American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:874–876 (2006)

The Serotonin 5-HT1D Receptor Gene and Attention-DeficitHyperactivity Disorder in Chinese Han SubjectsJun Li,1 Xia Zhang,1 Yufeng Wang,1* Rulun Zhou,1 Haobo Zhang,1 Li Yang,1 Bing Wang,1 and Stephen V. Faraone2

1Institute of Mental Health, Peking University (Peking University Sixth Hospital), Beijing 100083, China2Genetics Research Program and Department of Psychiatry and Behavioral Sciences,SUNY Upstate Medical University, Syracuse, New York

Attention-deficit/hyperactivity disorder (ADHD)is a heritable disease. Serotonin is one ofthe neurotransmitters involved in the etiology ofADHD. Serotonin-1D receptors are autoreceptorswhich can regulate the release of serotonin inbrain, so the HTR1D gene may be predisposing.The current study genotyped two variants ofHTR1D gene in 272 ADHD trios of Chineseethnicity, that is 1350T>C in the coding regionand 1236A>G in 30-UTR by the use of transmissiondisequilibrium test (TDT). The A allele of the1236A>G polymorphism exhibited both a trendtoward preferential transmission to ADHDprobands (x2¼3.815, P¼0.051) and a significantpreferential transmission to probands of ADHDC(x2¼4.198, P¼0.040). Additional polymorphi-sms in this gene need to be studied further.� 2006 Wiley-Liss, Inc.

KEY WORDS: attention-deficit/hyperactivity dis-order (ADHD); genetic polymorph-isms; TDT; HTR1D; serotonin

Please cite this article as follows: Li J, Zhang X, Wang Y,Zhou R, Zhang H, Yang L, Wang B, Faraone SV. 2006. TheSerotonin 5-HT1D Receptor Gene and Attention-DeficitHyperactivity Disorder in Chinese Han Subjects. Am JMed Genet Part B 141B:874–876.

INTRODUCTION

Attention-deficit/hyperactivity disorder (ADHD) is definedin the diagnostic and statistical manual of mental disorders,4th edition (DSM-IV) [AmericanPsychiatricAssociation, 1994]as developmental disorder exemplified by a persistent patternof inattention with or without hyperactivity or impulsivity.ADHD is estimated to affect 3–6% of school-age children[Faraone et al., 2003]. In China, prevalence of ADHD inschoolboys was about 5.8–6.1% [Shen et al., 1985; Leung et al.,

1996]. ADHD is themost commondisorder treated by pediatricpsychologists and psychiatrists. ADHD is clearly a significantrisk factor for school failure, emotional difficulties, poor peerrelationships in both adolescence and adulthood.

Family [Morrison and Stewart, 1971], twin [Thapar et al.,1995], and adoption [Cadoret, 1978] studies have providedevidence for a strong genetic component to ADHD. A recentreview of 20 twin studies suggested the heritability of ADHDwas about 0.76 [Faraone et al., 2005]. Although the mode ofinheritance of ADHD is not clear, the results of genetic etiologyof ADHD have implicated multiple genes, especially thoseinvolved in the catecholamine andmonoamine system, includ-ing dopamine, norepinephrine, and serotonin systems.

