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DRD2 and PPP1R1B (DARPP-32) polymorphisms independently confer increased risk for autism spectrum disorders… Hettinger, Joe A; Liu, Xudong; Hudson, Melissa L; Lee, Alana; Cohen, Ira L; Michaelis, Ron C; Schwartz, Charles E; Lewis, Suzanne M; Holden, Jeanette J May 4, 2012

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RESEARCH Open AccessDRD2 and PPP1R1B (DARPP-32) polymorphismsindependently confer increased risk for autismspectrum disorders and additively predictaffected status in male-only affected sib-pairfamiliesJoe A Hettinger1, Xudong Liu2,3, Melissa L Hudson2,3,4, Alana Lee2,3,4, Ira L Cohen4,5, Ron C Michaelis6,Charles E Schwartz7, Suzanne ME Lewis4,8,9 and Jeanette JA Holden1,2,3,4,10,11*AbstractBackground: The neurotransmitter dopamine (DA) modulates executive functions, learning, and emotionalprocessing, all of which are impaired in individuals with autism spectrum disorders (ASDs). Our previous findingssuggest a role for dopamine-related genes in families with only affected males.Methods: We examined two additional genes which affect DA function, the DRD2 and PPP1R1B (DARPP-32) genes,in a cohort of 112 male-only affected sib-pair families. Selected polymorphisms spanning these genes weregenotyped and both family-based and population-based tests were carried out for association analysis. Generaldiscriminant analysis was used to examine the gene-gene interactions in predicting autism susceptibility.Results: There was a significantly increased frequency of the DRD2 rs1800498TT genotype (P= 0.007) in affectedmales compared to the comparison group, apparently due to over-transmission of the T allele (P= 0.0003). Thefrequency of the PPP1R1B rs1495099CC genotype in affected males was also higher than that in the comparisongroup (P= 0.002) due to preferential transmission of the C allele from parents to affected children (P= 0.0009).Alleles rs1800498T and rs1495099C were associated with more severe problems in social interaction (P= 0.0002 andP= 0.0016, respectively) and communication (P= 0.0004 and P= 0.0046), and increased stereotypic behaviours(P= 0.0021 and P= 0.00072). General discriminant analysis found that the DRD2 and PPP1R1B genes additivelypredicted ASDs (P= 0.00011; Canonical R = 0.26) and explain ~7% of the variance in our families. All findingsremained significant following corrections for multiple testing.Conclusion: Our findings support a role for the DRD2 and PPP1R1B genes in conferring risk for autism in familieswith only affected males and show an additive effect of these genes towards prediction of affected status in ourfamilies.Keywords: Autism spectrum disorders, Dopamine receptors, DARPP-32, Association study, Candidate gene* Correspondence: holdenj@queensu.ca11Autism Research Program/Genetics and Genomics Research Laboratory,Ongwanada Resource Centre, 191 Portsmouth Ave, Kingston, ON, CanadaK7M 8A6Full list of author information is available at the end of the article© 2012 Hettinger et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Hettinger et al. Behavioral and Brain Functions 2012, 8:19http://www.behavioralandbrainfunctions.com/8/1/19IntroductionAutism spectrum disorders (ASDs) are characterized by re-petitive behaviours and interests, as well as deficiencies incommunication and social interaction. They are believedto be complex, polygenic disorders predominantly charac-terized by multifactorial inheritance [1], although Zhaoet al. [2] (2007) suggested that Mendelian inheritance mayapply to autism risk in a subgroup of families with affectedmales. To address the significant genetic heterogeneity andphenotypic variation seen among affected individuals,which has confounded the conclusive identification of can-didate genes for the majority of cases, we have been testinggenes for evidence of association with specific ASD endo-phenotypes in an effort to identify a subgroup within theASD population whose members share an underlyingpathophysiology.Abnormalities in neurotransmitter pathways can ac-count for the deficits seen in persons with ASDs. In con-trast to the attention which has been directed to thestudy of genes involved in the glutamate [3], GABA [3]and serotonin pathways [1], genes related to the synthe-sis, function and metabolism of dopamine (DA) havereceived little attention [4].We have argued [5] that genes in the dopaminergic(DAergic) pathway are excellent candidates based ontheir affect on ASD behaviours. DA modulates motorfunctions [6], cognitive processes (including executivefunctions [7] and learning [8]), and emotional regulation[9] - all of which are abnormal in individuals with autism[10-13]. DA also plays a role in social interactions [14]and the pathophysiology of stereotypies [15]; impair-ments in social interaction and the presence of increasedstereotypies are core features of autism. Furthermore,there is decreased DAergic activity in the medial pre-frontal cortex (PFC) in children with autism [16], andincreased levels of the major metabolite of DA, homova-nillic acid (HVA), in cerebrospinal fluid from affectedchildren compared to controls [17], indicating alteredDAergic function in these individuals.Based on our earlier findings on the dopamine β-hydroxylase (DBH) gene, which encodes the enzyme thatconverts DA to norepinephrine, in mothers from male-only affected sib-pair families [18], we have pursued acomprehensive study of DA-related genes in mothersand sons with ASDs. Since our initial study with DBH, inwhich we found an increased frequency of the 19-bp de-letion in mothers from male-only affected sib-pair fam-ilies [18], we identified a 3-marker risk haplotype in thedopamine D1 receptor (DRD1) gene [5] in our family co-hort having only affected sons. Here we report our find-ings on two other genes affecting DA levels