American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:473–476 (2006)

Contribution of 5-HT2A ReceptorGene -1438A>G Polymorphism to Outcome ofAttention-Deficit/Hyperactivity Disorder in AdolescentsJun Li,1 Chuanyuan Kang,1 Yufeng Wang,1* Rulun Zhou,1 Bing Wang,1 Lili Guan,1

Li Yang,1 and Stephen V. Faraone2

1Institute of Mental Health, Peking University (Peking University sixth hospital), Beijing, People’s Republic of China2Medical Genetics Research Program and Department of Psychiatry and Behavioral Sciences,SUNY Upstate Medical University, Syracuse, New York

Attention-deficit/hyperactivity disorder (ADHD)typically emerges before 7 years of age and maypersist into adolescence or adulthood. The ado-lescent outcome can be classified into fourtypes, including non-remission, syndromaticremission, symptomatic remission, and func-tional remission. Genetic factors are believed tocontribute to symptom stability and changeacross development, so adolescent outcome maybe a sub-phenotype for molecular genetic studiesof ADHD. Serotonin system genes are primecandidates for this sub-phenotype, since thedevelopment of this neurotransmitter systemparallels the course of ADHD. The current studyexamined the association between adolescentoutcome in ADHD and serotonin system genes,including the -1438A>G polymorphism of theserotonin 2A receptor gene (HTR2A) and the-759C>T polymorphism of the serotonin 2C recep-tor gene (HTR2C). The -1438A>G polymorphismwas found to be related to remission in ADHD,especially functional remission (P¼0.029). Due topotential phenotypic and etiologic heterogeneityin ADHD, the results of this study must bereplicated in additional samples before they canbe generalized to other populations.� 2006 Wiley-Liss, Inc.

KEY WORDS: serotonin; HTR2A; HTR2C; atten-tion-deficit/hyperactivity disor-der (ADHD); outcome; adolescent

Please cite this article as follows: Li J, Kang C, Wang Y,Zhou R, Wang B, Guan L, Yang L, Faraone SV.2006. Contribution of 5-HT2A Receptor Gene -1438A>GPolymorphism to Outcome of Attention-Deficit/Hyperactivity Disorder in Adolescents. Am J Med GenetPart B 141B:473–476.

INTRODUCTION

Attention-deficit/hyperactivity disorder (ADHD) is amongthe most common childhood psychiatric disorders, and ismarked by inappropriate levels of inattention, hyperactivity,and/or impulsivity. The disorder emerges early in childhood,usually before 7 years of age, and may persist into adolescenceor adulthood. The adolescent and young adult with ADHD is atrisk of school failure, emotional difficulties, poor peer relation-ships and even trouble with the law [Gittelman et al., 1985;Barkley et al., 1990], making ADHD a major public healthproblem.

Family, twin and adoption studies have demonstrated thatADHD is a highly heritable disorder with a mean heritabilityestimate of 75% [Faraone et al., 2005]. However, candidategene studies have produce inconsistent findings and aretherefore not conclusive regarding the genetic etiology ofADHD [Faraone et al., 2005]. One of the reasons for thisinconsistency may be the heterogeneity of ADHD [Faraoneet al., 1999]. A recently adopted approach is to focuson the association between genes and sub-phenotype orendophenotype of ADHD [Almasy and Blangero, 2001]. Sub-phenotype can decrease the heterogeneity of ADHD andendophenotype represents intermediate biological pheno-types, both of which may be closer to or more tightly mappedonto genes than is the full disorder. Several neuropsychological[Swansonet al., 2000], structural and functional neuroimaging[Castellanos et al., 1998] and electrophysiological [Loo et al.,2003] sub-phenotypes have been used in molecular geneticstudies of ADHD, and the results of these studies have provenvery informative in disentangling both the biological andgenetic basis of the illness.

Longitudinal studies of children diagnosedwithADHDhaveidentified age-dependent remission of symptoms in adoles-cence and young adulthood in many cases [Biederman et al.,2000]. Yet, most follow-up studies published during the pasttwo decades have consistently demonstrated that between 30and 50% children diagnosed with ADHD continue to displaysymptoms in adolescence and adulthood [Gittelman et al.,1985; Lambert et al., 1987]. Genetic factors are believed tocontribute to symptom stability versus change across develop-ment [Kuntsi et al., 2005], so it is reasonable to postulate thatthe adolescent outcome of ADHD is controlled at least in partby genetic factors. Persistence of ADHD into adolescence hasbeen proposed as a sub-phenotype previously [Faraone et al.,2000;Faraone, 2004a,b]; however, to ourknowledge, nogeneticassociation studies of adolescent outcome in ADHD have yetbeen reported.

The serotonin (5-HT) neurotransmitter system is a primecandidate pathway for ADHD, as it also shows some interest-ing age-dependent developmental patterns that parallel thetypical emergence of ADHD. For example, developmentalstudies in monkeys show increases in 5-HT receptor binding

Grant sponsor: Ministry of Science and Technology, China;Grant number: 2004BA720A20; Grant sponsor: Project of Scienceand Technology, Beijing; Grant number: Y0204003040831; Grantsponsor: Key Project for Clinical Faculty Foundation, Ministry ofHealth, China; Grant number: 2004-468.

