Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 918410 12 pageshttpdxdoiorg1011552013918410
Research ArticleCatecholaminergic Gene Variants Contribution in ADHD andAssociated Comorbid Attributes in the Eastern Indian Probands
Paramita Ghosh1 Kanyakumarika Sarkar2 Nipa Bhaduri3 Anirban Ray4 Keka Sarkar1
Swagata Sinha1 and Kanchan Mukhopadhyay1
1 Manovikas Biomedical Research and Diagnostic Centre 482 Madudah Plot I-24 Sector-J EM Bypass Kolkata 700107 India2Department of Biotechnology CT Institute of Pharmaceutical Sciences Jalandhar Panjab 140020 India3 Chembiotek TCG Lifesciences Kolkata 700091 India4Department of Psychiatry Chittaranjan National Medical College Kolkata 700020 India
Correspondence should be addressed to Kanchan Mukhopadhyay kanchanmvkyahoocom
Received 4 April 2013 Revised 7 August 2013 Accepted 12 August 2013
Academic Editor Xueyuan Cao
Copyright copy 2013 Paramita Ghosh et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Contribution of genes in attention deficit hyperactivity disorder (ADHD) has been explored in various populations and severalgenes were speculated to contribute small but additive effects We have assessed variants in four genes DDC (rs3837091 andrs3735273) DRD2 (rs1800496 rs1801028 and rs1799732) DRD4 (rs4646984 and rs4646983) and COMT (rs165599 and rs740603)in IndianADHDsubjects with comorbid attributes Cases were recruited following theDiagnostic and StatisticalManual forMentalDisorders-IV-TR after obtaining informed written consent DNA isolated from peripheral blood leukocytes of ADHD probands(119873 = 170) their parents (119873 = 310) and ethnically matched controls (119899 = 180) was used for genotyping followed by population-and family-based analyses by the UNPHASED program DRD4 sites showed significant difference in allelic frequencies by case-control analysis while DDC and COMT exhibited bias in familial transmission (119875 lt 005) rs3837091 ldquoAGAGrdquo rs3735273 ldquoArdquors1799732 ldquoCrdquo rs740603 ldquoGrdquo rs165599 ldquoGrdquo and single repeat alleles of rs4646984rs4646983 showed positive correlation with co-morbid characteristics (119875 lt 005) Multi dimensionality reduction analysis of case-control data revealed significant interactiveeffects of all four genes (119875 lt 0001) while family-based data showed interaction betweenDDCandDRD2 (119875 = 004)This first studyon these gene variants in Indo-Caucasoid ADHD probands and associated co-morbid conditions indicates altered dopaminergicneurotransmission in ADHD
1 Introduction
Attention deficit hyperactivity disorder (ADHD) is a neu-rodevelopmental disorder characterized by age inappropriateinattentiveness hyperactivity and impulsivity [1] Comorbid-ity is quite common in ADHDwith around 60ndash100 patientsexhibiting one or more co-morbid conditions Among differ-ent co-morbid characteristics oppositional defiant disorder(ODD) conduct disorder (CD) anxiety disorder (AD)depressive disorder mood disorder (MD) and learningdisabilities (LD) are of frequent occurrence [2] around 27of ADHD patients were reported to have ODD andor CDwhile 18 had AD The Diagnostic and Statistical ManualforMentalDisorders-IV-text revised (DSM-IV-TR) describesADHD children with ODD as unusually disobedient andhostile towards higher authority [1] A number of children
with ADHD (46) were also reported to have LD experienc-ing difficulty in reading spelling vocabulary arithmetic andwritten communication [2] this affects not only academicsbut also their social lives Common frontal lobe dysfunctionwas observed in both ADHD and LD patients [3]
Strong genetic basis of ADHD is supported by twinadoption or family-based studies [4 5] Amajor role of genesregulating neurotransmitters leading mainly to dopamine(DA) dysfunction has been postulated in the disease etiology(reviewed in [6]) Since DA activity is essential to the motorand cognitive functioning of the brain a wide range of neu-rological symptoms were speculated from malfunctioning ofeven a single part of the system [7]
Action of DA is mediated through DA receptors (DRD)grouped in two families based on the activation (D1-likereceptors DRD1 andDRD5) or inhibition (D2-like receptors
2 BioMed Research International
DRD2 DRD3 and DRD4) of adenylate cyclase in responseto ligand binding [8 9] The dopaminergic hypothesis ofADHD is based mostly on the malfunctioning of D2-likereceptors in the brain [10 11] The DRD4 gene encodingfor DA receptor 4 has been extensively studied and positiveassociations with ADHD were reported in Caucasian as wellas several non-Caucasian populations [5 12ndash15] The D2 andD3 receptors have been studied mostly in Caucasian andChinese populations revealing inconsistent findings [16ndash18]
Genes encoding for enzymes involved in the cate-cholaminergic system like catechol-O-methyl transferase(COMT) [19 20] dopamine decarboxylase (DDC) [21ndash23]dopamine beta hydroxylase [5 15] and monoamine oxidase[15 18 22 23] have also gained a lot of importance in explor-ing the etiological basis of ADHD Some of these geneticvariants have revealed significant association with ADHDassociated co-morbid disorders [24] and were speculated asthe reason for comorbidities being so common in Caucasiansubjects with ADHD [25]
In the Indo-Caucasoid ADHD probands DRD4 DAT1MAOA COMT and DBH gene variants showed significantassociation with the disorder [5] However till date neitherany report on DDC and DRD2 nor any information on thecontribution of gene variants inADHDassociated co-morbiddisorders was available in this particular ethnic group SinceADHD subjects frequently exhibit co-morbid behavioraldisorders including CD ODD and substance abuse [2 3]in the present study we have selected nine polymorphic sitesin four catecholaminergic genes DRD2 DRD4 DDC andCOMT which modulate function of DA The sites analyzedhave been explored in association with ADHD in EuropeanCaucasian and Han Chinese populations [12ndash14 18 22 25ndash28] or behavioral disorders [29 30] and analyzed for thefirst time for their contribution in the etiology of Indo-CaucasoidADHDprobands stratified on the basis of differentco-morbid disorders
2 Subjects and Methods
21 Study Subjects We recruited ADHD probands (119873 =170) from the out-patient department of Manovikas KendraRehabilitation and Research Institute for the HandicappedKolkata based on (a) DSM-IV-TR criteria [1] (b) hyper-activity level measured by Connersrsquo Parents and TeachersRating Scale [31] (c) intelligencedevelopmental quotientassessed byWechslerrsquos Intelligence Scale for Children [32] forchildren above five years and Developmental Screening Testfor children below 5 years [33] Mean age of probands was77 years plusmn 23 SD and male to female ratio was 10 1 Out of170 probands 143 were complete parent-proband trios 17 hadonly one parent and 10 were affected probands only Hyper-activeimpulsive (112) and inattentive (73) subtypes wereonly fewwhilemajority of the probands belonged to the com-bined subtype (815) Based on the presenting co-morbidsymptoms ADHD probands were subgrouped as ADHD-comorbidity ADHD+CD ADHD+LD ADHD+ODDand ADHD+MD Subjects suffering from only psychiatricproblems pervasive developmental disorders and any form
ofmental retardation (IQ le 80) including fragile X syndromewere excluded
A control group (119873 = 180 mean age 197 years plusmn 794 SDandmale to female ratio 10 3) evaluated following the DSM-IV-TR criteria [1] for ADHD was also recruited All theindividuals enlisted for the study belonged to the Indo-Caucasoid ethnic category For participation in the studyinformed written consent was obtained from the controlsand guardians of ADHD probands The study protocol wasapproved by the Institutional Human Ethical Committee
22 Selection of SNPs and Genotyping Nine polymorphicsites in four genes that is DDC (rs3837091 (AGAG InsrarrDel) and rs3735273 (GrarrA)) DRD2 (rs1800496 (CrarrT) rs1801028 (CrarrG) and rs1799732 (CrarrDel)) DRD4(rs4646984 (429 bp549 bp) and rs4646983 (286 bp298 bp))and COMT (rs740603 (GrarrA) and rs165599 (GrarrA)) wereselected based on their association with ADHD [12ndash14 1822 25ndash28] or behavioral disorders [29 30] in other ethnicgroups Functional role of these sites was obtained frompublished literature Sites without any published report wereanalyzed by F-SNP (httpcompbiocsqueensucaF-SNP)
Peripheral blood was collected from ADHD probandstheir parents and controls for isolation of genomic DNA [34]Details of oligonucleotide sequences and amplification pro-tocols are provided in Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552013918410
23 Statistical Analysis Data obtained was subjected toboth population-as well as family-based association analysesThe GENEPOP program (web version 34) (httpwbiomedwbiomedcurtineduaugenepop) was used to calculate alle-lic and genotypic frequencies followed by analyses for Hardy-Weinberg equilibrium (HWE) For case-control analysiswe have used the program COCAPHASE which is a partof a suite of programs UNPHASED [35] allelegenotypefrequencies of each marker obtained for the control indi-viduals were compared with these of the ADHD case groupand their parents For analysis of family-based transmis-sion Extended-transmission disequilibrium test (ETDT)[36] which is also a part of UNPHASED was used In thisprogram transmission from a single heterozygous (infor-mative) parent (duos) to an affected individual can be usedfor calculation Different groups with co-morbid charac-teristics were analyzed separately to find out associationwith the comorbidity Since numbers of cases were smallafter stratification based on co-morbid characteristics forthis analysis we have used the haplotype-based haplotyperelative risk (HHRR) program under the UNPHASEDtransmission from informative as well as noninformativeparents is taken into account for HHRR [37] Compar-isons were tested for multiple corrections (1000 itinerations)while running the UNPHASED Data showing significantassociation were further checked for power of the test byPiface version 172 [38] Odds ratio calculator was used tocalculate the odds ratio (OR) and its confidence interval(wwwhutchonnetConfidORhtm) Relative risk calculator
BioMed Research International 3
was used to calculate the relative risk (RR) and its con-fidence interval (httpwwwhutchonnetConfidRRhtm)While OR portrays the strength of association between twobinary data values compared symmetrically RR describesthe likelihood of developing disease in an exposed groupcompared to a nonexposed group