Early work implicated in ADHD studies has indicated lowcentral serotonin level in the development of ADHD [Rapoportet al., 1974]. Later, it was reported that low serotonin and itsmetabolites were associated with impulsivity and aggression[Linnoila et al., 1983]. The strongest evidence for the associa-tion between serotonin and ADHD came from the report byGainetdinov et al. [1999], which documented hyperactivityin mice lacking the gene encoding dopamine transporter(DAT-KO). When given psychostimulants, both activity anddopaminergic tonewereelevated inwild-typemice; in contrast,activity was substantially attenuated, while dopaminergictone was unperturbed in DAT-KO mice. This phenomenondemonstrated that dopamine was not the only factor con-tributing to the hyperactivity in these mice. When adminis-tered substances acted on a serotonin system, such asfluoxetine, quipazine, 5-hydroxytryptophan, and L-tryptophanto potentiate central serotonin, it markedly attenuated theactivity, with no effect on wild-type mice. These actions werepresumably due to increased extracellular concentration ofserotonin. All of these data demonstrated that serotonin playsa role in the etiology of ADHD, and subsequent researchfocused more attention to serotonin genes in the geneticcomponent of the etiology of ADHD. The serotonin transporterpromoter region (5-HTTLPR) [Manor et al., 2001; Fisher et al.,2002; Zoroglu et al., 2002] and serotonin receptor 1B gene(HTR1B) [Hawi et al., 2002; Quist et al., 2003] have repeatedlybeen reported to be associated with ADHD, especially whenrecently, pooled analyses suggested roles of 5-HTTLPR [Kentet al., 2002] and HTR1B to ADHD [Smoller et al., 2006]. As forthe tryptophan hydroxylase gene (TPH), no association withADHD was reported in Han Chinese population [Tang et al.,2001]. However, recently, two reports revealed positiveassociation of polymorphisms in the gene of TPH2, a secondform of TPH, and ADHD in European population [Sheehanet al., 2005; Walitza et al., 2005]. The work in our laboratoryfoundarole ofHTR1B inADHD[Li et al., 2005], but didnotfinda role of TPH in ADHD [Li et al., 2006].

Human serotonin-1D (HTR1D) and 1B receptors, shares77% amino acid sequence similarity and drug specificity [seeHartig et al., 1992 for review]. These two receptors were oncenamedas serotonin-1Dalphaand -1Dbeta.At least two reportshave demonstrated that HTR1Bmay contribute to the geneticetiology of ADHD [Hawi et al., 2002; Quist et al., 2003]. The

Jun Li and Xia Zhang contributed equally to this work.

Grant sponsor: Ministry of Science and Technology, China(2004BA720A20); Grant sponsor: Project of Science and Technol-ogy, Beijing (Y0204003040831); Grant sponsor: Key Project forClinical Faculty Foundation, Ministry of Health, China (2004-468).

*Correspondence to: Yufeng Wang, Institute of Mental Health,Peking University (Peking University sixth hospital), Beijing100083, China. E-mail: wangyf@bjmu.edu.cn

Received 15 December 2005; Accepted 28 April 2006

DOI 10.1002/ajmg.b.30364

� 2006 Wiley-Liss, Inc.

current study is the first to examine the potential role ofHTR1D in susceptibility towardADHD.HTR1Dwas located at1p34.3–36.3 and its coding receptor was expressed at basalganglia, hippocampus and some cortex, most of which werereported to be involved in ADHD.

MATERIALS AND METHODS

Subjects

Two hundred seventy-two nuclear families participated inthe present study. ADHD probands were recruited from theADHD outpatient clinic at the Child and Adolescent Psychia-tric Division of the Sixth Hospital, Peking University inBeijing, PRC. All probands were interviewed by at least twodifferent child psychiatrists with the aid of their biologicalparents and teacher and fulfilled DSM-IV diagnostic criteriafor ADHD [American Psychiatric Association, 1994]. All thesubjects involved were Han. Among these probands, 236 weremale and 36were female. The average agewas 10.5� 2.5 yearsand the average intelligence quotient (IQ) was 100.3� 14.0.One hundred nine (40.0%) probands met criteria for ADHDcombined type (ADHD-C), 17 (6.3%) met criteria for ADHDhyperactive-impulsive type (ADHD-HI), and 146 (53.7%) metcriteria for ADHD inattentive type (ADHD-I). Writteninformed consent was obtained from their parents. The studywas reviewed and approved by the Ethics Committee of thePeking University.