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

Received 31 August 2005; Accepted 23 February 2006

DOI 10.1002/ajmg.b.30320

� 2006 Wiley-Liss, Inc.

during infancy which peaks prior to the onset of puberty, andslowly decreases throughout adolescence and early adulthood[Lidow et al., 1991]. In humans, 6-year-olds were found to havegreaterbindingof 5-HT2 receptors relative tonewborns and13-to 14-year-olds [Biegon and Greuner, 1992]. A 2- to 3-yearfollowup study in 8- to 11-year-old boyswithADHD found thatcentral serotonin function decreased significantly with age[Pick et al., 1999].

Based on these clinical observations on the course of ADHDand the development of the serotonergic system, the presentstudy was designed to examine the potential role of 5-HT2receptor gene polymorphisms on the course of ADHD.Associations of these polymorphisms were examined for eachof the remission categories proposed by Keck et al. [1998],which include: (1) syndromatic remission, which refers to theloss of full diagnostic status; (2) symptomatic remission,which refers to the loss of partial diagnostic status; and (3)functional remission, which refers to the loss of partialdiagnostic status plus functional recovery. These categoriesare useful for clarifying components of the complex outcomesof ADHD, and ultimately may prove useful in derivinga better understanding of the genetic architecture of thedisorder itself.

MATERIALS AND METHODS

Subjects

Subjects consisted of 82 ADHD boys recruited from theADHD outpatient clinic at the Child and Adolescent Psychia-try Division of Sixth Hospital, Peking University in Beijing,PRC. Subjects were assessed at baseline and at follow-up 3–5 years later. All probands fulfilled DSM-IV diagnostic criteriafor ADHD based on interviews by at least two different childpsychiatrists with the aid of information from biologicalparents and teachers. All subjects were of Han Chinesedescent.Written informed consent was obtained from parents.The study was reviewed and approved by the Ethics Commit-tee ofPekingUniversity.At baseline, theage of subjects rangedfrom 7.5 to 13.5 years, with a mean age of 10.6� 1.6 years. Atfollow-up, the age of subjects ranged from 12.5 to 16.5 years,with a mean age of 14.4� 1.4 years. The mean durationbetween the two assessment was 3.8� 0.5 years. Fourteen(17.0%) children had been treated with methylphenidate orEEG biofeedback training for at least 6 months. However, nosubject had received treatment for the 6 months prior tothe study.

The interview instrument was the Clinical DiagnosticInterview Scales (CDIS) [Barkley, 1998a], a structured inter-view derived from the DSM-IV. Fifty-eight (70.7%) subjectsand their parents were directly interviewed, while 24 (29.3%)were interviewed by telephone. Both of the interviewersinvolved in the current study had master’s degrees inpsychiatry and were trained to high levels of inter-raterreliability.

Per the DSM-IV, symptoms of ADHD were divided into twoclusters (inattention and hyperactivity/impulsivity) and eachof the two clusters has nine symptoms. Based on the methoddescribed by Keck et al. [1998] and Biederman et al. [2000],syndromatic, symptomatic and functional remission weredefined as following: ‘‘syndromatic remission’’ referred tohaving four to five symptoms of each cluster; ‘‘ symptomaticremission’’ referred to having fewer than four symptoms of theeach cluster without functional recovery; and ‘‘functionalremission’’ referred to having fewer than four symptoms ofeach clusterplus functional recovery.Toassess functioning,weused a questionnaire requiring parents to give scores on theirchild’s functioning in five aspects, including school compe-tence, peer relationships, parent-child relationship, behavior,

and emotion. The score ranged from 0 to 100 and we definedscores �70 as indicating functional recovery.

Genotyping

Genomic DNA was extracted from whole blood by standardprocedures. Genetic polymorphisms, including the -1438A>Gpolymorphism of the serotonin receptor 2A gene (HTR2A,Genebank accession number: 3356) and the -759C>T poly-morphism of the serotonin receptor 2C gene (HTR2C,Genebank accession number: 3358), were genotyped underconditions previously reported [Quist et al., 2000; Yuan et al.,2000]. The 468 bp product of -1438A>G was digested by MspIand the alleles were separated in 2% agarose gel. Allele Awas not digested and allele G was digested into two fragmentsof 244 and 224 bp. The 252 bp product of -759C>Twas digestedby AciI and the alleles were separated in 3% agarose gel.Allele C was digested into 126bpþ 34bpþ 27bpþ 54bpþ 10bpor 126bpþ 34bpþ 81bpþ 10bp, allele T was digested into160bpþ 27bpþ 54bpþ 10bp or 160bpþ 81bpþ 10bp.