24 Epistatic Interaction Multifactor dimensionality reduc-tion (MDR) program [39] was used for analysis of the case-control data set Tuned ReliefF filter algorithm [40] was usedto screen noisy polymorphisms Since the number of affectedand unaffected individuals was not equal in the presentdataset balanced accuracy with random seed 1 was used toavoid spurious results due to chance divisions of the data[41] Then a naive Bayes classifier in the context of a 10-foldcross validation was used to estimate the testing accuracy ofeach one dimensional attribute of the 2-factor to 10-factormodels The cross-validation consistency (CVC) was alsocalculated which measures the number of times out of 10divisions of the data when the same best model was found[42]Themodelwith themaximum testing balanced accuracy(TBA) a CVC gt 5 out of 10 and a minimum predictionerror (PE)misclassification error for that comparison wasconsidered as the best model [42] Statistical significance (119875values) was calculated using a 1000-fold permutation testto compare observed testing accuracies with those expectedunder the null hypothesis of no association
For the family-based data we have analyzed only thetrio families by MDR phenomics version 10 [43] In absenceof any phenotype the MDR-pedigree disequilibrium test(MDR-PDT)was used for analysis [44] themissing genotypewas coded as ldquo3rdquo in the input file Statistical significance wascalculated after a 1000-fold permutation test 119875 values foreach statistic were obtained by fixed (FixP does not controlfor multiple tests) and nonfixed permutation tests (Non-FixPcontrolling for multiple testing)
3 Results
rs1800496 and rs1801028 were found to be nonpolymorphicafter analyzing 100 control subjects and 30 families withADHDprobands only the ldquoCrdquo variant was detected (Table 1)and we did not perform any further analysis for these sitesControl genotypes for rs165599 deviated marginally from theequilibrium (Table 2) while other sites studied obeyed theHWE in all the groups (Table 2)
Case-control analysis exhibited significantly higher fre-quency of the single repeat allele of rs4646983 inADHDcases(Table 1) Parents of ADHD probands showed higher allelicfrequencies for both rs4646983 and rs4646984 (Table 1)rs4646983 showed only a trend for higher significance(119875 = 009) which could be due to absence of homozygousgenotype of the single repeat variant in the control subjects(Table 2) Other sites failed to show any significant differencein allelic (Table 1) as well as genotypic frequencies (Table 2)
Family-based TDT analysis (Table 3) revealed significantbias in transmission of rs3837091 ldquoAGAGrdquo (119875 = 001 powersim75alpha at 5) Further analysis revealed that this biaswas
due to maternal overtransmission of the ldquoAGAGrdquo allele morespecifically to male probands (Table S2 119875 = 001 and powersim85 alpha at 5) rs740603 ldquoGrdquo (119875 = 002 power sim65alpha at 5) also showed a bias in transmission to ADHDcases (Table 3) this bias was due to paternal overtransmission(Table S2)
Haplotype analysis showed lower frequency of rs3837091-3735273 ldquoDel-Grdquo in ADHD cases (Table S3) which could beprimarily due to significant nontransmission (119875 = 0001power sim90 alpha at 5) of this haplotype from the par-ents (Table S4) The rs4646983-rs4646984 2R-2R haplotypewas present predominantly in control individuals (TableS3) Haplotype ldquoG-Ardquo of rs165599-rs740603 exhibited highertransmission (119875 = 004 power sim57 alpha at 5) to ADHDprobands (Table S4)
Major comorbidities observed in ADHD children fromeastern India are LD (44) ODD (33) CD (31) and MD(16) Substance abuse disorder tic disorder and AD werefound in only few cases and excluded from further analysisComparative analysis of ADHD probands subgrouped onthe basis of co-morbid characteristics revealed the followingobservations
31 DDC Bypopulation-based analysis we have noticed sig-nificant differences in ldquoAGAGrdquo allele frequency for rs3837091in ADHD comorbidity (Table 4) the ldquoAGAGAGAGrdquo geno-type was also overrepresented in this group (1205942 = 76 119875 =002) Further there was an overtransmission of the ldquoAGAGrdquoallele (119875 = 0006) (Table 5) which was principally paternalin nature (1205942 = 578 119875 = 002 power sim23 alpha at 5)On the other handmaternal overtransmission of the ldquoAGAGrdquoallele was significant in ADHD+CD (1205942 = 53 119875 = 002 andpower sim22 alpha at 5) In ADHD+MD ldquoAGAGAGAGrdquogenotype showed lower frequencies in parents as well asprobands as compared to the control population (1205942 = 264and 63119875 lt 00001 and 004 for probands and parents resp)
rs3735273 showed significant differences in allelic andgenotypic frequencies in ADHD+CD in comparison tocontrols ldquoArdquo allele and ldquoAArdquo genotype frequencies were higherin probands (1205942 = 65 and 123 119875 = 001 and 0002 resp) ORwas also high in this group (217)
Family-based analysis failed to show any significant biasin transmission for rs3735273 (Table 5)
32 DRD2 Population-based analysis (Table 4) revealed sig-nificant differences in allelic and genotypic frequencies forrs1799732 the ldquoCrdquo allele (1205942 = 464119875 = 003) and ldquoCCrdquo geno-type were overrepresented in ADHD+LD (1205942 = 968 119875 =0008) as well as in ADHD+MD (genotypic 1205942 = 586 119875 =005) with a noticeably high OR Family-based analysesshowed overtransmission of the ldquoCrdquo allele (Table 5) toADHD+LD (1205942 = 749 119875 = 0006 OR = 633 power =79 120572 at 5) Other comorbidities failed to show anysignificant contribution (Tables 4 and 5)
33 DRD4 In ADHD+CD the ldquosingle repeatrdquo (1R) alleleof rs4646984 showed higher frequencies (119875 = 004) as
4 BioMed Research International
Table 1 Comparative analysis of allelic frequencies in ADHD probands their parents and controls
(051ndash154)049NB significant P values are presented in bold
compared to the control population (Table 4) along withsignificant (119875 = 002) familial overtransmission (Table 5)
rs4646983 also showed significant differences in allelic(1R) and genotypic (1R1R) frequencies in ADHD+CD (1205942 =470 and 778 119875 = 003 and 002 resp) ADHD+ODD(1205942 = 475 136 119875 = 003 0001 resp) and ADHD+MD(1205942 = 429 and 209 119875 = 004 and 0001 resp) bypopulation-based analysis (Table 4) the OR was above 2 inall the co-morbid groups Family-based analysis showed lackof any transmission bias (Table 5) though OR was high inADHD+CD and ADHD+MD it could be due to a widevariation in confidence interval
34 COMT The rs165599 ldquoGrdquo allele was found to be signif-icantly overrepresented in ADHD+LD cases (Table 4) andtheir parents (1205942 = 421 119875 = 004 power = 54120572 at 5)Furthermore in ADHD+LD ldquoGGrdquo genotype showed higherfrequencies as compared to the control population (1205942 =101119875 = 0006) Lack of any associationwas noticed for otherco-morbid conditions (Table 4)
For rs740603 (Table 4) the ldquoGrdquo allele (1205942 = 389 119875 =004) and ldquoGGrdquo genotype (1205942 = 835 119875 = 0015 power =82 120572 at 5) were overrepresented in ADHD+ODD whencompared to control In ADHD+MD also ldquoGrdquo allele (1205942 =714 119875 = 0007) and ldquoGGrdquo genotype (1205942 = 178 119875 lt 00001power = 98 120572 at 5) showed significant overrepresen-tation Statistically significant overtransmission of the ldquoGrdquoallele (119875 = 003 power = 57 120572 at 5) from parents toADHD+MD was also noticed (Table 5) For this site both
population- and family-based data showed high OR inADHD+MD
35 Epistatic Interaction Gene-gene interaction analysis byMDR describes percentage of entropy (information gainmdashIG) by each factor or by 2-way interaction nodes indicateindependent main effect while connecting lines betweenthe nodes indicate interactive effect contributed by pairwisecombinations All the positive values indicate a gain in effectwhereas negative values indicate redundancy or lack of anysynergistic effect In the present study positive nodal IGvalues obtained by case-control analysis indicate significantmain effect of rs3735273 followed by rs3837091 rs1799732rs4646984 and rs740603 in ADHD (Figure 1) MDR analysisof case-control data revealed strong interaction (TBA =0755 CVC = 10 119875 lt 0000) between rs3837091 rs1799732rs4646984 and rs740603 (summarized in Table S5 only thebest models are shown)
Gene-gene interaction analysis using family-based data(Table 6) revealed significant interaction between rs3837091and rs1799732 only after correction for multiple testing (119875 =004)
In ADHD comorbidity group rs3837091 exhibited inde-pendent main effect followed by rs3735273 rs1799732rs4646983 and rs740603 (Figure S1A) Though interactionbetween rs3837091 rs17997332 rs740603 showed a trend tobe significant (119875 = 0008) the CVC value was insignificant(Table S6)
For ADHD+CD (Figure S1B) we have noticed sig-nificant main effect of rs3837091 followed by rs3735273
BioMed Research International 5
Table 2 Genotypic frequencies in controls compared with that of ADHD probands and their parents
Table 3 Analysis of allelic transmission from parents to probands (119873 = 170)
Site Allele Transmitted () Not Transmitted () 1205942 (P value) Relative Risk (95 CI)
rs3837091 Del 035 065 664 (001) 054 (040ndash073)AGAG 065 035
rs3735273 G 047 053 022 (063) 089 (067ndash117)A 053 047
rs1799732 C 053 047 021 (065) 128 (085ndash149)Del 047 053
rs4646984 1 R 052 048 010 (075) 108 (082ndash143)2 R 048 052
rs4646983 1 R 049 051 002 (089) 096 (073ndash127)2 R 051 049
rs165599 G 046 054 059 (044) 085 (064ndash113)A 054 046
rs740603 G 062 038 524 (002) 163 (122ndash219)A 038 062
NB significant P values are presented in bold
6 BioMed Research International
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
DRD2 DRD3 and DRD4) of adenylate cyclase in responseto ligand binding [8 9] The dopaminergic hypothesis ofADHD is based mostly on the malfunctioning of D2-likereceptors in the brain [10 11] The DRD4 gene encodingfor DA receptor 4 has been extensively studied and positiveassociations with ADHD were reported in Caucasian as wellas several non-Caucasian populations [5 12ndash15] The D2 andD3 receptors have been studied mostly in Caucasian andChinese populations revealing inconsistent findings [16ndash18]
Genes encoding for enzymes involved in the cate-cholaminergic system like catechol-O-methyl transferase(COMT) [19 20] dopamine decarboxylase (DDC) [21ndash23]dopamine beta hydroxylase [5 15] and monoamine oxidase[15 18 22 23] have also gained a lot of importance in explor-ing the etiological basis of ADHD Some of these geneticvariants have revealed significant association with ADHDassociated co-morbid disorders [24] and were speculated asthe reason for comorbidities being so common in Caucasiansubjects with ADHD [25]
In the Indo-Caucasoid ADHD probands DRD4 DAT1MAOA COMT and DBH gene variants showed significantassociation with the disorder [5] However till date neitherany report on DDC and DRD2 nor any information on thecontribution of gene variants inADHDassociated co-morbiddisorders was available in this particular ethnic group SinceADHD subjects frequently exhibit co-morbid behavioraldisorders including CD ODD and substance abuse [2 3]in the present study we have selected nine polymorphic sitesin four catecholaminergic genes DRD2 DRD4 DDC andCOMT which modulate function of DA The sites analyzedhave been explored in association with ADHD in EuropeanCaucasian and Han Chinese populations [12ndash14 18 22 25ndash28] or behavioral disorders [29 30] and analyzed for thefirst time for their contribution in the etiology of Indo-CaucasoidADHDprobands stratified on the basis of differentco-morbid disorders