andfunction, the dopamine D2 receptor (DRD2) and proteinphosphatase 1, regulatory subunit 1B (PPP1R1B) genes,and results of tests for gene-gene interactions.The DRD2 gene comprises eight exons [19] and mapsto 11q22-q23 [20]. It encodes the dopamine D2 receptorwhich, in addition to its role in postsynaptic neurons,acts as an autoreceptor mediating DA synthesis [21] andneurotransmission [22] in DAergic neurons. The dopa-mine D2 receptor is involved in the DAergic modulationof executive functions [23], reversal learning [24] andemotional processing [25]. Drd2−/− mice have abnormalgait similar to that of individuals with Parkinson disease[26], and the administration of antipsychotic medications(e.g., risperidone, a dopamine D2 receptor antagonist)has proven efficacious in treating symptoms associatedwith ASDs [27].The PPP1R1B gene, located at chromosome 17q12 andcomprising 7 exons (http://www.ncbi.nlm.nih.gov; Gen-eID 84152), encodes DARPP-32, which is expressed indopaminoceptive (DAceptive) neurons [28] and mediatesthe effects of both D1 and D2 dopamine receptor classes[29]. For example, dopamine D2 receptor antagonist-induced catalepsy in rats is attenuated in Ppp1r1b−/−mice [30] and knockout mice are impaired in reversallearning [31]. Genetic [32,33] and immunoblot [34] stud-ies showed an association of PPP1R1B with altered PFCDARPP-32 protein levels in schizophrenia and bipolardisorder, two conditions for which DA dysfunction is evi-dent and which exhibit comorbidity with autism [35].There are two previous studies which examined theDRD2 gene as a candidate gene for autism. The first [36]reported an increased frequency of the TaqI A1 allele inpersons with autism (N= 33) compared to controls(N= 314), whereas the second [37] found no evidence fortransmission disequilibrium of an intragenic microsatel-lite in 39 affected sib-pair families. No association studieshave examined the role of PPP1R1B as a candidate genefor ASDs.Based on our hypothesis that DA-related genes are im-portant in male-only affected sib-pair families [18], weexamined four markers at the DRD2 locus that are com-monly used to investigate possible associations betweenDAergic function and behavioural abnormalities [38],and three polymorphisms at the PPP1R1B locus to deter-mine whether there was an association of these DA-related genes with autism.Materials and methodsSubjectsThe 112 affected sib-pair families and the comparisongroup (N= 443) were previously described [5]. Briefly,the study group included 28 families from Canada [18], 5from the South Carolina Autism Project [39], and 79families obtained through the Autism Genetic ResourceExchange (AGRE) in the United States [40]. All familieshave two or more children with either autism or anASD, including Asperger syndrome and pervasiveHettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 2 of 13http://www.behavioralandbrainfunctions.com//8/1/19developmental disorder (PDD) variants. This study wasapproved by the Queen’s University Research EthicsBoard and written informed consent was obtained fromparents of all participating families from Canada andSouth Carolina, and through AGRE [40].All 443 samples from the comparison group (blood-spots from anonymous newborns taken for the purposeof PKU testing and made available from the OntarioMinistry of Health) were available for the study of thePPP1R1B locus. Two hundred and fifty-three comparisongroup samples were used for the DRD2 gene studies.There were no significant differences in the allele fre-quencies for DRD2 (P= 0.57–0.93) and PPP1R1B(P= 0.28–0.91) markers in males and females from therespective comparison cohorts and thus our comparisoncohorts included both males and females. Althoughcomprehensive information regarding psychiatric and be-havioural disorders is not available for the comparisongroup, we do not expect the prevalence of ASDs in thiscomparison group to be greater than that in the generalpopulation, or approximately 1/110 [41].Marker amplification and genotypingDRD2Four polymorphisms (rs1799732 Ins/Del, rs1079597 G/A, rs1800498 T/C and rs1800497 C/T) were studied atthe DRD2 locus (Figure 1). The DRD2 gene contains fourhaplotype blocks [Haploview 4.1; available at http://hap-map.ncbi.nlm.nih.gov] [42]. Three of the blocks are small(<4 kb) and one block is 20 kb in size. One of four poly-morphisms used in this study, rs1079597, is part of theHapMap dataset. both this SNP and and rs1800498 arelocated within the larger haplotype block and wereexamined in previous studies of other neuropsychiatricdisorders. Primer sequences, PCR and digestion condi-tions are shown in Table 1. All PCR reactions were car-ried out using 5 ng of template DNA; digestion productswere separated on either 2% (rs1079597, rs1800498 andrs1800497) or 2.5% (rs1799732) agarose gels and visua-lized using ethidium bromide and UV illumination. Inorder to minimize genotyping errors, DNAs fromaffected individuals and family members were randomlyarranged on 96-well plates, and all results were inde-pendently scored and tabulated by two persons.PPP1R1BThree polymorphisms (rs1495099 G/C, rs907094 T/Cand rs3764352 A/G) were examined in the PPP1R1Bgene (Figure 2). These variants were chosen from theNCBI dbSNP Build 121 database from the Human Gen-ome Project (available at http://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi) based on the following criteria:the markers span the PPP1R1B locus, they have minorallele frequencies (MAFs) of approximately 20%, andalleles at rs907094 and rs3764352 are associated withchanges in DARPP-32 mRNA expression and measuresof cognitive performance [32]. The PPP1R1B locus has asingle haplotype block which includes rs907094 andrs3764352 as haplotype-tagged SNPs (htSNPs). PCRreactions were carried out using 5 ng of template DNAand amplicons were digested using conditions shown inTable 1. All digestion products were separated on 2%agarose gels and visualized using ethidium bromide andUV illumination. In order to minimize genotyping errors,DNAs from affected individuals and family memberswere randomly arranged on 96-well plates, and all resultswere independently scored and tabulated by twopersons.0|00.13|0.07 0.82 |0.69 0|0 0.25|0.28 0.15|0.18 rs1799732 rs1079597 rs1800498 rs1800497| |65.6kbFigure 1 Illustration of the DRD2 locus. A schematic showing gene structure, marker positions and measures of linkage disequilibrium betweenrs1799732, rs1079597, rs1800498 and rs1800497 listing r2 of the comparison group (N=244) followed by r2 of parents from families (N=213).Legend:▪ exon; intron; □ untranslated region.