Statistics

The w2 test was used to compare allele frequencies betweengroups of remitted children and non-remitted children.Genotype frequencies were not compared due to the relativelylow numbers of subjects with each genotype. Statisticaltests were conducted using SPSS software (version 11.0) forWindows.

RESULTS

Of the 82 total children in the sample, 41 (50%) showed noremission, 41 (50%) met criteria for remission: 14 (17%) metcriteria for syndromatic remission, 7 (9%) met criteria forsymptomatic remission, and 20 (24%) met criteria for func-tional remission.

Relative to children showingno remission ofADHD, childrenwith any type of remission had a higher frequency of the Aallele relative to non-remitted subjects; however, this trend didnot reach statistical significance (P¼ 0.061). Children withfunctional remission (P¼ 0.029) had higher frequencies of theAallele of the -1438A>Gpolymorphism ofHTR2A (Table I).Nosignificant differences in allele frequencies of the -759C>Tpolymorphism of HTR2C were observed among any of theremitted or non-remitted subjects (Table II).

DISCUSSION

In the course of ADHD, adolescence is an importantperiod when symptoms of approximately half of patients maydecline or even disappear entirely [Gittelman et al., 1985;Lambert et al., 1987; Faraone et al., 2006]. Many factors mayinfluence this trajectory, including environmental and socialfactors, which have been examined previously [Biedermanet al., 1996; Barkley, 1998b]. Although genetic influences on

TABLE I. Comparison of Allele Frequencies of the HTR2A-1438A>G Polymorphism Between Remitted and

Nonremitted Children

Phenotypic category N G A w2a Pa

Remission 82 36 46 3.514 0.061Syndromatic remission 28 14 14 0.618 0.432Symptomatic remission 14 7 7 0.356 0.551Functional remission 40 15 25 4.764 0.029a

Non-remission 82 48 34 — —

aTest statistics and P-values are relative to subjects with non-remission.

474 Li et al.

the emergence of the disorder are well established, biologicalinfluences on the course of the disorder during adolescence andyoung adulthood have not been as often considered. To ourknowledge, the current study is the first to focus on thecontribution of genetic polymorphisms to adolescent outcomeinADHD. Although the current sample size is relatively small,we have identified a relatively strong influence (oddsratios� 2.0) of the A allele of the HTR2A -1438A>G poly-morphism on various stages of remission in ADHD, especiallyfunctional remission.

The 5-HT2A receptor gene is an important candidate genefor ADHD due to the interaction of the serotonin system withthe dopamine system. Several associations between ADHDand serotonin system genes have been reported [Kent et al.,2002; Li et al., 2004; Smoller et al., 2005]. The -1438A>Gpolymorphism and the 102T>C polymorphism (which is inhigh linkage disequilibrium with -1438A>G) of HTR2A havebeen studied previously for a role in ADHD; however, nopositive associations were found [Quist et al., 2000; Zorogluet al., 2003]. It is proposed that most longitudinal studies thatassay 5-HT levels mainly reflect 5-HT2A receptor function[Biegon and Greuner, 1992; Pick et al., 1999]. Based on thesestudies and the conclusion that 5-HTactivity is age-dependent,wehypothesized that the 5-HT2A receptor genemight relate toremission of ADHD, and the current study has providedevidence for such a relationship. Among the three types ofremission, functional remission (or full recovery)may have thestrongest genetic foundation, which may be why it wasobserved to have the strongest relationship to the -1438A>Gpolymorphism.

The derivation and validation of appropriate sub-pheno-types and endophenotypes is believed to be one bottleneck ofgenetic studies; thus, a main contribution of the current studymay be the use of adolescent outcome of ADHD as one suchvalid sub-phenotype [Faraone et al., 2000; Faraone, 2004a,b].However, the advantages and disadvantages of this sub-phenotype need further investigation. Another gene involvedin the current study was HTR2C which, due to its physicallocation on chromosome Xq24, only allowed for hemizygousexamination of association of this gene with ADHD outcome.Thus, very few T alleles were observed, and this may haveseverely constrained the power to detect any associationbetween this polymorphism and remittance of ADHD in thissample.

Besides the relatively small sample size, the main limita-tions of the current study is that we used a self-reportquestionnaire to assess functioning, which can only give ageneral sense of functioning in these children; however, thisapproach has some precedent. For example, Biederman et al.[2000] used the Global Assessment of Functioning Scale anddefined scores higher than 60 as ‘‘no functional impairment.’’The differences between these scales and the thresholdsapplied may be one reason for a higher level of functionalrecovery observed in the current study relative to thatwork.As

with any allelic association detected in a small sample from arelatively homogeneous population, these results must bereplicated by other groups before these results can be acceptedand generally applied. This is especially relevant to our studygiven that we did not correct for multiple comparisons.Ultimately, if this HTR2A polymorphism can be verified as apredictor of functional remission in ADHD, this gene may findsome utility in the clinic for formulating treatment strategiesand establishing prognoses.

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aTest statistics and P-values are relative to subjects with non-remission.

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