2 Subjects and Methods
21 Study Subjects We recruited ADHD probands (119873 =170) from the out-patient department of Manovikas KendraRehabilitation and Research Institute for the HandicappedKolkata based on (a) DSM-IV-TR criteria [1] (b) hyper-activity level measured by Connersrsquo Parents and TeachersRating Scale [31] (c) intelligencedevelopmental quotientassessed byWechslerrsquos Intelligence Scale for Children [32] forchildren above five years and Developmental Screening Testfor children below 5 years [33] Mean age of probands was77 years plusmn 23 SD and male to female ratio was 10 1 Out of170 probands 143 were complete parent-proband trios 17 hadonly one parent and 10 were affected probands only Hyper-activeimpulsive (112) and inattentive (73) subtypes wereonly fewwhilemajority of the probands belonged to the com-bined subtype (815) Based on the presenting co-morbidsymptoms ADHD probands were subgrouped as ADHD-comorbidity ADHD+CD ADHD+LD ADHD+ODDand ADHD+MD Subjects suffering from only psychiatricproblems pervasive developmental disorders and any form
ofmental retardation (IQ le 80) including fragile X syndromewere excluded
A control group (119873 = 180 mean age 197 years plusmn 794 SDandmale to female ratio 10 3) evaluated following the DSM-IV-TR criteria [1] for ADHD was also recruited All theindividuals enlisted for the study belonged to the Indo-Caucasoid ethnic category For participation in the studyinformed written consent was obtained from the controlsand guardians of ADHD probands The study protocol wasapproved by the Institutional Human Ethical Committee
22 Selection of SNPs and Genotyping Nine polymorphicsites in four genes that is DDC (rs3837091 (AGAG InsrarrDel) and rs3735273 (GrarrA)) DRD2 (rs1800496 (CrarrT) rs1801028 (CrarrG) and rs1799732 (CrarrDel)) DRD4(rs4646984 (429 bp549 bp) and rs4646983 (286 bp298 bp))and COMT (rs740603 (GrarrA) and rs165599 (GrarrA)) wereselected based on their association with ADHD [12ndash14 1822 25ndash28] or behavioral disorders [29 30] in other ethnicgroups Functional role of these sites was obtained frompublished literature Sites without any published report wereanalyzed by F-SNP (httpcompbiocsqueensucaF-SNP)
Peripheral blood was collected from ADHD probandstheir parents and controls for isolation of genomic DNA [34]Details of oligonucleotide sequences and amplification pro-tocols are provided in Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552013918410
23 Statistical Analysis Data obtained was subjected toboth population-as well as family-based association analysesThe GENEPOP program (web version 34) (httpwbiomedwbiomedcurtineduaugenepop) was used to calculate alle-lic and genotypic frequencies followed by analyses for Hardy-Weinberg equilibrium (HWE) For case-control analysiswe have used the program COCAPHASE which is a partof a suite of programs UNPHASED [35] allelegenotypefrequencies of each marker obtained for the control indi-viduals were compared with these of the ADHD case groupand their parents For analysis of family-based transmis-sion Extended-transmission disequilibrium test (ETDT)[36] which is also a part of UNPHASED was used In thisprogram transmission from a single heterozygous (infor-mative) parent (duos) to an affected individual can be usedfor calculation Different groups with co-morbid charac-teristics were analyzed separately to find out associationwith the comorbidity Since numbers of cases were smallafter stratification based on co-morbid characteristics forthis analysis we have used the haplotype-based haplotyperelative risk (HHRR) program under the UNPHASEDtransmission from informative as well as noninformativeparents is taken into account for HHRR [37] Compar-isons were tested for multiple corrections (1000 itinerations)while running the UNPHASED Data showing significantassociation were further checked for power of the test byPiface version 172 [38] Odds ratio calculator was used tocalculate the odds ratio (OR) and its confidence interval(wwwhutchonnetConfidORhtm) Relative risk calculator
BioMed Research International 3
was used to calculate the relative risk (RR) and its con-fidence interval (httpwwwhutchonnetConfidRRhtm)While OR portrays the strength of association between twobinary data values compared symmetrically RR describesthe likelihood of developing disease in an exposed groupcompared to a nonexposed group
24 Epistatic Interaction Multifactor dimensionality reduc-tion (MDR) program [39] was used for analysis of the case-control data set Tuned ReliefF filter algorithm [40] was usedto screen noisy polymorphisms Since the number of affectedand unaffected individuals was not equal in the presentdataset balanced accuracy with random seed 1 was used toavoid spurious results due to chance divisions of the data[41] Then a naive Bayes classifier in the context of a 10-foldcross validation was used to estimate the testing accuracy ofeach one dimensional attribute of the 2-factor to 10-factormodels The cross-validation consistency (CVC) was alsocalculated which measures the number of times out of 10divisions of the data when the same best model was found[42]Themodelwith themaximum testing balanced accuracy(TBA) a CVC gt 5 out of 10 and a minimum predictionerror (PE)misclassification error for that comparison wasconsidered as the best model [42] Statistical significance (119875values) was calculated using a 1000-fold permutation testto compare observed testing accuracies with those expectedunder the null hypothesis of no association
For the family-based data we have analyzed only thetrio families by MDR phenomics version 10 [43] In absenceof any phenotype the MDR-pedigree disequilibrium test(MDR-PDT)was used for analysis [44] themissing genotypewas coded as ldquo3rdquo in the input file Statistical significance wascalculated after a 1000-fold permutation test 119875 values foreach statistic were obtained by fixed (FixP does not controlfor multiple tests) and nonfixed permutation tests (Non-FixPcontrolling for multiple testing)
3 Results
rs1800496 and rs1801028 were found to be nonpolymorphicafter analyzing 100 control subjects and 30 families withADHDprobands only the ldquoCrdquo variant was detected (Table 1)and we did not perform any further analysis for these sitesControl genotypes for rs165599 deviated marginally from theequilibrium (Table 2) while other sites studied obeyed theHWE in all the groups (Table 2)
Case-control analysis exhibited significantly higher fre-quency of the single repeat allele of rs4646983 inADHDcases(Table 1) Parents of ADHD probands showed higher allelicfrequencies for both rs4646983 and rs4646984 (Table 1)rs4646983 showed only a trend for higher significance(119875 = 009) which could be due to absence of homozygousgenotype of the single repeat variant in the control subjects(Table 2) Other sites failed to show any significant differencein allelic (Table 1) as well as genotypic frequencies (Table 2)
Family-based TDT analysis (Table 3) revealed significantbias in transmission of rs3837091 ldquoAGAGrdquo (119875 = 001 powersim75alpha at 5) Further analysis revealed that this biaswas
due to maternal overtransmission of the ldquoAGAGrdquo allele morespecifically to male probands (Table S2 119875 = 001 and powersim85 alpha at 5) rs740603 ldquoGrdquo (119875 = 002 power sim65alpha at 5) also showed a bias in transmission to ADHDcases (Table 3) this bias was due to paternal overtransmission(Table S2)
Haplotype analysis showed lower frequency of rs3837091-3735273 ldquoDel-Grdquo in ADHD cases (Table S3) which could beprimarily due to significant nontransmission (119875 = 0001power sim90 alpha at 5) of this haplotype from the par-ents (Table S4) The rs4646983-rs4646984 2R-2R haplotypewas present predominantly in control individuals (TableS3) Haplotype ldquoG-Ardquo of rs165599-rs740603 exhibited highertransmission (119875 = 004 power sim57 alpha at 5) to ADHDprobands (Table S4)
Major comorbidities observed in ADHD children fromeastern India are LD (44) ODD (33) CD (31) and MD(16) Substance abuse disorder tic disorder and AD werefound in only few cases and excluded from further analysisComparative analysis of ADHD probands subgrouped onthe basis of co-morbid characteristics revealed the followingobservations
31 DDC Bypopulation-based analysis we have noticed sig-nificant differences in ldquoAGAGrdquo allele frequency for rs3837091in ADHD comorbidity (Table 4) the ldquoAGAGAGAGrdquo geno-type was also overrepresented in this group (1205942 = 76 119875 =002) Further there was an overtransmission of the ldquoAGAGrdquoallele (119875 = 0006) (Table 5) which was principally paternalin nature (1205942 = 578 119875 = 002 power sim23 alpha at 5)On the other handmaternal overtransmission of the ldquoAGAGrdquoallele was significant in ADHD+CD (1205942 = 53 119875 = 002 andpower sim22 alpha at 5) In ADHD+MD ldquoAGAGAGAGrdquogenotype showed lower frequencies in parents as well asprobands as compared to the control population (1205942 = 264and 63119875 lt 00001 and 004 for probands and parents resp)
rs3735273 showed significant differences in allelic andgenotypic frequencies in ADHD+CD in comparison tocontrols ldquoArdquo allele and ldquoAArdquo genotype frequencies were higherin probands (1205942 = 65 and 123 119875 = 001 and 0002 resp) ORwas also high in this group (217)
Family-based analysis failed to show any significant biasin transmission for rs3735273 (Table 5)
32 DRD2 Population-based analysis (Table 4) revealed sig-nificant differences in allelic and genotypic frequencies forrs1799732 the ldquoCrdquo allele (1205942 = 464119875 = 003) and ldquoCCrdquo geno-type were overrepresented in ADHD+LD (1205942 = 968 119875 =0008) as well as in ADHD+MD (genotypic 1205942 = 586 119875 =005) with a noticeably high OR Family-based analysesshowed overtransmission of the ldquoCrdquo allele (Table 5) toADHD+LD (1205942 = 749 119875 = 0006 OR = 633 power =79 120572 at 5) Other comorbidities failed to show anysignificant contribution (Tables 4 and 5)
33 DRD4 In ADHD+CD the ldquosingle repeatrdquo (1R) alleleof rs4646984 showed higher frequencies (119875 = 004) as
4 BioMed Research International
Table 1 Comparative analysis of allelic frequencies in ADHD probands their parents and controls
(051ndash154)049NB significant P values are presented in bold
compared to the control population (Table 4) along withsignificant (119875 = 002) familial overtransmission (Table 5)