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 3 of 13http://www.behavioralandbrainfunctions.com//8/1/19Statistical analysesPrior to carrying out analyses, Mendelian errors werechecked in the family cohort using the FBAT program,v1.5.5 [44]. DRD2 marker data on five families andPPP1R1B marker data on two families were excludedfrom the analyses due to identified Mendelian errors.Single gene analyses were performed as previouslydescribed [5]. To avoid allele and genotype frequencydistortion from using related individuals in case–controlcomparisons, one affected individual was selected at ran-dom from each family using SPSS v14.0 (SPSS, Chicago,IL) with the same cohort of randomly chosen individualsused for single marker allele and genotype frequencycomparisons for all polymorphisms as well as generaldiscriminant analyses. Family-based association tests(FBAT), including quantitative disequilibrium tests(QTDT), were done using FBAT v1.5.5. Because FBATv1.5.5 can accommodate multiple affected individualsfrom each family, all affected individuals including thoseused for case–control comparisons, were included forFBAT and QTDT analyses. The domains, ‘reciprocal so-cial interaction’, ‘communication’ and ‘repetitive stereo-typed behaviours’ used for QTDT analyses were derivedfrom the total scores from the ‘Qualitative AbnormalitiesTable 1 PCR and digestion conditions for DRD2 and PPP1R1B markers used in this studyDRD2 Marker(5′! 3′)1Primers [MgCl2] Annealing # ofcyclesRestrictiontemperature enzyme (U)2rs1799732 Ins/Del F 5′-GAGAAGACTGGCGAGCAGAC-3′ 1.5 mM 63°C 35 BstNI (0.05)R 5′-CCACCAAAGGAGCTGTACCT-3′rs1079597 G/A3 F 5′-GATACCCACTTCAGGAAGTC-3′ 1.0 mM 55°C 34 TaqI (0.4)R 5′-CAGTAAAGAACTAGGAGTCAG-3′rs1800498 T/C3 F 5′-CCCAGCAGGGAGAGGGAGTA-3′ 1.0 mM 55°C 34 TaqI (0.4)R 5′-GACAAGTACTTGGTAAGCATG-3′rs1800497 C/T3 F 5′-CCGTCGACGGCTGGCCAAGTTGTCTA-3′ 1.0 mM 58°C 34 TaqI (0.4)R 5′-CCGTCGACCCTTCCTGAGTGTCATCA-3′PPP1R1B Marker(5′!3′)Primers [MgCl2] Annealingtemperature# ofcyclesRestrictionenzyme (U)2rs1495099 G/C F 5′-TTGTTGCTGAGCTGAGATGC-3′ 1.0 mM 60°C 35 PvuII (0.3)R 5′-CTCCAGGGAAATGCACAAAG-3′rs907094 T/C F 5′-ACCTGATTGGGAGAGGGACT-3′ 1.0 mM 60°C 34 MseI (0.3)R 5′-GTAAGCTGAGGGGCCTGTG-3′rs3764352 A/G F 5′-CTGTTTTGGAGGGGTCTCAG-3′ 1.0 mM 60°C 35 BccI (0.3)R 5′-TGGGAATACTGAAGAGTCAACC-3′1Rs1799732 Ins/Del, rs1079597 G/A, rs1800498 T/C, and rs1800497 C/T previously known as −141 C Ins/Del, TaqI B, TaqI D, and TaqI A, respectively.2Restriction enzymes obtained from New England Biolabs, Pickering, ON, Canada.3Primer sequences have been reported previously [43].0.67|0.72 0.68|0.73 0.940.97rs1495099 rs907094 rs3764352| |9.7kbFigure 2 Illustration of the PPP1R1B locus. A schematic showing gene structure, marker positions and measures of linkage disequilibriumbetween rs1495099, rs907094 and rs3764352 listing r2 of the comparison group (N=435) followed by r2 of parents from families (N=216).Legend: ▪ exon; intron; □ untranslated region.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 4 of 13http://www.behavioralandbrainfunctions.com//8/1/19in Reciprocal Social Interaction’ (A1 to A4), ‘QualitativeAbnormalities in Communication’ (B1, B2(V), B3(V) andB4) and ‘Restricted, Repetitive, and Stereotyped Patternsof Behaviour’ (C1 to C4) subdomains in the ADI-R diag-nostic algorithm [45].General Discriminant Analysis (GDA) was used toevaluate the predictive value of our single gene findingsin discriminating between individuals with and withoutautism, as well as to test for evidence of interactioneffects between genes. Genotypes were coded as categor-ical variables and GDA was performed using Statistica9.1 [Statsoft, Tulsa, OK, USA].Corrections for multiple comparisonsThe contribution of a single gene to autism susceptibilityis predicted to be relatively small and thus difficult to de-tect statistically. Although corrections for multiple test-ing must be made in genetic association studies,Bonferroni correction is thought to be too stringent, witha high risk for rejecting true significant findings. Thefalse discovery rate (FDR) approach [46] is a compromisebetween not correcting for multiple comparisons, whichis too lax, and Bonferroni adjustments, which are toostrict. FDR has two methods, the Benjamini and Hoch-berg (BH) method and the Benjamini and Liu method(BL) [46]. The BH method is appropriate for correctingfor both independent and positively-dependent compari-sons [47], and thus is appropriate for genetic studiesusing polymorphisms.FDR corrections were performed separately for singlegene case–control and family-based comparisons, as wellas GDA findings, using an initial FDR threshold of 0.050.ResultsLinkage disequilibrium of polymorphisms at the DRD2and PPP1R1B lociHigh r2 measures of linkage disequilibrium (LD) wereobserved between DRD2 polymorphisms rs1079597 andrs1800497, with no LD between rs1799732 andrs1079597 and rs1800497 (Figure 1). R-squared measuresbetween PPP1R1B markers showed high LD (r2> 0.9)between rs907094 and rs3764352, and lower LD betweenrs1495099 and rs907094 and rs3764352 (Figure 2).Case–control comparisonsAll four