Dopamine D4 Receptor and Tyrosine Hydroxylase Genes in Bipolar Disorder

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Molecular Psychiatry (2002) 7, 860–866  2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Dopamine D4 receptor and tyrosine hydroxylase genes in bipolar disorder: evidence for a role of DRD4 P Muglia1, A Petronis1, E Mundo1, S Lander2, T Cate1 and JL Kennedy1

1Neurogenetics Section, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Toronto, Canada; 2Health Sciences Centre, Room PZ202, University of Manitoba, Winnipeg, Manitoba, Canada

The involvement of the mesocorticolimbic dopamine system in behaviors that are compro- mised in patients with mood disorder has led to the investigation of dopamine system genes as candidates for bipolar disorder. In particular, the functional VNTRs in the exon III of the dopamine D4 (DRD4) and in intron I of the tyrosine hydroxylase (TH) genes have been investigated in numerous association studies that have produced contrasting results. Likewise, linkage studies in multiplex bipolar families have shown both positive and negative results for markers in close proximity to DRD4 and TH on 11p15.5. We performed a linkage disequilibrium analysis of the DRD4 and TH VNTRs in a sample of 145 nuclear families comprised of DSM- IV bipolar probands and their biological parents. An excess of transmissions and non transmissions was observed for the DRD4 4- and 2-repeat alleles respectively. The biased transmission showed a parent of origin effect (POE) since it was derived almost exclusively from the maternal meiosis (4-repeat allele maternally transmitted 40 times vs 20 times non-transmitted; ␹2 = 6.667; df = 1; P = 0.009; while paternally transmitted 26 times vs 21 times non- transmitted; ␹2 = 0.531; df = 1; P = 0.46). The analysis of TH did not reveal biased transmission of intron I VNTR alleles. Although replication of our study is necessary, the fact that DRD4 exhibit POE and is located on 11p15.5, in close proximity to a cluster of imprinted genes, suggests that genomic imprinting may be operating in bipolar disorder. Molecular Psychiatry (2002) 7, 860–866. doi:10.1038/sj.mp.4001098 Keywords: genetics; psychiatry; complex disease; chromosome 11; parent of origin effect; genomic imprinting

Introduction logical and behavioral studies in humans continues to support a dopaminergic role in the etiology of bipolar Family, twin and adoption studies demonstrated evi- disorder.15 The short arm of chromosome 11 on p15.5 dence for the importance of genetic factors in bipolar harbors two dopamine system genes, ie the dopamine disorder.1 The mode of inheritance does not follow D4 receptor (DRD4) and the tyrosine hydroxylase (TH) Mendelian rules and multiple genes2 with complex genes, and these genes have been extensively investi- gene–gene3 and gene–environment interactions are gated in bipolar disorder.16–25 likely to be involved. The gene for the dopamine D4 receptor is highly Different linkage studies have investigated the short polymorphic containing a functional variable number arm of chromosome 11 in bipolar families since the of tandem repeats (VNTR) in the third exon that enco- ﬁrst evidence for linkage in an Old Order Amish kin- des for the longest intracellular loop of the recep- dred.4 This ﬁnding however was not replicated in the tor.26,27 The polymorphism consists of 2–10 imperfect reanalysis of the families, after the disease occurred in tandem repeats of 48 bp, each repeat encoding for 16 some individuals who were unaffected at the time of amino acids. Nineteen different repeat sequences have the primary analysis.5 Further linkage studies of 11p been described that lead to 25 different haplotypes in several data sets of bipolar families have failed to coding for 18 different predicted amino acid show consistent results.6–14 sequences.28 In vitro studies have found different phar- Preliminary evidence for the involvement of dopa- macological properties for the D4 receptors coded by mine pathways in bipolar disorder arose from animal the 2, 4, 7 and 10 repeat alleles.26,29 Tyrosine studies, and more recently evidence from pharmaco- hydroxylase converts tyrosine to DOPA and is the rate- limiting enzyme in the synthesis of catecholamines. Its gene contains a microsatellite in Intron I constituted by