rs4646983 also showed significant differences in allelic(1R) and genotypic (1R1R) frequencies in ADHD+CD (1205942 =470 and 778 119875 = 003 and 002 resp) ADHD+ODD(1205942 = 475 136 119875 = 003 0001 resp) and ADHD+MD(1205942 = 429 and 209 119875 = 004 and 0001 resp) bypopulation-based analysis (Table 4) the OR was above 2 inall the co-morbid groups Family-based analysis showed lackof any transmission bias (Table 5) though OR was high inADHD+CD and ADHD+MD it could be due to a widevariation in confidence interval
34 COMT The rs165599 ldquoGrdquo allele was found to be signif-icantly overrepresented in ADHD+LD cases (Table 4) andtheir parents (1205942 = 421 119875 = 004 power = 54120572 at 5)Furthermore in ADHD+LD ldquoGGrdquo genotype showed higherfrequencies as compared to the control population (1205942 =101119875 = 0006) Lack of any associationwas noticed for otherco-morbid conditions (Table 4)
For rs740603 (Table 4) the ldquoGrdquo allele (1205942 = 389 119875 =004) and ldquoGGrdquo genotype (1205942 = 835 119875 = 0015 power =82 120572 at 5) were overrepresented in ADHD+ODD whencompared to control In ADHD+MD also ldquoGrdquo allele (1205942 =714 119875 = 0007) and ldquoGGrdquo genotype (1205942 = 178 119875 lt 00001power = 98 120572 at 5) showed significant overrepresen-tation Statistically significant overtransmission of the ldquoGrdquoallele (119875 = 003 power = 57 120572 at 5) from parents toADHD+MD was also noticed (Table 5) For this site both
population- and family-based data showed high OR inADHD+MD
35 Epistatic Interaction Gene-gene interaction analysis byMDR describes percentage of entropy (information gainmdashIG) by each factor or by 2-way interaction nodes indicateindependent main effect while connecting lines betweenthe nodes indicate interactive effect contributed by pairwisecombinations All the positive values indicate a gain in effectwhereas negative values indicate redundancy or lack of anysynergistic effect In the present study positive nodal IGvalues obtained by case-control analysis indicate significantmain effect of rs3735273 followed by rs3837091 rs1799732rs4646984 and rs740603 in ADHD (Figure 1) MDR analysisof case-control data revealed strong interaction (TBA =0755 CVC = 10 119875 lt 0000) between rs3837091 rs1799732rs4646984 and rs740603 (summarized in Table S5 only thebest models are shown)
Gene-gene interaction analysis using family-based data(Table 6) revealed significant interaction between rs3837091and rs1799732 only after correction for multiple testing (119875 =004)
In ADHD comorbidity group rs3837091 exhibited inde-pendent main effect followed by rs3735273 rs1799732rs4646983 and rs740603 (Figure S1A) Though interactionbetween rs3837091 rs17997332 rs740603 showed a trend tobe significant (119875 = 0008) the CVC value was insignificant(Table S6)
For ADHD+CD (Figure S1B) we have noticed sig-nificant main effect of rs3837091 followed by rs3735273
BioMed Research International 5
Table 2 Genotypic frequencies in controls compared with that of ADHD probands and their parents
Table 3 Analysis of allelic transmission from parents to probands (119873 = 170)
Site Allele Transmitted () Not Transmitted () 1205942 (P value) Relative Risk (95 CI)
rs3837091 Del 035 065 664 (001) 054 (040ndash073)AGAG 065 035
rs3735273 G 047 053 022 (063) 089 (067ndash117)A 053 047
rs1799732 C 053 047 021 (065) 128 (085ndash149)Del 047 053
rs4646984 1 R 052 048 010 (075) 108 (082ndash143)2 R 048 052
rs4646983 1 R 049 051 002 (089) 096 (073ndash127)2 R 051 049
rs165599 G 046 054 059 (044) 085 (064ndash113)A 054 046
rs740603 G 062 038 524 (002) 163 (122ndash219)A 038 062
NB significant P values are presented in bold
6 BioMed Research International
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
was used to calculate the relative risk (RR) and its con-fidence interval (httpwwwhutchonnetConfidRRhtm)While OR portrays the strength of association between twobinary data values compared symmetrically RR describesthe likelihood of developing disease in an exposed groupcompared to a nonexposed group
24 Epistatic Interaction Multifactor dimensionality reduc-tion (MDR) program [39] was used for analysis of the case-control data set Tuned ReliefF filter algorithm [40] was usedto screen noisy polymorphisms Since the number of affectedand unaffected individuals was not equal in the presentdataset balanced accuracy with random seed 1 was used toavoid spurious results due to chance divisions of the data[41] Then a naive Bayes classifier in the context of a 10-foldcross validation was used to estimate the testing accuracy ofeach one dimensional attribute of the 2-factor to 10-factormodels The cross-validation consistency (CVC) was alsocalculated which measures the number of times out of 10divisions of the data when the same best model was found[42]Themodelwith themaximum testing balanced accuracy(TBA) a CVC gt 5 out of 10 and a minimum predictionerror (PE)misclassification error for that comparison wasconsidered as the best model [42] Statistical significance (119875values) was calculated using a 1000-fold permutation testto compare observed testing accuracies with those expectedunder the null hypothesis of no association
For the family-based data we have analyzed only thetrio families by MDR phenomics version 10 [43] In absenceof any phenotype the MDR-pedigree disequilibrium test(MDR-PDT)was used for analysis [44] themissing genotypewas coded as ldquo3rdquo in the input file Statistical significance wascalculated after a 1000-fold permutation test 119875 values foreach statistic were obtained by fixed (FixP does not controlfor multiple tests) and nonfixed permutation tests (Non-FixPcontrolling for multiple testing)
3 Results
rs1800496 and rs1801028 were found to be nonpolymorphicafter analyzing 100 control subjects and 30 families withADHDprobands only the ldquoCrdquo variant was detected (Table 1)and we did not perform any further analysis for these sitesControl genotypes for rs165599 deviated marginally from theequilibrium (Table 2) while other sites studied obeyed theHWE in all the groups (Table 2)
Case-control analysis exhibited significantly higher fre-quency of the single repeat allele of rs4646983 inADHDcases(Table 1) Parents of ADHD probands showed higher allelicfrequencies for both rs4646983 and rs4646984 (Table 1)rs4646983 showed only a trend for higher significance(119875 = 009) which could be due to absence of homozygousgenotype of the single repeat variant in the control subjects(Table 2) Other sites failed to show any significant differencein allelic (Table 1) as well as genotypic frequencies (Table 2)
Family-based TDT analysis (Table 3) revealed significantbias in transmission of rs3837091 ldquoAGAGrdquo (119875 = 001 powersim75alpha at 5) Further analysis revealed that this biaswas
due to maternal overtransmission of the ldquoAGAGrdquo allele morespecifically to male probands (Table S2 119875 = 001 and powersim85 alpha at 5) rs740603 ldquoGrdquo (119875 = 002 power sim65alpha at 5) also showed a bias in transmission to ADHDcases (Table 3) this bias was due to paternal overtransmission(Table S2)
Haplotype analysis showed lower frequency of rs3837091-3735273 ldquoDel-Grdquo in ADHD cases (Table S3) which could beprimarily due to significant nontransmission (119875 = 0001power sim90 alpha at 5) of this haplotype from the par-ents (Table S4) The rs4646983-rs4646984 2R-2R haplotypewas present predominantly in control individuals (TableS3) Haplotype ldquoG-Ardquo of rs165599-rs740603 exhibited highertransmission (119875 = 004 power sim57 alpha at 5) to ADHDprobands (Table S4)
Major comorbidities observed in ADHD children fromeastern India are LD (44) ODD (33) CD (31) and MD(16) Substance abuse disorder tic disorder and AD werefound in only few cases and excluded from further analysisComparative analysis of ADHD probands subgrouped onthe basis of co-morbid characteristics revealed the followingobservations
31 DDC Bypopulation-based analysis we have noticed sig-nificant differences in ldquoAGAGrdquo allele frequency for rs3837091in ADHD comorbidity (Table 4) the ldquoAGAGAGAGrdquo geno-type was also overrepresented in this group (1205942 = 76 119875 =002) Further there was an overtransmission of the ldquoAGAGrdquoallele (119875 = 0006) (Table 5) which was principally paternalin nature (1205942 = 578 119875 = 002 power sim23 alpha at 5)On the other handmaternal overtransmission of the ldquoAGAGrdquoallele was significant in ADHD+CD (1205942 = 53 119875 = 002 andpower sim22 alpha at 5) In ADHD+MD ldquoAGAGAGAGrdquogenotype showed lower frequencies in parents as well asprobands as compared to the control population (1205942 = 264and 63119875 lt 00001 and 004 for probands and parents resp)
rs3735273 showed significant differences in allelic andgenotypic frequencies in ADHD+CD in comparison tocontrols ldquoArdquo allele and ldquoAArdquo genotype frequencies were higherin probands (1205942 = 65 and 123 119875 = 001 and 0002 resp) ORwas also high in this group (217)
Family-based analysis failed to show any significant biasin transmission for rs3735273 (Table 5)
32 DRD2 Population-based analysis (Table 4) revealed sig-nificant differences in allelic and genotypic frequencies forrs1799732 the ldquoCrdquo allele (1205942 = 464119875 = 003) and ldquoCCrdquo geno-type were overrepresented in ADHD+LD (1205942 = 968 119875 =0008) as well as in ADHD+MD (genotypic 1205942 = 586 119875 =005) with a noticeably high OR Family-based analysesshowed overtransmission of the ldquoCrdquo allele (Table 5) toADHD+LD (1205942 = 749 119875 = 0006 OR = 633 power =79 120572 at 5) Other comorbidities failed to show anysignificant contribution (Tables 4 and 5)
33 DRD4 In ADHD+CD the ldquosingle repeatrdquo (1R) alleleof rs4646984 showed higher frequencies (119875 = 004) as
4 BioMed Research International
Table 1 Comparative analysis of allelic frequencies in ADHD probands their parents and controls
(051ndash154)049NB significant P values are presented in bold
compared to the control population (Table 4) along withsignificant (119875 = 002) familial overtransmission (Table 5)
rs4646983 also showed significant differences in allelic(1R) and genotypic (1R1R) frequencies in ADHD+CD (1205942 =470 and 778 119875 = 003 and 002 resp) ADHD+ODD(1205942 = 475 136 119875 = 003 0001 resp) and ADHD+MD(1205942 = 429 and 209 119875 = 004 and 0001 resp) bypopulation-based analysis (Table 4) the OR was above 2 inall the co-morbid groups Family-based analysis showed lackof any transmission bias (Table 5) though OR was high inADHD+CD and ADHD+MD it could be due to a widevariation in confidence interval
34 COMT The rs165599 ldquoGrdquo allele was found to be signif-icantly overrepresented in ADHD+LD cases (Table 4) andtheir parents (1205942 = 421 119875 = 004 power = 54120572 at 5)Furthermore in ADHD+LD ldquoGGrdquo genotype showed higherfrequencies as compared to the control population (1205942 =101119875 = 0006) Lack of any associationwas noticed for otherco-morbid conditions (Table 4)