markers of DRD2 were in HWE in the compari-son and family cohorts with the exception of rs1799732and rs1800498 in affected males (P= 0.009 and P= 0.012,respectively); all markers were in HWE in the parentsfrom these families. As shown in Table 2, there was anincreased frequency of the rs1800498 TT genotype(P= 0.007) in affected males (43.4% versus 28.7% in thecomparison group), which remained significant followingFDR correction. No significant differences in genotypefrequencies of the other three markers were seen be-tween cases and the comparison group (P= 0.32–0.51)(Table 2). Separate analyses using the extended-transmis-sion disequilibrium test (ETDT) showed that significantover-transmission of the rs1800498 T allele was not frommothers (21 transmitted, 11 untransmitted; χ2 = 3.125,df = 1, P= 0.077) but was from fathers (26 transmitted,12 untransmitted; χ2 = 5.158, df = 1, P= 0.023) to affectedsons. No differences in rs1800498 T allele or rs1800498TT genotype frequencies were found between mothers(P= 0.69 and P= 0.26, respectively) or fathers (P= 0.90and P= 0.65, respectively) and the comparison group(data not shown).All three PPP1R1B markers were in HWE in the com-parison group; none were in HWE in the cohort ofaffected individuals (P= 0.008, P= 0.033 and P= 0.033,respectively), although all markers were in HWE in theparents (data not shown). As shown in Table 2, thers1495099 CC (P= 0.002), rs907094 CC (P= 0.028) andrs3764352 GG (P= 0.025) genotype frequencies wereincreased in affected males (22.0%, 14.5% and 14.5%, re-spectively) relative to the comparison group (9.9%, 6.9%and 6.7%, respectively). Findings on alleles of these poly-morphisms were similar, with the minor allele frequen-cies of all three markers, rs1495099 C, rs907094 C andrs3764352 G, being increased in the affected males rela-tive to the comparison group (P= 0.001, P= 0.014 andP= 0.021, respectively, all remained significant followingFDR correction; data not shown).Because we hypothesize that maternal effects includinggenetic factors may contribute to autism susceptibility insome autism families [18], we compared frequencies ofrs1495099 C alleles and rs1495099 CC genotypes be-tween mothers (33.2% and 12.7%, respectively) and thecomparison group (28.7% and 9.9%, respectively) butfound no significant differences (P= 0.19 and P= 0.39, re-spectively; data not shown).Family-based association testsFBAT showed that the DRD2 rs1800498 T allele wasover-transmitted to affected males (P= 0.0003; significantfollowing FDR correction), while no evidence of prefer-ential allele transmission was found for the other threemarkers (P= 0.16–0.94) (Table 3). The rs1799732 Ins -rs1079597 G - rs1800498 T - rs1800497 C (Ins-G-T-C)haplotype, consisting of the major alleles for all fourmarkers, was over-transmitted to affected males but witha P-value (P= 0.0009; data not shown) slightly higherthan that observed with rs1800498 T alone (P= 0.0003).It should be noted that the additive model was used inFBAT for polymorphisms at the DRD2 locus because ofthe increased CT and TT genotype frequencies found forrs1800498.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 5 of 13http://www.behavioralandbrainfunctions.com//8/1/19For PPP1R1B, a recessive model was used in FBAT be-cause of the increased frequency of rs1495099 CC,rs907094 CC and rs3764352 GG genotypes found inaffected individuals compared to the comparison cohort.Family-based association analyses and FDR-based correc-tions showed significant over-transmission of rs1495099C (P= 0.00092) but not of rs907094 C (P= 0.11) orrs3764352 G (P= 0.09) (Table 3). The rs1495099 C -rs907094 C - rs3764352 G (C-C-G) haplotype was notsignificantly over-transmitted to affected males(P= 0.031; not significant following FDR correction; datanot shown) compared to that of rs1495099 C alone(P= 0.00092).Genotype-phenotype associationsWe next used quantitative transmission disequilibriumtests (QTDT) to determine whether the extent of impair-ment in the core behaviours was more pronounced inaffected males with the risk allele. The DRD2 rs1800498T allele was associated with more severe impairments inreciprocal social interaction (P= 0.0002), verbal commu-nication (P= 0.0004), and repetitive and stereotypedbehaviours (P= 0.0021); these findings remained signifi-cant following corrections for multiple comparisons.The PPP1R1B rs1495099 C allele was associated withhigher ADI-R domain scores (more severe problems) inaffected males for social interaction (P= 0.0016), nonver-bal communication (P= 0.0046), and stereotyped beha-viours (P= 0.00072) (Table 4), with strong evidence forassociation shown by multivariate QTDT betweenrs1495099 C and the combined effect of all three ADI-Rsubdomains (P= 0.00042; data not shown). All findingswere significant following FDR-based corrections.General discriminant analysesWe used GDA to determine whether DA-related genespredict ASD susceptibility in affected males compared tothe comparison group and to test for gene-gene interac-tions. Tests were performed based on our single genefindings for DRD2 and PPP1R1B from this study, andTable 2 Marker genotype frequencies at the DRD2 and PPP1R1B loci in the comparison group and males with ASD fromaffected sib-pair families1DRD2 Genotype FDRthreshold2rs1799732 N Ins/Ins Del/Ins Del/Del χ2 (df = 2) P1Comparison group 238 188 (79.0%) 46 (19.3%) 4 (1.7%)Affected males3 109 89 (81.7%) 16 (14.7%) 4 (3.7%) 2.253 0.32 0.025rs1079597 N G/G A/G A/A χ2 (df = 2) PComparison group 244 168 (68.9%) 71 (29.1%) 5 (2.0%)Affected males3 105 70 (66.7%) 30 (28.6%) 5 (4.8%) 1.944 0.38 0.038rs1800498 N T/T C/T C/C χ2 (df = 2) PComparison group 244 70 (28.7%) 130 (53.3%) 44 (18.0%)Affected males3 106 46 (43.4%) 38 (35.8%) 22 (20.8%) 9.790 0.007 0.013rs1800497 N C/C T/C T/T χ2 (df = 2) PComparison group 245 164 (66.9%) 69 (28.2%) 12 (4.9%)Affected males3 107 65 (60.7%) 35 (32.7%) 7 (6.5%) 1.333 0.51 0.050PPP1R1B Genotype FDRthreshold2rs1495099 N C/C C/G G/G χ2 (df = 2) P1Comparison group 434 43 (9.9%) 163 (37.6%) 228 (52.5%)Affected males3 109 24 (22.0%) 39 (35.8%) 46 (42.2%) 12.273 0.002 0.0083rs907094 N C/C C/T T/T χ2 (df = 2) PComparison group 434 30 (6.9%) 146 (33.6%) 258 (59.4%)Affected males3 110 16 (14.5%) 38 (34.5%) 56 (50.9%) 7.176 0.028 0.033rs3764352 N G/G A/G A/A χ2 (df = 2) PComparison group 434 29 (6.7%) 152 (35.0%) 253 (58.3%)Affected males3 110 16 (14.5%) 38 (34.5%) 56 (50.9%) 7.408 0.025 0.0251P-values less than 0.05 are in bold and P-values which remain significant following false-discovery rate (FDR) corrections for multiple comparisons are underlined.2P-value≤ FDR threshold is significant.3One affected individual was randomly chosen from each family.