Genotyping

Genomic DNA was extracted from whole blood bystandard procedures. 1350T>Cpolymorphismwas genotypedusing the conditions described by Ozaki et al. [1995].1236A>G polymorphism was amplified with forward primer50-TCC TGT AAC CTC ATC GGG ATT G-30 and backwardprimer 50-CGA GAG GGC AAG ACT CCG TC-30. Afterinitial denaturation at 958C for 4 min, 35 cycles were carriedout at 968C for 30 sec, 538C for 30 sec, and 728C for 30 sec,followed by a final step of elongation at 728C for 7 min. PCRproducts (10 ml) were digested with five units of EcoRV (MBIFermentas, Vilnius, Lithuania) in 16 ml reaction volumes for12 hr at 378C. The digested PCR products were run on a 2.5%agarose gel at 100 V for 1.5 hr, then the agarose gel into watercontaining ethidium bromide for half an hour. Finally, theywere analyzedwithGelDoc 2000 imaging system to detect andrecord the genotype of each sample. After digestion, the Avariant was cut into two fragments of 66 and 170 bp, while theG variant was uncut and was 236 bp. Genotypes were read bytwo persons, both of whom were blind to the identity of thesamples.

Statistics

The transmission disequilibrium test (TDT) [Spielman et al.,1993] was performed to test for linkage and association of thetwo polymorphisms with ADHD.

RESULTS

Table I shows the results of TDT analysis for 1350T>C. Nobiased transmission was observed in either the total set ofADHD trios (w2¼ 1.190, P¼ 0.275) or within the categoricalsub-types of ADHD trios.

Table II shows the results of TDT analysis for 1236A>G. Atendency of overtransmission of A allele to ADHD probandswas observed (w2¼ 3.815, P¼ 0.051). When families weredivided into three groups according to the diagnostic sub-typesof probands, significant transmission bias was observed infamilies with ADHD-C probands (w2¼ 4.198, P¼ 0.040); how-ever, no significant transmission bias was observed in trioswith ADHD-I probands (w2¼ 0.637, P¼ 0.425). Results for theADHD and ADHD-C trios were statistically insignificant bythe Holm–Bonferroni criterion.

DISCUSSION

The current study is the first to explore the relationshipbetween HTR1D and ADHD. Serotonin-1D receptors areautoreceptors which can regulate the release of serotonin inbrain [Maura et al., 1993]. Therefore, variants inHTR1D genemayplaya role inADHDbydisrupting this process.Ozaki et al.[1995] screened the coding region of HTR1D by SSCP andfound 1350T>C, which is synonymous. Bergen et al. [2003]genotyped four polymorphisms in HTR1D, three of which,except 1350T>C, exhibited significant transmission disequi-librium, including 2190A>G, �628T>C, and �1123T>C.

The 1236A>G (rs623988) is located in the 30-UTR and thecurrent study was the first to genotype it in ADHD samples.TDT analysis showed evidence of an association between thisvariant and ADHD, especially for ADHD-C probands. Since1236A>G could not change the function ofHTR1D, it may notbe the true risk-conferring allele; it may be in linkagedisequilibrium (LD) with the true functional variant, perhapswithin theHTR1D gene itself, or possibly in the vicinity of thegene. 1236A>G is in very strong LD with 2190A>G (datafrom our own laboratory) which was also located at the 30-UTRof HTR1D. From the study of Bergen et al. [2003], we can seethat 2190A>G was in very significant LD with two poly-morphisms (�628T>C and �1123T>C) in the promoter ofHTR1D; therefore, 1236A>G was also in LD with these twopromoter polymorphisms which may change the expression ofHTR1D. Further study is needed to study the relationshipbetween promoter polymorphisms and ADHDand to ascertain

TABLE I. The Number of Alleles of 1350T>C Polymorphism Transmitted or not TransmittedFrom Heterozygous Parents to ADHD Offspring

Phenotype Number of trios Alleles

T/NT

w2 PT NT

ADHD 272 C 64 78 1.190 0.275T 78 64

ADHD-C 109 C 23 34 1.754 0.185T 34 23

ADHD-I 146 C 36 38 0.014 0.907T 38 36

T, transmitted; NT, not transmitted.

5-HT1D Polymorphism and ADHD 875

the function of these variants, so that the role of HTR1D inADHDmay be further clarified.

In summary, the current study indicated an associationbetween HTR1D and ADHD; however, this result should bereplicated in both additional Han Chinese samples andsamples from other ethnic populations as well.