For rs740603 (Table 4) the ldquoGrdquo allele (1205942 = 389 119875 =004) and ldquoGGrdquo genotype (1205942 = 835 119875 = 0015 power =82 120572 at 5) were overrepresented in ADHD+ODD whencompared to control In ADHD+MD also ldquoGrdquo allele (1205942 =714 119875 = 0007) and ldquoGGrdquo genotype (1205942 = 178 119875 lt 00001power = 98 120572 at 5) showed significant overrepresen-tation Statistically significant overtransmission of the ldquoGrdquoallele (119875 = 003 power = 57 120572 at 5) from parents toADHD+MD was also noticed (Table 5) For this site both
population- and family-based data showed high OR inADHD+MD
35 Epistatic Interaction Gene-gene interaction analysis byMDR describes percentage of entropy (information gainmdashIG) by each factor or by 2-way interaction nodes indicateindependent main effect while connecting lines betweenthe nodes indicate interactive effect contributed by pairwisecombinations All the positive values indicate a gain in effectwhereas negative values indicate redundancy or lack of anysynergistic effect In the present study positive nodal IGvalues obtained by case-control analysis indicate significantmain effect of rs3735273 followed by rs3837091 rs1799732rs4646984 and rs740603 in ADHD (Figure 1) MDR analysisof case-control data revealed strong interaction (TBA =0755 CVC = 10 119875 lt 0000) between rs3837091 rs1799732rs4646984 and rs740603 (summarized in Table S5 only thebest models are shown)
Gene-gene interaction analysis using family-based data(Table 6) revealed significant interaction between rs3837091and rs1799732 only after correction for multiple testing (119875 =004)
In ADHD comorbidity group rs3837091 exhibited inde-pendent main effect followed by rs3735273 rs1799732rs4646983 and rs740603 (Figure S1A) Though interactionbetween rs3837091 rs17997332 rs740603 showed a trend tobe significant (119875 = 0008) the CVC value was insignificant(Table S6)
For ADHD+CD (Figure S1B) we have noticed sig-nificant main effect of rs3837091 followed by rs3735273
BioMed Research International 5
Table 2 Genotypic frequencies in controls compared with that of ADHD probands and their parents
Table 3 Analysis of allelic transmission from parents to probands (119873 = 170)
Site Allele Transmitted () Not Transmitted () 1205942 (P value) Relative Risk (95 CI)
rs3837091 Del 035 065 664 (001) 054 (040ndash073)AGAG 065 035
rs3735273 G 047 053 022 (063) 089 (067ndash117)A 053 047
rs1799732 C 053 047 021 (065) 128 (085ndash149)Del 047 053
rs4646984 1 R 052 048 010 (075) 108 (082ndash143)2 R 048 052
rs4646983 1 R 049 051 002 (089) 096 (073ndash127)2 R 051 049
rs165599 G 046 054 059 (044) 085 (064ndash113)A 054 046
rs740603 G 062 038 524 (002) 163 (122ndash219)A 038 062
NB significant P values are presented in bold
6 BioMed Research International
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
(051ndash154)049NB significant P values are presented in bold
compared to the control population (Table 4) along withsignificant (119875 = 002) familial overtransmission (Table 5)
rs4646983 also showed significant differences in allelic(1R) and genotypic (1R1R) frequencies in ADHD+CD (1205942 =470 and 778 119875 = 003 and 002 resp) ADHD+ODD(1205942 = 475 136 119875 = 003 0001 resp) and ADHD+MD(1205942 = 429 and 209 119875 = 004 and 0001 resp) bypopulation-based analysis (Table 4) the OR was above 2 inall the co-morbid groups Family-based analysis showed lackof any transmission bias (Table 5) though OR was high inADHD+CD and ADHD+MD it could be due to a widevariation in confidence interval
34 COMT The rs165599 ldquoGrdquo allele was found to be signif-icantly overrepresented in ADHD+LD cases (Table 4) andtheir parents (1205942 = 421 119875 = 004 power = 54120572 at 5)Furthermore in ADHD+LD ldquoGGrdquo genotype showed higherfrequencies as compared to the control population (1205942 =101119875 = 0006) Lack of any associationwas noticed for otherco-morbid conditions (Table 4)
For rs740603 (Table 4) the ldquoGrdquo allele (1205942 = 389 119875 =004) and ldquoGGrdquo genotype (1205942 = 835 119875 = 0015 power =82 120572 at 5) were overrepresented in ADHD+ODD whencompared to control In ADHD+MD also ldquoGrdquo allele (1205942 =714 119875 = 0007) and ldquoGGrdquo genotype (1205942 = 178 119875 lt 00001power = 98 120572 at 5) showed significant overrepresen-tation Statistically significant overtransmission of the ldquoGrdquoallele (119875 = 003 power = 57 120572 at 5) from parents toADHD+MD was also noticed (Table 5) For this site both
population- and family-based data showed high OR inADHD+MD
35 Epistatic Interaction Gene-gene interaction analysis byMDR describes percentage of entropy (information gainmdashIG) by each factor or by 2-way interaction nodes indicateindependent main effect while connecting lines betweenthe nodes indicate interactive effect contributed by pairwisecombinations All the positive values indicate a gain in effectwhereas negative values indicate redundancy or lack of anysynergistic effect In the present study positive nodal IGvalues obtained by case-control analysis indicate significantmain effect of rs3735273 followed by rs3837091 rs1799732rs4646984 and rs740603 in ADHD (Figure 1) MDR analysisof case-control data revealed strong interaction (TBA =0755 CVC = 10 119875 lt 0000) between rs3837091 rs1799732rs4646984 and rs740603 (summarized in Table S5 only thebest models are shown)
Gene-gene interaction analysis using family-based data(Table 6) revealed significant interaction between rs3837091and rs1799732 only after correction for multiple testing (119875 =004)
In ADHD comorbidity group rs3837091 exhibited inde-pendent main effect followed by rs3735273 rs1799732rs4646983 and rs740603 (Figure S1A) Though interactionbetween rs3837091 rs17997332 rs740603 showed a trend tobe significant (119875 = 0008) the CVC value was insignificant(Table S6)
For ADHD+CD (Figure S1B) we have noticed sig-nificant main effect of rs3837091 followed by rs3735273
BioMed Research International 5
Table 2 Genotypic frequencies in controls compared with that of ADHD probands and their parents
Table 3 Analysis of allelic transmission from parents to probands (119873 = 170)
Site Allele Transmitted () Not Transmitted () 1205942 (P value) Relative Risk (95 CI)
rs3837091 Del 035 065 664 (001) 054 (040ndash073)AGAG 065 035
rs3735273 G 047 053 022 (063) 089 (067ndash117)A 053 047
rs1799732 C 053 047 021 (065) 128 (085ndash149)Del 047 053
rs4646984 1 R 052 048 010 (075) 108 (082ndash143)2 R 048 052
rs4646983 1 R 049 051 002 (089) 096 (073ndash127)2 R 051 049
rs165599 G 046 054 059 (044) 085 (064ndash113)A 054 046
rs740603 G 062 038 524 (002) 163 (122ndash219)A 038 062
NB significant P values are presented in bold
6 BioMed Research International
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
Table 3 Analysis of allelic transmission from parents to probands (119873 = 170)
Site Allele Transmitted () Not Transmitted () 1205942 (P value) Relative Risk (95 CI)
rs3837091 Del 035 065 664 (001) 054 (040ndash073)AGAG 065 035
rs3735273 G 047 053 022 (063) 089 (067ndash117)A 053 047
rs1799732 C 053 047 021 (065) 128 (085ndash149)Del 047 053
rs4646984 1 R 052 048 010 (075) 108 (082ndash143)2 R 048 052
rs4646983 1 R 049 051 002 (089) 096 (073ndash127)2 R 051 049
rs165599 G 046 054 059 (044) 085 (064ndash113)A 054 046
rs740603 G 062 038 524 (002) 163 (122ndash219)A 038 062
NB significant P values are presented in bold
6 BioMed Research International
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
Table 4 Case-control analysis of allelic frequencies in ADHD probands with various co-morbidities
Site
ADHD comorbidity(119873 = 42)
ADHD + CD(119873 = 33)
ADHD + LD(119873 = 42)
ADHD + ODD(119873 = 24)
ADHD +MD(119873 = 20)
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 467(003)
197(106ndash365)
000(077)
109(061ndash194)
020(066)
114(064ndash204)
000(088)
096(054ndash170)
344(006)
059(033ndash103)
rs3735273 000(100)
1(053ndash190)
649(001)
217(119ndash397)
041(052)
123(066ndash229)
000(087)
095(050ndash181)
302(008)
171(093ndash316)
rs1799732 002(089)
11(047ndash254)
059(044)
151(061ndash371)
464(003)
284(097ndash829)
021(064)
085(038ndash189)
196(016)
284(097ndash829)
rs4646984 012(073)
111(059ndash209)
439(004)
191(105ndash351)
167(020)
147(079ndash273)
118(028)
141(076ndash262)
0003(095)
1(053ndash190)
rs4646983 041(052)
142(055ndash369)
470(003)
252(104ndash611)
316(008)
203(082ndash503)
475(003)
252(104ndash611)
429(004)
288(120ndash688)
rs165599 203(015)
146(083ndash260)
055(046)
124(070ndash220)
421(004)
172(097ndash304)
008(078)
091(050ndash164)
001(091)
095(053ndash172)
rs740603 041(052)
117(067ndash204)
009(076)
112(065ndash196)
003(088)
108(062ndash188)
389(004)
176(10ndash309)
714(0007)
251(141ndash447)
NB significant P values are presented in bold
Table 5 Analysis of allelic transmission in ADHD probands with different co-morbidities
Site ADHD comorbidity ADHD + CD ADHD + LD ADHD + ODD ADHD +MD
1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI) 1205942 (P) OR
(95 CI)
rs3837091 760(0006)
035(025ndash050)
543(001)
061(045ndash082)
067(041)
072(054ndash096)
053(047)
069(052ndash093)
000(100)
100(076ndash132)
rs3735273 004(084)
092(006ndash1483)
017(068)
092(070ndash122)
014(070)
085(064ndash112)
136(024)
203(149ndash277)
009(076)
122(092ndash162)
rs1799732 037(055)
069(020ndash229)
044(051)
156(042ndash587)
749(0006)
633(135ndash2968)
070(040)
063(021ndash190)
022(064)
155(024ndash985)
rs4646984 017(068)
084(038ndash189)
514(002)
258(112ndash593)
141(024)
16(073ndash35)
013(072)
088(043ndash178)
160(020)
052(019ndash144)
rs4646983 170(019)
050(017ndash144)
181(017)
253(062ndash1063)
032(057)
138(045ndash421)
090(034)
185(051ndash667)
111(029)
322(032ndash3289)
rs165599 164(020)
160(078ndash329)
164(020)
058(025ndash134)
003(086)
107(052ndash219)
127(026)
060(025ndash146)
159(021)
053(019ndash144)
rs740603 100(032)
150(068ndash327)
035(055)
079(036ndash172)
148(022)
066(033ndash130)
048(049)
137(056ndash339)
446(003)
273(106ndash703)
NB significant P values are presented in bold
Table 6 Gene-gene interaction analyzed by MDRPDT usingfamily-based data of all ADHD cases
Two-locus model MDR-PDT FixP NonFixP[1 3] 4627 0002 004[1 5] 4326 0003 008[3 5] 4454 0002 00721mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 5mdashrs4646983 6mdashrs165599 and 7mdashrs740603 No of attributes = 7 MDR-PDT MDR-pedigreedisequilibrium test FixP does not control for multiple tests Non FixPcontrolling for multiple testing
rs4646983 rs1799732 and rs4646984 No interaction wasnoticed between the sites for this group (Table S6)
In ADHD+LD rs3837091 showed significant maineffects followed by rs3735273 rs1799732 rs4646983 andrs4646984 (Figure S1C) In this group also no significantinteraction between the sites was noticed (Table S6)