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 6 of 13http://www.behavioralandbrainfunctions.com//8/1/19our previous findings with DRD1 [5]. DRD2 rs1800498genotypes and PPP1R1B rs1495099 genotypes each sig-nificantly contributed to prediction of ASDs in our fam-ilies (P= 0.0063 and P= 0.00086, respectively), as well aswhen weighted and tested together (P= 0.00011; Canon-ical R = 0.26) (Table 5). We generated a classificationmatrix to determine the percent correct classification ofindividuals with and without autism based on DRD2rs1800498 and PPP1R1B rs1495099 genotypes, andfound that 97% of individuals from our comparison co-hort and 13% of individuals with autism were correctlyclassified (the analysis was based on the a priori baselinefrequency of 70% of individuals without autism and 30%of individuals with autism). Using the weighted scores topredict group membership, we found that 72% of controlindividuals and 64% of affected individuals were pre-dicted correctly using DRD2 and PPP1R1B genotypes.However, while ~7% of the variance was explained withDRD2 rs1800498 and PPP1R1B rs1495099 genotypes,addition of our previously identified DRD1 rs265981–rs4532–rs686 haplotypes to the analyses did not result ina significant improvement in the overall canonical cor-relation (Canonical R = 0.27).Adding all possible two-way interactions betweenDRD2 rs1800498 and PPP1R1B rs1495099 genotypes, aswell as comparisons between DRD1 rs265981–rs4532–rs686 haplotypes and DRD2 rs1800498 genotypes, andDRD1 rs265981–rs4532–rs686 haplotypes and PPP1R1Brs1495099 genotypes, we found no evidence for gene-gene interactions (P= 0.35–0.75; data not shown).DiscussionOur model for the involvement of the DA pathway in de-termining some of the core deficits of ASDs is based onearlier results implicating the DBH gene as a maternaleffect locus, and on our hypothesis that autism suscepti-bility is determined by a combination of fetal susceptibil-ity genes and fetal gender as well as maternal effectsincluding maternal genetic factors [18]. Following ourfindings with the DRD1 gene [5], and as part of ourTable 3 FBAT of marker allele transmissions under an additive model at the DRD2 locus and FBAT of marker alleletransmissions under a recessive model at the PPP1R1B locus in affected sib-pair families1DRD2 # Observed Expected Z P2 FDRFam threshold3rs1799732Ins 31 90.0 84.0 1.4 0.16 0.030Del 31 38.0 44.0 −1.4 0.16rs1079597G 56 149.0 148.0 0.17 0.86 0.040A 56 71.0 72.0 −0.17 0.86rs1800498T 73 185.0 160.0 3.6 0.0003 0.010C 73 115.0 140.0 −3.6 0.0003rs1800497C 63 171.0 170.5 0.08 0.94 0.050T 63 89.0 89.5 −0.08 0.94PPP1R1B #FamObserved Expected Z P2 FDRthreshold3rs1495099G 52 42.0 43.8 −0.4 0.72 0.043C 34 39.0 26.3 3.3 0.00092 0.0071rs907094T 52 46.0 48.3 −0.5 0.66 0.036C 24 25.0 19.8 1.6 0.11 0.029rs3764352A 54 48.0 49.8 −0.3 0.73 0.050G 26 27.0 21.3 1.7 0.09 0.0211All affected individuals were included in the analyses.2P-values less than 0.05 are in bold and P-values which remain significant following false-discovery rate (FDR) corrections for multiple comparisons are underlined.3P-value≤ FDR threshold is significant.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 7 of 13http://www.behavioralandbrainfunctions.com//8/1/19investigation to determine whether other DA-relatedgenes are significant factors in the etiology of ASDs, wefound evidence for association of the DRD2 andPPP1R1B genes with autism in affected males from mul-tiple-incidence families.We found an increased frequency of the DRD2rs1800498 TT genotype (P= 0.007) in affected males(43.4%) compared to the comparison group (28.7%)(Table 2), and the rs1800498 T allele was over-transmit-ted to affected children (P= 0.0003) (Table 3). Thers1800498 risk allele was associated with more severeimpairments in social interaction (P= 0.0002), verbalcommunication (P= 0.0004) and stereotyped behaviours(P= 0.0021) in affected males (Table 4), and thers1800498 TT genotype was associated with an increasedrisk for ASD with an OR of 1.9 [95% CI: 1.5–2.5]. Inaddition, we examined three polymorphisms at thePPP1R1B locus and identified the rs1495099 C allele as arecessive risk allele for susceptibility to ASDs in male-only affected sib-pair families. The CC genotype fre-quency was increased in affected males (22.0%) relativeto the comparison group (9.9%, P= 0.002), and family-based association tests using FBAT with a recessivemodel showed distorted allele transmission with over-transmission of this allele in families (P= 0.00092)(Table 3). This allele was associated with greater impair-ments in social interaction (P= 0.0016) and nonverbalcommunication (P= 0.0046), and more severe stereo-typed behaviours (P= 0.00072), core features of ASDs. Fi-nally, the rs1495099 CC genotype was associated with anincreased risk for ASD with an OR of 2.6 [95% CI = 1.9–3.6]. All findings were significant following FDR-basedcorrections for multiple comparisons.Functional effects of DRD2 and PPP1R1B risk alleles ongene