 8-Hydroxyquinoline-b -D -glucoside .6ע.3.7ע.4 1.6ע.8.002.012.302Helicin
1.7ע.9 1.8ע.8
2.9ע.9.017.025.796hTAS2R16gene (figs.4and 5),it is very likely that the alcoholism-susceptibility locus detected in the prent study is within hTAS2R16.
The K172N substitution rs846664is located in the extracellular loop 2between transmembrane domains 45(fig.3).In G protein–coupled receptors,including TAS2Rs,this domain has been associated with ligand binding (Adler et al.2000;Pronin et al.2004).Moreover ,experimental evidence has demonstrated that the extra-cellular loop 2is involved in the activation of the bitter-receptor hTAS2R43by its agonist 6–nitrosaccha-rin (Pronin et al.2004),which suggests that the K172N substitution in the extracellular loop 2may alter recep-tor signaling/taste perception.To directly test whether K172N and/or R222H influence hTAS2R16function,we expresd cDNAs coding for the three hTAS2R16haplotypes N172ϩR222,N172ϩH222,and K172H222in HEK293T cells,and we performed functional assays with four bitter-taste agonists as described where (Bufe et al.2002;Sora
nzo et al.2005).We not analyze the fourth predicted haplotype (K172R222)becau it was not obrved in the entire COGA