ADHD+MD cases (Figure S1D) exhibited significantmain effect for rs3837091 followed by rs4646983 rs740603rs4646984 and rs1799732 Two locus interaction analysesrevealed lack of significant interaction (Table S6)
In the ADHD+ODD (Figure S1E) independent maineffects were observed for rs3837091 followed by rs740603rs165599 rs3735273 and rs1799732 Positive values for thecorresponding connecting lines among DDC (rs3837091 andrs3735273) DRD2 (rs1799732) and COMT (rs165599 andrs740603) indicated interaction between the sites for thisgroup (Figure S1E) Strong interaction betweenDDCDRD2
BioMed Research International 7
7568
minus363
minus823004
minus1621
minus811
12243minus672
1996
minus2289
41203
minus485
32155
minus628
22435
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
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[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
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[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
Figure 1 Two-way gene-gene interaction analyzed for different sitesusing case-control dataset All the positive IG values in the nodesindicate independentmain effect of all themarkers All the lineswithnegative IG values indicate redundancy or lack of any synergisticinteraction between the markers 1mdashrs3837091 2mdashrs3735273 3mdashrs1799732 4mdashrs4646984 and 7mdashrs740603
and COMT was also documented from significant 119875 valuesand CVC = 10 (Table S6)
Analysis of family-based data by MDR-PDT failed toshow any statistically significant result in any of these groupsafter corrections formultiple testing (Non-Fix119875 gt 005 TableS7)
4 Discussion
In the present investigation on Indo-Caucasoid populationassociation of nine gene variants with ADHD and its associ-ated co-morbid features were explored rs3837091 rs1801028rs4646984 rs4646983 rs740603 and rs165599 have beeninvestigated previously in different ethnic groups for associ-ation with ADHD [12ndash14 18 22 25ndash28] Association studieshave also shown contribution of rs3735273 and rs1799732 innicotine and alcohol dependence respectively [29 30] SinceADHD related behavioral attributes and conduct problemswere reported to share a common genetic etiology andnicotine as well as alcohol addiction is often detected inadults with ADHD [2 19 20 45] we have analyzed thesesites for the first time in association with ADHD in theIndo-Caucasoid probands independent allelic associationsor transmission of different variants were noticed in subjectswithADHDADHD+CDADHD+LDADHD+ODD andADHD+MD
41 DDC Enzyme encoded by the DDC gene catalyzesbiosynthesis of three crucial neurotransmitters (1) decar-boxylation of L-34 dihydroxyphenylalanine (L-DOPA) to
dopamine (2) 5-hydroxytryptophan (5HTP) to serotoninand (3) L-tryptophan to tryptamine Both DA and serotoninneurotransmitter systems have been reported to be alteredin ADHD [24] making DDC a good candidate gene for thedisorder Functional brain imaging studies showed increasedDDC activity in the midbrains of ADHD children anddecreased activity in the prefrontal regions in ADHD adults[46] Genome-wide association scan confirmed associationof DDC with ADHD in a number of Caucasian populations[21] In the Chinese Han population rs3837091 AGAG inser-tiondeletion in the exon 1 of DDC showed association withADHD inattentive subtype [18] In Spanish ADHD casesDDC variants showed association with both childhood andadultADHD[22] while in IrishADHDsubjects amarginallysignificant overtransmission was reported [25] rs3735273was investigated earlier in association with nicotine depen-dence [29] In the present investigation while rs3735273failed to show significant differences rs3837091 ldquoAGAGrdquoallele showed higher transmission in ADHD probands withconcomitant lower occurrence and transmission of haplotypecontaining the Del allele Further analysis showed that thiswas due to highermaternal transmission of the ldquoAGAGrdquo allelespecifically to male probands Cases stratified on the basisof comorbidity revealed significant association of rs3837091ldquoAGAGrdquo and rs3735273 ldquoArdquo with ADHD-comorbidity andADHD+CD respectively Bias in parental transmission ofthe ldquoAGAGrdquo variant was also observed paternal in ADHD-comorbidity and maternal in ADHD+CD In ADHD+MDthe ldquoAGAGAGAGrdquo genotype showed lower frequencies infamilies with ADHD probands In silico analysis of rs3837091and rs3735273 by F-SNP failed to show any alteration infunction of the DDC gene Based on the biased maternaltransmission wemay infer that rs3837091may have some rolein the etiology of ADHD especially in male probands andcould be the reason for higher occurrence of ADHD+CDIt can be speculated that rs3837091 is in association withanother functional site in DDC and further investigation iswarranted to find out the actual role of DDC in the etiologyof ADHD
42 DRD2 Pharmacological intervention of several neu-ropsychiatric and neurologic disorders essentially relies onthemodulation of function of theDRD2 receptor SNPs in theDRD2 gene have shown association with ADHD in probandsfrom Finland [26] Associations have also been reported inBrazilian [29] as well as Spanish [47] schizophrenics andArabian addicts [48] Since this gene may play a role inbehavioral attributes we have explored association of threefunctional variants rs1800496 rs1801028 and rs1799732withADHD A proline to serine substitution at codon 309 causedby CgtT transition rs1800496 was predicted to play rolein protein coding splicing regulation and posttranslationalmodification (F-SNP) An earlier report also hypothesizedthat this substitution may cause impairment in modulatingadenylate cyclase activity [49] However the ldquoArdquo allele fre-quency was reported to be very low (0002) in the Caucasianpopulation [50] In the exon 7 rs1801028 a CgtG transitionaltering the 311 codon causes a serine to cysteine substitution
8 BioMed Research International
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
the Cys311 variant was reported to have decreased affinity forDA [49] This variant was also found to alter protein codingsplicing regulation and posttranslational modification (F-SNP) In the Caucasian population frequency of the ldquoGrdquoallele was found to be 003 [50] and association analysis withADHD failed to show any significance [51] In the presentinvestigation on Indo-Caucasoid population both rs1800496and rs1801028weremonomorphic for the wild type ldquoCrdquo alleleand thus no association with ADHD could be ascertained
Another functional variant in the DRD2 -141C InsDelvariant rs1799732 alters transcriptional activity of the pro-moter thus regulating expression of the receptor [52] andhas been reported to influence D2 receptor density in thestriatum [53] Response to antipsychotic drugs was alsofound to be affected by rs1799732 [54] While no publishedliterature on association of this variant with ADHD wasobserved the -141C insertion allele showed association withalcohol dependence in Indian males [30] Frequency ofthe ldquoDelrdquo allele was reported to be 014 in the Caucasianpopulation [50] which is comparable with the frequencyobtained in the present study on the Indo-Caucasoid pop-ulation (012) Our pioneering analysis on rs1799732 inassociation with ADHD revealed nominal bias for the ldquoCrdquoallele in the probands by both population- and family-based analyses along with statistically significant occurrenceand transmission inADHD+LDMaternal overtransmissionwas also noticed in ADHD+LD group Further the ldquoCCrdquogenotype showed statistically significant higher occurrence inADHD+LD and ADHD+MD On the basis of the presentdata it may be inferred that rs1799732 could be important forthe etiology of ADHD associated LD and MD and may turnout to be useful for pharmacological as well as psychologicalinterventions that directly hit specific neurophysiologicalmechanisms compromised in ADHD probands
43 DRD4 DRD4 receptor is predominantly expressed inthe frontal lobe regions of the brain a region thought to beinvolved in the etiology of ADHD [3] Association studiesalso indicate DRD4 as a candidate gene for ADHD [5 12ndash15 21] Extensive work has been done on the exon 3 48 bpvariable number of tandem repeats and meta-analysis ofmore than 30 published reports revealed that the higherrepeat variant (7R) that reduces sensitivity to DA increasesrisk for the disorder [15 21] In the Indo-Caucasoid ADHDprobands we have also observed significant association of thehigher repeats [5] Another repeat variant rs4646984 locatedabout 12 kb upstream of the initiation codon and affectingtranscriptional activity of the promoter [14] showed nominalassociation of the duplicated allele in Caucasian populationfrom Norway Spain [55] and USA [14 56] On the otherhand haplotypes containing the single repeat allele haveshown higher frequency in Caucasian ADHD probands fromHungary [13] A study on ADHD subjects from Taiwan alsoshowed negative association with the duplicated allele [57]A 12 bp repeat variant near the junction of the extracellulardomain of the receptor speculated to alter agonist bindingand signal transduction [58] was also studied in limitednumber of Indo-Caucasoid ADHD (119873 = 70) and Italian
delusional disorder patients (119873 = 59) respectively [12 59] Inthe present investigation we have replicated analysis of thesetwo repeat polymorphisms association of the single repeatalleles of rs4646983 was noticed with ADHD Cases withcomorbidities like CD ODD and MD showed significantlyhigher frequency of the single repeat variant rs4646984single repeat allele also showed associationwithADHD+CD(OR = 258) Over representation of the double repeat (2R)allele of rs4646983 (119875 = 004 power = 54 120572 at 005) alongwith higher frequency of the 2R-2R haplotype in controlsamples (119875 = 005) indicates some protective role of thisallele in the studied population Whether this diversity inallelic association in absence of any allelic flip is due to adifference in association with the disorder or is generated dueto type I error in different studied population merits furtherinvestigation in large cohort of subjects