expressionOur findings at the DRD2 and PPP1R1B loci may reflectthe functional effects of unidentified risk variants in LDwith rs1800498 at DRD2 and rs1495099 at PPP1R1B.Functional analyses of these markers have not beenreported but in silico analyses performed using PupaSuite[available at http://pupasuite.bioinfo.cipf.es/] [48] did notidentify any putative functional role for these polymorph-isms while analyses using FASTSNP [available at http://fastsnp.ibms.sinica.edu.tw/pages/input_CandidateGen-eSearch.jsp] [49] predicted a ‘very low-to-low’ effect forrs1800498 at DRD2 as an intronic enhancer and a ‘verylow-to-medium’ effect for rs1495099 at PPP1R1B as aregulatory region/intronic enhancer. Meyer-Lindenberget al. [32] (2007) identified a common 7-marker PPP1R1Bhaplotype that was associated with increased DARPP-32mRNA expression and improved performance on mea-sures of working memory and cognitive flexibility. Thishaplotype included the T and A alleles of rs907094 andrs3764352 respectively, while a haplotype containing theminor alleles at these loci (i.e. rs907094 C and rs3754352G) was associated with decreased mRNA expression inpost-mortem brain. Houlihan et al. [50] (2009) screenedthis 7-marker haplotype to test PPP1R1B as a genetic de-terminant of cognitive ageing and found that rs907094 CTable 4 QTDT of rs1800498 alleles under an additive model at the DRD2 locus and QTDT of rs1495099 alleles under arecessive model at the PPP1R1B locus in affected sib-pair families1ADI-RSubdomainDRD2 # Observed Expected Z P2 FDRrs1800498 Fam threshold3Social T 56 2909.0 2452.5 3.7 0.0002 0.017Interaction C 56 1557.0 2013.5 −3.7 0.0002Verbal T 46 1364.0 1103.5 3.6 0.0004 0.033Communication C 46 666.0 926.5 −3.6 0.0004Stereotyped T 56 876.0 754.5 3.1 0.0021 0.050Behaviours C 56 502.0 623.5 −3.1 0.0021ADI-R Subdomain PPP1R1Brs1495099#FamObserved Expected Z P2 FDRthreshold3Social C 19 480.0 283.5 3.2 0.0016 0.017Interaction G 36 535.0 579.5 −0.5 0.59 0.042Nonverbal C 10 108.0 52.3 2.8 0.0046 0.025Communication G 20 130.0 123.3 0.2 0.82 0.050Stereotyped C 19 142.0 79.8 3.4 0.00072 0.0083Behaviours G 36 176.0 200.3 −0.9 0.38 0.0331All affected males were included for QTDT analyses.2P-values less than 0.05 are in bold and P-values which remain significant following false-discovery rate (FDR) corrections for multiple comparisons are underlined.3P-value≤ FDR threshold is significant.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 8 of 13http://www.behavioralandbrainfunctions.com//8/1/19and rs3754352 G alleles were associated with decreasedcognitive ability. However, our findings do not support anassociation of either rs907094 C or rs3764352 G with aut-ism in our family cohort. Unfortunately, becausers1495099 was not included in these studies, its functionalrole is not known. With respect to the DRD2 locus, thers1800498 polymorphism was found to be in low LD withrs1799732, a functional variant in the DRD2 promoter[51], in our comparison group and parents from families(Figure 1). To investigate whether alleles from rs1799732are contributing as a risk factor for autism susceptibility inour families, comparisons between family-based tests ofrs1800498 and rs1799732 alleles considered separately(P=0.0003 and P=0.16, respectively), and haplotypes con-taining alleles from both rs1800498 and rs1799732,showed that the observed over-transmission in families isderived from rs1800498, and not because of the rs1799732polymorphism (data not shown). However, only 31 familieswere informative for rs1799732 compared to 73 familiesfor rs1800498, so we cannot determine from these findingswhether alleles from the functional variant rs1799732 arecontributing to autism susceptibility in our families. An-other genetic variant at the DRD2 locus, rs1076560GT, hasbeen associated with altered mRNA isoform expression[52,53] and differences in striatal post-synaptic D2 recep-tor abundance [54]. Bertolini et al. [53] (2009) found incontrol and schizophrenia cohorts (N=114 and N=91, re-spectively) that individuals heterozygous for the minor “T”allele perform worse in the N-back test of working mem-ory but only at a high level of difficulty (2-back) comparedto individuals homozygous for the major “G” allele whileZhang et al. [52] (2007) found using healthy subjects(N=117) that heterozygous individuals performed worseat higher attentional loads in the variable attentional con-trol (VAC) task, and had increased activity as measuredusing BOLD fMRI in PFC and striatum compared to indi-viduals homozygous for the major allele. However, the truecontribution of this variant to DAergic function and cogni-tion is unclear as no genotype effects of this polymorphismto overall working memory performance and fMRI activitywere also reported [52,54]. Nevertheless, both rs1076560and rs1079597 are in high LD in the HapMap CEU panel(r2> 0.9) and are found in the same 20 kb haplotype blockas rs1800498. However, no information is available regard-ing LD between rs1076560 and rs1800498. It is of interestthat low LD (r2< 0.3) was found between rs1800498 andrs1079597 in our comparison group and parents fromfamilies (Figure 1). Additional families informative at theseDRD2 loci are needed to determine whether these func-tional variants or another functional polymorphism in LDwith rs1800498 is responsible for the increased risk forautism. In addition, functional analyses and resequencingof both genes in affected individuals with this risk haplo-type at DRD2 or the risk allele at PPP1R1B are required.Pathophysiological contributions of DRD2 and PPP1R1B torisk for autismThe QTDT results support an association of the DRD2and PPP1R1B loci with autism (Table 4). A role for theDRD2 gene in autism susceptibility is suggested by thefact that antipsychotic medications, which prevent dopa-mine D2 receptor activation, improve