REFERENCES

American Psychiatric Association. 1994. Diagnostic and statistical manualof mental disorders. 4th edition. (DSM-IV). Washington, DC: AmericanPsychiatric Association.

Bergen AW, van den Bree MB, Yeager M, Welch R, Ganjei JK, Haque K,Bacanu S, Berrettini WH, Grice DE, Goldman D, Bulik CM, Klump K,FichterM,HalmiK,KaplanA, StroberM, Treasure J,Woodside B,KayeWH. 2003. Candidate genes for anorexia nervosa in the 1p33-36 linkageregion: Serotonin 1D and delta opioid receptor loci exhibit significantassociation to anorexia nervosa. Mol Psychiatry 4:397–406.

Cadoret RJ. 1978. Psychopathology in adopted-away offspring of biologicparents with antisocial behavior. Arch Gen Psychiatry 2:176–184.

Faraone SV, Sergeant J, Gillberg C, Biederman J. 2003. The worldwideprevalence of ADHD: Is it an American condition? World Psychiatry2:104–113.

Faraone SV, Perlis RH, Doyle AE, Smoller JW, Goralnick J, Holmgren MA,et al. 2005.Molecular genetics of attention deficit hyperactivity disorder.Biol Psychiatry 57:1313–1323.

FisherSE,FrancksC,McCracken JT,McGoughJJ,MarlowAJ,MacPhie IL,Newbury DF, Palmer CGS,Woodward JA, Del’HommeM, Cantwell DP,Nelson SF, Monaco AP, Smalley SL. 2002. A genomewide scan for lociinvolved in attention-deficit/hyperactivity disorder. Am J Hum Genet70:1183–1196.

Gainetdinov RR, Wetsel WC, Jones SR, et al. 1999. Role of serotonin in theparadoxical calming effect of psychostimulants onhyperactivity. Science285(15):397–401.

Hartig PR, Branchek TA, Weinshank RL. 1992. A subfamily of 5-HT1Dreceptor genes. Trends Pharmacol Sci 4:152–159.

Hawi Z, Dring M, Kirley A, Foley D, Kent L, Craddock N, Asherson P,Curran S, Gould A, Richards S, Lawson D, Pay H, Turic D, Langley K,Owen M, O’Donovan M, Thapar A, Fitzgerald M, Gill M. 2002.Serotonergic system and attention deficit hyperactivity disorder(ADHD): A potential susceptibility locus at the 5-HT(1B) receptor genein 273 nuclear families from a multi-centre sample. Mol Psychiatry7:718–725.

Kent L, Doerry U, Hardy E, Parmar R, Gingell K, Hawi Z, Kirley A, LoweN,Fitzgerald M, Gill M, Craddock N. 2002. Evidence that variation at theserotonin transporter gene influences susceptibility to attention deficithyperactivity disorder (ADHD): Analysis and pooled analysis. MolPsychiatry 7:908–912.

Leung PWL, Luk SL, Ho TP, Taylor E, Mak FL, Bacon-Shone J. 1996. Thediagnosis and prevalence of hyperactivity in Chinese schoolboys. BrJ Psychiatry 168:486–496.

Li J,WangY, ZhouR, ZhangH,Yang L,WangB,Khan S, Faraone SV. 2005.Serotonin 5-HT1B receptor gene and attention deficit hyperactivitydisorder in Chinese Han subjects. Am J Med Genet Part B Neuropsy-chiatr Genet 132B:59–63.

Li J, Wang Y, Zhou R, Zhang H, Yang L, Wang B, Faraone SV. 2006.Association between tryptophan hydroxylase gene polymorphisms andattention deficit hyperactivity disorder inChineseHanpopulation. AmJMed Genet B Neuropsychiatr Genet 41:126–129.

Linnoila M, Virkkunen M, Scheinin M, et al. 1983. Low cerebrospinal fluid5-hydroxyindoleacetic acid concentration differentiates impulsive fromnonimpulsive violent behavior. Life Sci 33:2609–2614.