44 COMT COMT helps in the metabolism of DAadrenalin and norepinephrine and has been implicated in theetiology of substance abuse schizophrenia and novelty seek-ing as well as ADHD A number of investigations have beencarried out on a functional variant ValMet polymorphismat codon 158 [19 20] and studies in Indo-Caucasoid ADHDprobands [5] as well as meta-analysis failed to support anyassociation [60] A G gt A substitution rs740603 in the intron1 of COMT gene predicted to alter transcriptional regulation(F-SNP) though failed to show any association with ADHDin Caucasian subjects from Finland [26] and Ireland [20] ahaplotype consisting of the ldquoArdquo allele was reported to provideprotection towards nicotine dependence in the African-American population (119875 = 00005) [61] Another G gt Atransition rs165599 at the 31015840UTR of COMT predicted toaffect gene expression [62] showed association with ADHDand obsessive compulsive disorder in Jews from Israel [27]On the other hand in British Caucasian ADHD childrenrs165599 revealed no significant association [63] The G-A haplotype consisting of rs4680-rs165599 showed higheroccurrence in patients with anxiety spectrum phenotypes[64] Sexually dimorphic effects of COMT haplotypes inboys and girls [65] and strong association with severity ofhyperactivity symptoms [66] have also been reported Ouranalysis revealed statistically significant bias in transmissionof the rs740603 ldquoGrdquo allele to ADHD and ADHD+MDprobands the biased transmission was paternal in nature(119875 = 003) while maternal transmission to male probandswas nominal only (119875 = 009) Marginally significant higheroccurrence of the ldquoGrdquo was also observed in ADHD+ODDby population-based analysis Higher occurrence of the ldquoGrdquoallele as well as ldquoGGrdquo genotype of rs165599was also noticed inADHD+LD probands On the other hand haplotype analy-sis showed a nominal bias in overtransmission of rs165599-rs740603 ldquoG-Ardquo (119875 = 004) which failed to be significantby case-control comparison Earlier investigators reported anassociation of rs165599 ldquoArdquo with anxiety spectrum disorder[64] Since only a few Indian ADHD probands reportedanxiety disorder further investigation in extended numberof samples is warranted to find out whether protectionto anxiety is conferred by the rs165599 ldquoGrdquo allele in this
BioMed Research International 9
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
population Moreover contribution of the rs740603 ldquoGrdquo inADHDalsomerits further exploration based on earlier reportof protection to nicotine dependence [61]
45 Epistatic Interaction In an earlier investigation on theIndo-Caucasoid ADHD probands we have noticed additiveeffects of DBH rs1108580 and DRD4 rs1800955 while theDRD4 exon 3 VNTR DAT1 31015840UTR and intron 8 VNTRMAOA u-VNTR rs6323 COMT rs4680 rs362204 DBHrs1611115 and rs1108580 were found to exert strong indepen-dent effects [5] Investigation on young adults from USArevealed lack of significant interaction between DRD4 andDAT1 (SLC6A3) while monoaminergic system genes showedsignificant interaction with ADHD symptoms [67] On theother hand an interaction between DRD2-DRD4 was foundto be associated with development of CD and adult antisocialbehavior in males [68] In a more recent study no epistaticinteractionwas found betweenCOMT andDRD4 [69] Alter-natively an interaction between functional variants in DRD2and COMT was found to hamper working memory [70]In the present investigation interactive effect of DRD2 andCOMT was noticed in ADHD+ODD while in other groupsindependent main effects of these sites were observed Statis-tically significant interaction of DDC rs3837091 with DRD2rs1799732 DRD4 rs4646984 and COMT rs740603 was alsonoticed by population-based analysis Further interaction ofDDC rs3837091 with DRD2 rs1799732 was strong in familieswith ADHD probands the 119875 value remained statisticallysignificant even after correction for multiple testing DRD4and DDC also exhibited significant main effects Whileboth DDC and COMT are important for neurotransmittermetabolism COMTalso plays vital roles in catecholestrogensand catechol-containing flavonoids Furthermore ADHD ishypothesized to be caused by an interaction of differentgenetic as well as environmental factors It may be quiteprobable that the variants we found to be associated withADHD have relatively small effect sizes keeping with themultifactorial polygenic etiology of ADHD [15 17 18 21]Theother question that remains to be answered is whether thetraits of ADHD are affected by haploinsufficiency for someof these alleles
Altered dopaminergic neurotransmission is implicatedin ADHD based on the presenting clinical features ofprobands available animal models and pharmacotherapeu-tics [3ndash6 10 46] In the present study on Indo-CaucasoidADHD probands both population- and family-based anal-yses revealed higher transmission as well as independenteffect of DRD2 rs1799732 ldquoCrdquo allele Decreased frequency ofthe rs1799732 ldquoDelrdquo allele was speculated to contribute to anelevated DRD2 density leading to DA hyperactivity [71] Invivo experiments in mice showed that DRD2 over expressionin the striatum impacts DA levels rates of DA turnoverand activation of D1 receptors in the prefrontal cortex thebrain structuremainly associatedwithworkingmemory [72]Further altered expression ofDRD2 andCOMT was found tohamper workingmemory a trait affected in ADHDprobands[70] On the basis of the above observations we infer thatthe eastern Indian ADHD probands may have an altered DAsignaling
5 Conclusion
This association analysis on Indo-Caucasoid subjects withADHD explored gene variants studied for association withdifferent behavioral disorders In this preliminary investiga-tion with limited number of ADHD probands we have alsostudied association with different co-morbid conditions thatare frequently observed in ADHD patients The suggestedreason for these comorbidities to be so common in ADHDsubjects was hypothesized to be due to sharing of a numberof gene variants [24] As a support to the aforesaid fact wehave noticed higher frequencies and bias in transmissionof DDC DRD2 DRD4 and COMT variants in individu-als with ADHD and those exhibiting different co-morbidconditions In our earlier investigation in this ethnic groupwe have observed a trend for alteration in dopaminergicneurotransmission in ADHD probands [5 12] The presentstudy also indicates involvement of gene variants whichmay hamper catecholaminergic neurotransmission Furtherinvestigation on functional behavioral and environmentalattributes incorporating larger sample sizes is warranted tounderstand the complex disease etiology
Authorsrsquo Contribution
Paramita Ghosh and Kanyakumarika Sarkar equally contrib-uted to this work
Acknowledgments
The authors are thankful to the volunteers for participationin the study The research was sponsored partly by theDepartment of Science andTechnologyGovernment of India(SRCSI172009) Fellowships provided to Paramita Ghosh(Indian Council of Medical Research India) and Kanyaku-marika Sarkar and Nipa Bhaduri (Council of Scientific andIndustrial Research India) are also acknowledged
References
[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders Washington DC USA 4th edi-tion 2000
[2] K Larson S A Russ R S Kahn and N Halfon ldquoPatterns ofcomorbidity functioning and service use for US children withADHD 2007rdquo Pediatrics vol 127 no 3 pp 462ndash470 2011
[3] J W Lazar and Y Frank ldquoFrontal systems dysfunction in chil-dren with attention-deficithyperactivity disorder and learningdisabilitiesrdquo Journal of Neuropsychiatry and Clinical Neuro-sciences vol 10 no 2 pp 160ndash167 1998
[4] CM Freitag andWRetz ldquoFamily and twin studies in attention-deficit hyperactivity disorderrdquo Key Issues in Mental Health vol176 pp 38ndash57 2010
[5] M Das A D Bhowmik N Bhaduri et al ldquoRole of gene-genegene-environment interaction in the etiology of eastern IndianADHD probandsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 2 pp 577ndash587 2011
[6] S DiMaio N Grizenko and R Joober ldquoDopamine genes andattention-deficit hyperactivity disorder a reviewrdquo Journal ofPsychiatry and Neuroscience vol 28 no 1 pp 27ndash38 2003
10 BioMed Research International
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
[7] E F Coccaro S L Hirsch andM A Stein ldquoPlasma homovanil-lic acid correlates inversely with history of learning problems inhealthy volunteer and personality disordered subjectsrdquo Psychi-atry Research vol 149 no 1ndash3 pp 297ndash302 2007
[8] O Civelli J R Bunjow and D K Grandy ldquoMolecular diversityof the dopamine receptorsrdquo Annual Review of Pharmacologyand Toxicology vol 32 pp 281ndash307 1993
[9] J A Gingrich and M G Caron ldquoRecent advances in themolecular biology of dopamine receptorsrdquo Annual Review ofNeuroscience vol 16 pp 299ndash231 1993
[10] X Fan M Xu and E J Hess ldquoD2 dopamine receptor subtype-mediated hyperactivity and amphetamine responses in a modelof ADHDrdquo Neurobiology of Disease vol 37 no 1 pp 228ndash2362010
[11] P Shaw M Gornick J Lerch et al ldquoPolymorphisms of thedopamine D4 receptor clinical outcome and cortical structurein attention-deficithyperactivity disorderrdquo Archives of GeneralPsychiatry vol 64 no 8 pp 921ndash931 2007
[12] N Bhaduri M Das S Sinha et al ldquoAssociation of dopamineD4 receptor (DRD4) polymorphisms with attention deficithyperactivity disorder in Indian populationrdquo American Journalof Medical Genetics B vol 141 no 1 pp 61ndash66 2006
[13] E Kereszturi O Kiraly Z Csapo et al ldquoAssociation betweenthe 120-bp duplication of the dopamine D4 receptor gene andattention deficit hyperactivity disorder genetic and molecularanalysesrdquo American Journal of Medical Genetics B vol 144 no2 pp 231ndash236 2007
[14] J T McCracken S L Smalley J J McGough et al ldquoEvidencefor linkage of a tandem duplication polymorphism upstream ofthe dopamine D4 receptor gene (DRD4) with attention deficithyperactivity disorder (ADHD)rdquo Molecular Psychiatry vol 5no 5 pp 531ndash536 2000
[15] T Banaschewski K Becker S Scherag B Franke and DCoghill ldquoMolecular genetics of attention-deficithyperactivitydisorder an overviewrdquo European Child and Adolescent Psychia-try vol 19 no 3 pp 237ndash257 2010
[16] I D Waldman and I R Gizer ldquoThe genetics of attention deficithyperactivity disorderrdquo Clinical Psychology Review vol 26 no4 pp 396ndash432 2006
[17] E Mick and S V Faraone ldquoGenetics of attention deficithyperactivity disorderrdquo Child and Adolescent Psychiatric Clinicsof North America vol 17 no 2 pp 261ndash284 2008
[18] L Guan B Wang Y Chen et al ldquoA high-density single-nucleotide polymorphism screen of 23 candidate genes inattention deficit hyperactivity disorder suggesting multiplesusceptibility genes amongChineseHan populationrdquoMolecularPsychiatry vol 14 no 5 pp 546ndash554 2009
[19] P J Carpentier A Arias Vasquez M Hoogman et alldquoShared and unique genetic contributions to attention deficithyperactivity disorder and substance use disorders a pilot studyof six candidate genesrdquo European Neuropsychopharmacologyvol 23 pp 448ndash457 2013
[20] Z Hawi N Matthews E Barry et al ldquoA high density linkagedisequilibrium mapping in 14 noradrenergic genes evidenceof association between SLC6A2 ADRA1B and ADHDrdquo Psy-chopharmacology (Berl) vol 225 pp 895ndash902 2013
[21] J Lasky-Su B M Neale B Franke et al ldquoGenome-wide associ-ation scan of quantitative traits for attention deficit hyperactiv-ity disorder identifies novel associations and confirms candidategene associationsrdquo American Journal of Medical Genetics B vol147 no 8 pp 1345ndash1354 2008