the core symptomsof ASDs [55]. Postsynaptic D2 receptors and presynapticD2 autoreceptors are involved in the DAergic modula-tion of cognitive and emotional processes that areimpaired in individuals with autism [56,57]. Thus, func-tional polymorphisms which affect receptor availability(e.g. altered gene expression), either postsynaptically onDAceptive neurons or presynaptically on DAergic neu-rons, may contribute to the impairments found in indivi-duals with autism.DARPP-32 mediates the downstream effects of dopa-mine receptor activation, and thus plays an importantrole in the modulation of DA-related processes whichare abnormal in individuals with autism. Unlike dopa-mine receptors, which can be studied using systemic orlocal administration of ligands, DARPP-32 is found inthe cytoplasm of DAceptive neurons and there are fewstudies which have examined its role in DA-modulatedprocesses and behaviours. One such study by Hotte et al.[58] (2006) found that administration of D1 receptorantagonists in mice caused deficits in working memorywhich coincided with decreased levels of phosphory-lated-DARPP-32 in the PFC. Deficiencies in workingTable 5 General discriminant analysis of DRD2 rs1800498 and PPP1R1B rs1495099 genotypes towards prediction ofASDs in affected males1Eigenvalue Canonical Wilk’s χ2 df P3 FDRR Lambda F2 threshold4DRD2 - PPP1R1B 0.07 0.26 0.94 23.27 4 0.00011 0.017DRD2 0.97 5.14 343 0.0063 0.050PPP1R1B 0.96 7.20 343 0.00086 0.0331One affected individual was randomly chosen from each family.2Chi-statistic reported for additive test of DRD2 and PPP1R1B and F statistic reported for single gene tests.3P-values less than 0.05 are in bold and P-values which remain significant following false-discovery rate (FDR) corrections for multiple comparisons are underlined.4P-value≤ FDR threshold is significant.Hettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 9 of 13http://www.behavioralandbrainfunctions.com//8/1/19memory [59] and impairments in reversal learning [10]are found in individuals with autism. Both Drd2 −/−mice and Ppp1r1b −/− mice exhibit impairments in re-versal learning compared to wild-type mice [31,60].The role of DARPP-32 in mediating the DA-relatedchanges to neuronal excitability necessary for memory andlearning was shown in a study by Calabresi et al. [61](2000). These authors were unable to induce long-term po-tentiation (LTP) and long-term depression (LTD), twoforms of synaptic plasticity, in the striatum of Ppp1r1b −/−mice. DA has a role in synaptic plasticity in both the stri-atum [62] and amygdala [63], subcortical structures thatare important for regulating emotional behaviours [64] andsocial interactions [65], and are implicated in the patho-physiology of repetitive behaviours [66].Effects between dopamine-related genes and risk for ASDBased on our findings in this study and our previousfindings [5], single-gene analyses showed evidence forassociation of DRD1, DRD2 and PPP1R1B with autism inmale-only affected sib-pair families. We performed gen-eral discriminant analysis using Statistica v9.1 to predictASD susceptibility in affected males, and to test forevidence of gene-gene interactions of DA-related genesand ASDs. We found that DRD2 rs1800498 genotypesand PPP1R1B rs1495099 genotypes significantly contrib-uted to prediction of ASDs in our families when testedseparately and together (P= 0.0063–0.00011), whichaccounted for ~7% of the variance. These results weresignificant following corrections for multiple compari-sons (Table 5). We also found that 97% of individualsfrom our comparison cohort and 13% of individuals withautism were correctly classified while 72% of controlindividuals and 64% of affected individuals were pre-dicted correctly using a weighted additive combinationof DRD2 and PPP1R1B genotypes. The inclusion ofDRD1 rs265981–rs4532–rs686 haplotypes into the ana-lysis did not significantly change our findings (data notshown), and we found no evidence for gene-gene inter-actions (data not shown).Our findings suggest that single genes individually con-fer risk of autism susceptibility in our families and, al-though there was no evidence for gene-gene interactionsbetween DA-related genes and autism susceptibility perse, we did find evidence that DRD2 and PPP1R1B to-gether significantly contribute to ASD prediction. Thus,while both genes independently confer risk of autismsusceptibility, there is a cumulative effect towards pre-dicting whether an individual has the condition. Further-more, based on our findings from GDA analysis wefound an effect size of ~7%. While this effect size issmall, it is comparable to those reported in similar stud-ies [4]. These generally small effect sizes likely reflect thehighly heterogeneous nature of ASDs.Parent-of-origin effects of DRD1 and DRD2 alleles ondevelopmentThe increased transmission of the DRD1 rs265981 C–rs4532 A –rs686 T (C-A-T) haplotype from mothers(P= 0.029) [5], and of the DRD2 rs1800498 T allele fromfathers (P= 0.023), to affected sons suggests that imprint-ing effects of these genes are also important risk factorsfor ASDs. A role for imprinting has been proposed forseveral brain-related disorders, including autism [67].There is evidence of imprinting of genes from neuro-transmitter pathways implicated in ASDs such as the 5-hydroxytryptamine receptor 2A (HTR2A) gene in theserotoninergic pathway [68].The dopamine D1 and D2 receptors are expressed inhuman placentae [69,70] and fetal brains [71,72]. PlacentalD1 and D2 receptors are involved in DA-mediated releaseof opioids [73] and lactogen [74] respectively, which arerequired for fetal development and growth . Both dopamineD1 and D2 receptors are involved in brain development.Dopamine D2 receptors expressed in fetal brain induceneurite outgrowth and axon elongation while dopamine D1receptors inhibit neurite outgrowth