Manor I, Eisenberg J, Tyano S, Sever Y, Cohen H, Ebstein RP, Kotler M.2001. Family-based association study of the serotonin transporterpromoter region polymorphism (5-HTTLPR) in attention deficit hyper-activity disorder. Am J Med Genet 105:91–95.

Maura G, Thellung S, Andrioli GC, Ruelle A, Raiteri M. 1993. Release-regulating serotonin 5-HT1D autoreceptors in human cerebral cortex.J Neurochem 3:1179–1182.

Morrison JR, Stewart MA. 1971. A family study of the hyperactive childsyndrome. Biol Psychiatry 3:189–195.

Ozaki N, Lappalainen J, Dean M, Virkkunen M, Linnoila M, Goldman D.1995. Mapping of the serotonin 5-HT1D alpha autoreceptor gene(HTR1D) on chromosome 1 using a silent polymorphism in the codingregion. Am J Med Genet 2:162–164.

Quist JF,BarrCL, SchacharR,RobertsW,MaloneM,TannockR,BasileVS,Beitchman J, Kennedy JL. 2003. The serotonin 5-HT1B receptor geneand attention deficit hyperactivity disorder. Mol Psychiatry 8:98–102.

Rapoport J, Quinn P, Scribanu N, et al. 1974. Platelet serotonin ofhyperactive school age boys. Br J Psychiatry 125:138–140.

SheehanK,LoweN,KirleyA,Mullins C, FitzgeraldM,GillM,Hawi Z. 2005.Tryptophanhydroxylase 2 (TPH2) gene variants associatedwithADHD.Mol Psychiatry 10:944–949.

Shen YC, Wang YF, Yang XL. 1985. An epidemiological investigation ofminimal brain dysfunction in six elementary schools in Beijing. J ChildPsychol Psychiatry 26:777–788.

Smoller JW, Biederman J, Arbeitman L, Doyle AE, Fagerness J, Perlis RH,Sklar P, Faraone SV. 2006. Association between the 5HT1B receptorgene (HTR1B) and the inattentive subtype of ADHD. Biol Psychiatry59:460–467.

Spielman RS,McGinnis RE, EwensWJ. 1993. Transmission test for linkagedisequilibrium: The insulin gene region and insulin-dependent diabetesmellitus (IDDM). Am J Hum Genet 3:506–516.

Tang G, Ren D, Xin R, Qian Y, Wang D, Jiang S. 2001. Lack of associationbetween the tryptophan hydroxylase gene A218C polymorphism andattention-deficit hyperactivity disorder in Chinese Han population. AmJ Med Genet 105:485–488.

Thapar A, Hervas A, McGuffin P. 1995. Childhood hyperactivity scores arehighly heritable and show sibling competition effects: Twin studyevidence. Behav Genet 6:537–544.

Walitza S, Renner TJ, Dempfle A, Konrad K, Wewetzer Ch, Halbach A,Herpertz-Dahlmann B, Remschmidt H, Smidt J, Linder M, Flierl L,KnolkerU,Friedel S, SchaferH,GrossC,HebebrandJ,WarnkeA,LeschKP. 2005. Transmission disequilibrium of polymorphic variants in thetryptophan hydroxylase-2 gene in attention-deficit/hyperactivity dis-order. Mol Psychiatry 10:1126–1132.

ZorogluSS,ErdalME,Alasehirli B,ErdalN, Sivasli E, TutkunH,SavasHA,Herken H. 2002. Significance of serotonin transporter gene 5-HTTLPRand variable number of tandemrepeat polymorphism inattentiondeficithyperactivity disorder. Neuropsychobiology 45:176–181.

TABLE II. The Number of Alleles of 1236A>G Polymorphism Transmitted or Not TransmittedFrom Heterozygous Parents to ADHD Offspring

Phenotype Number of trios Alleles

T/NT

w2 PT NT

ADHD 272 A 169 134 3.815 0.051G 134 169

ADHD-C 109 A 75 51 4.198 0.040G 51 75

ADHD-I 146 A 84 73 0.637 0.425G 73 84

T, transmitted; NT, not transmitted.

876 Li et al.