[22] M Ribases J A Ramos-Quiroga A Hervas et al ldquoExplorationof 19 serotoninergic candidate genes in adults and children withattention-deficithyperactivity disorder identifies associationfor 5HT2A DDC andMAOBrdquoMolecular Psychiatry vol 14 no1 pp 71ndash85 2009
[23] Q-J Qian J Liu Y-F Wang L Yang L-L Guan and SV Faraone ldquoAttention deficit hyperactivity disorder comorbidoppositional defiant disorder and its predominately inattentivetype evidence for an association with COMT but notMAOA inaChinese samplerdquoBehavioral and Brain Functions vol 5 article8 2009
[24] D E Comings R Gade-Andavolu N Gonzalez et al ldquoCom-parison of the role of dopamine serotonin and noradrenalinegenes in ADHD ODD and conduct disorder multivariateregression analysis of 20 genesrdquo Clinical Genetics vol 57 no 3pp 178ndash196 2000
[25] A Kirley Z Hawi G Daly et al ldquoDopaminergic system genesin ADHD toward a biological hypothesisrdquo Neuropsychophar-macology vol 27 no 4 pp 607ndash619 2002
[26] E S Nyman M N Ogdie A Loukola et al ldquoADHD candidategene study in a population-based birth cohort association withDBH andDRD2rdquo Journal of the AmericanAcademy of Child andAdolescent Psychiatry vol 46 no 12 pp 1614ndash1621 2007
[27] E Michaelovsky D Gothelf M Korostishevsky et al ldquoAsso-ciation between a common haplotype in the COMT generegion and psychiatric disorders in individuals with 22q112DSrdquoInternational Journal of Neuropsychopharmacology vol 11 no 3pp 351ndash363 2008
[28] JWuH XiaoH Sun L Zou and LQ Zhu ldquoRole of dopaminereceptors in ADHD a systematic meta-analysisrdquo MolecularNeurobiology vol 45 pp 605ndash620 2012
[29] H Zhang Y Ye X Wang J Gelernter J Z Ma and MD Li ldquoDOPA decarboxylase gene is associated with nicotinedependencerdquo Pharmacogenomics vol 7 no 8 pp 1159ndash11662006
[30] P Prasad A Ambekar and M Vaswani ldquoDopamine D2 recep-tor polymorphisms and susceptibility to alcohol dependence inIndian males a preliminary studyrdquo BMC Medical Genetics vol11 no 1 article 24 2010
[31] C K Conners Connersrsquo Rating ScalesmdashRevised Multi-HealthSystems Toronto Canada 1997
[32] D Wechsler Wechsler Intelligence Scale for Children ManualPsychological Corporation SanAntonio Tex USA 3rd edition1991
[33] J Bharat Raj ldquoAIISH norms on SFB with Indian childrenrdquoJournal of All India Institute of Speech and Hearing vol 2 pp34ndash39 1971
[34] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988
[35] F Dudbridge ldquoPedigree disequilibrium tests for multilocushaplotypesrdquo Genetic Epidemiology vol 25 no 2 pp 115ndash1212003
[36] R S Spielman R E McGinnis and W J Ewens ldquoTransmis-sion test for linkage disequilibrium the insulin gene regionand insulin-dependent diabetes mellitus (IDDM)rdquo AmericanJournal of Human Genetics vol 52 no 3 pp 506ndash516 1993
[37] J D Terwilliger and J Ott ldquoA haplotype-based ldquoHaplotype Rel-ative Riskrdquo approach to detecting allelic associationsrdquo HumanHeredity vol 42 no 6 pp 337ndash346 1992
[38] R V Lenth ldquoStatistical power calculationsrdquo Journal of animalscience vol 85 no 13 pp E24ndashE29 2007
BioMed Research International 11
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
[39] J H Moore J C Gilbert C-T Tsai et al ldquoA flexible computa-tional framework for detecting characterizing and interpretingstatistical patterns of epistasis in genetic studies of humandisease susceptibilityrdquo Journal ofTheoretical Biology vol 241 no2 pp 252ndash261 2006
[40] J H Moore and B C White ldquoTuning relief for genome-wide genetic analysisrdquo in Evolutionary ComputationMachineLearning and Data Mining in Bioinformatics vol 4447 ofLecture Notes in Computer Science pp 166ndash175 Springer BerlinGermany 2007
[41] M D Ritchie L W Hahn N Roodi et al ldquoMultifactor-dimen-sionality reduction reveals high-order interactions amongestrogen-metabolism genes in sporadic breast cancerrdquo Ameri-can Journal of Human Genetics vol 69 no 1 pp 138ndash147 2001
[42] L W Hahn M D Ritchie and J H Moore ldquoMultifactordimensionality reduction software for detecting gene-gene andgene-environment interactionsrdquo Bioinformatics vol 19 no 3pp 376ndash382 2003
[43] H Mei M L Cuccaro and E R Martin ldquoMultifactor dimen-sionality reduction-phenomics a novel method to capturegenetic heterogeneity with use of phenotypic variablesrdquo Ameri-can Journal of HumanGenetics vol 81 no 6 pp 1251ndash1261 2007
[44] E R Martin M D Ritchie L Hahn S Kang and J HMoore ldquoA novel method to identify gene-gene effects in nuclearfamilies the MDR-PDTrdquo Genetic Epidemiology vol 30 no 2pp 111ndash123 2006
[45] A Thapar R Harrington and P McGuffin ldquoExamining thecomorbidity of ADHD-related behaviours and conduct prob-lems using a twin study designrdquo British Journal of Psychiatryvol 179 pp 224ndash229 2001
[46] M Ernst A J Zametkin J A Matochik D Pascualvaca P HJons and R M Cohen ldquoHigh midbrain [18F]DOPA accumu-lation in children with attention deficit hyperactivity disorderrdquoAmerican Journal of Psychiatry vol 156 no 8 pp 1209ndash12151999
[47] M J Parsons I Mata M Beperet et al ldquoA dopamine D2 recep-tor gene-related polymorphism is associated with schizophre-nia in a Spanish population isolaterdquo Psychiatric Genetics vol 17no 3 pp 159ndash163 2007
[48] L N AL-Eitan S A Jaradat G K Hulse and G K Tay ldquoCus-tom genotyping for substance addiction susceptibility genes inJordanians of Arab descentrdquo BMC Research Notes vol 5 article497 2012
[49] A Cravchik D R Sibley and P V Gejman ldquoFunctional analysisof the humanD2 dopamine receptormissense variantsrdquo Journalof Biological Chemistry vol 271 no 42 pp 26013ndash26017 1996
[50] A Doehring A Kirchhof and J Lotsch ldquoGenetic diagnostics offunctional variants of the human dopamine D2 receptor generdquoPsychiatric genetics vol 19 no 5 pp 259ndash268 2009
[51] R D Todd and E A Lobos ldquoMutation screening of thedopamine D2 receptor gene in attention-deficit hyperactivitydisorder subtypes preliminary report of a research strategyrdquoAmerican Journal ofMedical Genetics B vol 114 no 1 pp 34ndash412002
[52] T Arinami M Gao H Hamaguchi andM Toru ldquoA functionalpolymorphism in the promoter region of the dopamine D2receptor gene is associated with schizophreniardquo Human Molec-ular Genetics vol 6 no 4 pp 577ndash582 1997
[53] M J Arranz and J De Leon ldquoPharmacogenetics and phar-macogenomics of schizophrenia a review of last decade ofresearchrdquoMolecular Psychiatry vol 12 no 8 pp 707ndash747 2007
[54] J-P Zhang T Lencz and A K Malhotra ldquoD2 receptor geneticvariation and clinical response to antipsychotic drug treatmenta meta-analysisrdquo American Journal of Psychiatry vol 167 no 7pp 763ndash772 2010
[55] C Sanchez-MoraM RibasesM Casas et al ldquoExploringDRD4and its interaction with SLC6A3 as possible risk factors foradult ADHD a meta-analysis in four European populationsrdquoAmerican Journal of Medical Genetics B vol 156 no 5 pp 600ndash612 2011
[56] V Kustanovich J Ishii L Crawford et al ldquoTransmission dis-equilibrium testing of dopamine-related candidate gene poly-morphisms in ADHD confirmation of association of ADHDwith DRD4 and DRD5rdquo Molecular Psychiatry vol 9 no 7 pp711ndash717 2004
[57] K-J Brookes X Xu C-K Chen Y-S Huang Y-Y Wu andP Asherson ldquoNo evidence for the association of DRD4 withADHD in a Taiwanese population within-family studyrdquo BMCMedical Genetics vol 6 article 31 2005
[58] P Seeman C Ulpian G Chouinard et al ldquoDopamine D4receptor variant D4GLYCINE194 in Africans but not in Cau-casians no association with schizophreniardquo American Journalof Medical Genetics vol 54 no 4 pp 384ndash390 1994
[59] M Catalano M Nobile E Novelli M M Nothen and ESmeraldi ldquoDistribution of a novel mutation in the first exon ofthe human dopamine D4 receptor gene in psychotic patientsrdquoBiological Psychiatry vol 34 no 7 pp 459ndash464 1993
[60] D K L Cheuk and V Wong ldquoMeta-analysis of associationbetween a catechol-O-methyltransferase gene polymorphismand attention deficit hyperactivity disorderrdquo Behavior Geneticsvol 36 no 5 pp 651ndash659 2006
[61] J Beuten T J Payne J ZMa andMD Li ldquoSignificant associa-tion of catechol-O-methyltransferase (COMT) haplotypes withnicotine dependence in male and female smokers of two ethnicpopulationsrdquo Neuropsychopharmacology vol 31 no 3 pp 675ndash684 2006
[62] N J Bray P R Buckland N M Williams et al ldquoA haplotypeimplicated in schizophrenia susceptibility is associated withreduced COMT expression in human brainrdquo American Journalof Human Genetics vol 73 no 1 pp 152ndash161 2003
[63] D Turic H Williams K Langley M Owen A Thaparand M C OrsquoDonovan ldquoA family based study of catechol-O-methyltransferase (COMT) andAttentionDeficit HyperactivityDisorder (ADHD)rdquoAmerican Journal ofMedical Genetics B vol133 no 1 pp 64ndash67 2005
[64] J M Hettema S-S An J Bukszar et al ldquoCatechol-O-methyltransferase contributes to genetic susceptibility sharedamong anxiety spectrumphenotypesrdquoBiological Psychiatry vol64 no 4 pp 302ndash310 2008
[65] M Karayiorgou C Sobin M L Blundell et al ldquoFamily-based association studies support a sexually dimorphic effectof COMT and MAOA on genetic susceptibility to obsessive-compulsive disorderrdquo Biological Psychiatry vol 45 no 9 pp1178ndash1189 1999
[66] H Halleland A J Lundervold A Halmoslashy J Haavik and SJohansson ldquoAssociation between catechol O-methyltransferase[COMT] haplotypes and severity of hyperactivity symptoms inadultsrdquo American Journal of Medical Genetics B vol 150 no 3pp 403ndash410 2009
[67] L C Bidwell M E Garrett F J McClernon et al ldquoA prelim-inary analysis of interactions between genotype retrospectiveADHD symptoms and initial reactions to smoking in a sample
12 BioMed Research International
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
of young adultsrdquo Nicotine and Tobacco Research vol 14 no 2pp 229ndash233 2012
[68] K M Beaver J P Wright M DeLisi et al ldquoA gene timesgene interaction between DRD2 and DRD4 is associated withconduct disorder and antisocial behavior in malesrdquo Behavioraland Brain Functions vol 3 article 30 2007
[69] S Heinzel T Dresler C G Baehne et al ldquoCOMTtimesDRD4 epis-tasis impacts prefrontal cortex function underlying responsecontrolrdquo Cerebral Cortex vol 23 pp 1453ndash1462 2013
[70] H Xu C B Kellendonk E H Simpson et al ldquoDRD2 C957Tpolymorphism interacts with the COMT Val158Met poly-morphism in human working memory abilityrdquo SchizophreniaResearch vol 90 no 1ndash3 pp 104ndash107 2007
[71] P J Harrison and D R Weinberger ldquoSchizophrenia genesgene expression and neuropathology on the matter of theirconvergencerdquo Molecular Psychiatry vol 10 no 1 pp 40ndash682005
[72] C Kellendonk E H Simpson H J Polan et al ldquoTransientand selective overexpression of dopamine D2 receptors in thestriatum causes persistent abnormalities in prefrontal cortexfunctioningrdquo Neuron vol 49 no 4 pp 603ndash615 2006