in cortical neuron dif-ferentiation [75], with the opposite effects found in striatalneuron differentiation [76]. There is no evidence for thekind of parent-of-origin-specific DNA methylation at theDRD1 or DRD2 loci in either human placentae or fetalbrains that is suggestive of imprinting [77]. Further, a reviewof the ‘imprinted gene and parent-of-origin effect’ database[available at http://igc.otago.ac.nz/home.html] [78] did notyield any evidence supporting imprinting at either locus.The possibility remains, however, that imprinting of thesegenes may occur during a very narrow developmentalperiod or in a specific subpopulation of brain cells, as hasbeen demonstrated for the Ube3a gene in mice [79]. Itshould be noted that the evidence presented for parent-of-origin effects in ASDs is based on three markers in theDRD1 gene and one marker in the DRD2 gene and thus,more polymorphisms need to be studied to confirm ourfindings.ConclusionsOur results strongly support a role for the DRD2 andPPP1R1B genes in susceptibility to autism spectrum beha-viours in males from affected sib-pair families in whichthere are only affected males, and especially those maleswhere there are severe impairments in social interaction(DRD2 and PPP1R1B), verbal communication (DRD2),nonverbal communication (PPP1R1B) and stereotypedbehaviours (DRD2 and PPP1R1B). Our results also supportan additive effect of the DRD2 and PPP1R1B genes in pre-dicting ASDs in our families. However, we recognize thatlarge genome-wide association studies have not implicatedthese genes in autism susceptibility, but those studies didnot analyse male-only affected sib-pair families separatelyHettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 10 of 13http://www.behavioralandbrainfunctions.com//8/1/19from other families, and there is no information about thelevel of impairments in the core characteristics of ASDs.Further studies are needed using additional similar familycohorts and sequencing of the DRD2 and PPP1R1B genesin individuals with the risk alleles to identify functionalpolymorphisms that could be associated with the clinicalfindings in order to further develop our model for an asso-ciation of DA-related gene function and susceptibility toASD behaviours.Competing interestsThe authors declare they have no competing interests.Authors’ contributionsJJAH and JAH conceived and designed the study. JAH was a graduatestudent and this study is part of his PhD research. JAH and XL carried outthe molecular analyses. MLH and AL manage the study subjects andphenotype data. ILC, RCM, CES, and MESL participated in the evaluation ofthe subjects. JAH, XL, and ILC performed the data analyses. JAH wrote theinitial draft of the manuscript and all authors read and participated in thewriting and approval of the final manuscript.AcknowledgmentsThis work was supported by research grants from the Ontario Mental HealthFoundation (OMHF; JJAH, PI), the Canadian Institutes for Health Research(CIHR; #43820, JJAH, PI) and the Canada Foundation for Innovation (#7939) tothe Autism Spectrum Disorders Canadian-American Research Consortium(ASD-CARC) (JJAH, PI; www.asdcarc.com; www.AutismResearch.ca) andfunded in part by a grant from the South Carolina Department of Disabilitiesand Special Needs (SCDDSN)(CES). This work was also supported by aresearch studentship from the OMHF to JAH. JAH was a trainee with theCIHR/Autism Speaks STIHR Interdisciplinary Inter-Institute Autism SpectrumDisorders Training Program (PI: JJAH) (www.AutismTraining.ca). MESL is aCareer Scholar supported by the Michael Smith Foundation for HealthResearch. The authors are very grateful to all the families who participated inthis research and we gratefully acknowledge the resources provided by theAutism Genetic Resource Exchange (AGRE) Consortium and the participatingAGRE families. AGRE is a program of Cure Autism Now and is supported, inpart, by grant MH64547 from the National Institute of Mental Health toDaniel H. Geschwind (PI).Author details1Department of Physiology, Queen’s University, Kingston, ON, Canada.2Queen’s Genetics and Genomics Lab at Ongwanada, Ongwanada ResourceCentre, Kingston, ON, Canada. 3Department of Psychiatry, Queen’s University,Kingston, ON, Canada. 4Autism Spectrum Disorders – Canadian-AmericanResearch Consortium, Kingston, ON, Canada. 5Department of Psychology andGeorge A. Jervis Clinic, New York State Institute for Basic Research inDevelopmental Disabilities, Staten Island, NY, USA. 6Department of Biology,Western Carolina University, Cullowhee, North Carolina, USA. 7Center forMolecular Studies, Greenwood Genetic Center, Greenwood, South Carolina,USA. 8Department of Medical Genetics, University of British Columbia,Vancouver, BC, Canada. 9B.C. Child and Family Research Institute, Vancouver,BC, Canada. 10Centre for Neuroscience Studies, Queen’s University, Kingston,ON, Canada. 11Autism Research Program/Genetics and Genomics ResearchLaboratory, Ongwanada Resource Centre, 191 Portsmouth Ave, Kingston, ON,Canada K7M 8A6.Received: 13 June 2011 Accepted: 25 February 2012Published: 4 May 2012References1. Bacchelli E, Maestrini E: Autism spectrum disorders: molecular geneticadvances. Am J Med Genet C Semin Med Genet 2006, 142C:13–23.2. 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HumMol Genet 2003, 12:837–847.doi:10.1186/1744-9081-8-19Cite this article as: Hettinger et al.: DRD2 and PPP1R1B (DARPP-32)polymorphisms independently confer increased risk for autismspectrum disorders and additively predict affected status in male-onlyaffected sib-pair families. Behavioral and Brain Functions 2012 8:19.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitHettinger et al. Behavioral and Brain Functions 2012, 8:19 Page 13 of 13http://www.behavioralandbrainfunctions.com//8/1/19


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