Correspondence: Dr JL Kennedy, Neurogenetics Section, Clarke a tetranucleotide repeat of a (TCAT)n motif Site, Centre for Addiction and Mental Health, Department of Psy- (HUMTH01) that can be repeated five to 10 times.30 The chiatry, University of Toronto, 250 College Street R-30, Toronto, Ontario, Canada M5T 1R8. E-mail: James KennedyȰcamh.net 10 repeat alleles can be present in two different 31 Received 3 December 2001; revised 14 January 2002; accepted 31 sequence variants. A functional in vitro study of the January 2002 tyrosine hydroxylase gene has suggested that the DRD4 and TH in bipolar disorder P Muglia et al 861 microsatellite HUMTH01 may be implicated in the subjects, written informed consent was obtained from regulation of the gene expression.32 each patient and their respective parents who agreed The inconsistencies shown across the linkage studies to participate in the study. The diagnoses were deter- that investigated the short arm of chromosome 11 in mined after assembling available medical records and bipolar disorder do not allow us to exclude the the Structured Clinical Interview for DSM-IV, Axis I involvement of genes with a small contribution to the Disorders.38 The family interview for genetic study disease (eg DRD4 and TH) because of the low power of (FIGS) was part of the assessment of the families and linkage analysis to detect genes with partial contri- it was administered to the probands and to their bio- bution to disease.33 The majority of the association logical relatives. The interviews were conducted by an studies that have investigated the role of DRD4 in experienced clinical research assistant. The diagnosis bipolar disorder did not show evidence for associ- was based on consensus of at least two psychiatrists ation.18–22,25 However, a large sample of bipolar dis- (JLK, EM). order patients has shown association with delusional symptoms in bipolar patients,23 although the grouping Laboratory methods of the DRD4 exon III alleles obscured the effect of the Blood samples were drawn from patients and relatives single alleles. In a similar fashion, the association stud- and DNA samples were extracted from whole blood ies of HUMTH01 in bipolar disorder produced both following standard high salt procedures. The 48-base positive and negative association.14,17 However, pair VNTR region in the third exon of DRD4 was ampli- because DRD4 and TH association studies have mainly fied using PCR techniques with primers and conditions adopted case-control study designs and because of the previously published.28 The PCR products containing limited use of family-based strategy in small sample the DRD4 48-bp repeat were visualized using gel sizes,16,24 the involvement of DRD4 and TH in bipolar electrophoresis performed in 3.5% agarose prepared disorder remains a question. The case-control with ethidium bromide and 1 × TBE (Tris, boric acid, approach, in fact, is associated with the risk of produc- EDTA). We used an ABI Prism 310 (Applied Biosys- ing spurious results because of the difference in allele tems, Foster City, CA, USA) for the fragment analysis frequencies across different populations34 and this has of the TH tetranucleotide VNTR using a PCR product been shown for DRD4 exon III VNTR and HUMTH01 obtained with 5Ј fluorescently labeled primer with alleles.31,35 The family-based association strategy has sequence previously published.39 The TH genotypes the advantage of having a lower chance of false posi- were assigned using GENOTYPER 2.5 software that tives (type I errors) due to population stratification,36 compared allele size with the size standard TAMRA has good power to detect genes with partial effect when (Applied Biosystems, Warrington, UK). The subjects compared to linkage strategies, and allows the separate were genotyped blind to their affection status and fam- analysis of maternally and paternally transmitted ily structure. The eighteen 2-repeat alleles of the DRD4 alleles. The separation of the data into analysis of 48-bp VNTR heterozygous proband mothers that were maternal and paternal meioses may help in dis- informative for the TDT analysis were sequenced. To tinguishing true association from false positive.37 In the increase the fidelity of the PCR product for sequencing, present study we performed a family-based association the PCRs were performed using the same conditions study of the DRD4 48-bp repeat VNTR and the TH used for the genotyping assay, however instead of Taq intron I tetranucleotide repeat VNTR in a sample com- polymerase, native Pfu polymerase (Stratagene) was prised of subjects suffering from DSM-IV bipolar dis- used.40 Each of the 2-repeat alleles was sequenced order and their biological parents. twice and the sequences were compared to the available published sequences for the 48-bp DRD4 exon III VNTR.28 Methods Subjects Statistical methods The sample consisted of 154 patients with a DSM-IV The PEDMANAGER program software (v0.9) was used diagnosis of bipolar disorder (104 Bipolar I; 40 Bipolar to check genotypes according to the family structure in II; eight Schizoaffective bipolar type disorder; two order to exclude the presence of parent-proband Bipolar Disorder Not Otherwise Specified) and their incompatibility and to calculate the allele frequencies. biological relatives that make up 145 nuclear families. We tested our nuclear families for the presence of devi- Nine of the families have two affected probands. The ation from equal transmission of alleles from hetero- probands were 103 (66.9%) females and 51 males zygous parents using the Transmission Disequilibrium (33.1%) and were predominantly (97%) of Caucasian Test for mutiallelic polymorphisms as implemented by origin with a mean age of 36.25 (SD =±9.48, range 13– Sham and Curtis41 in the ETDT package (E-TDT, v1.8). 62 years) and with a mean age of onset of the disorder This approach tests the null hypothesis of no associ- of 18.54 (SD =±7.08; range 5–40 years). The patients ation by the classical McNemar test statistic for trans- were recruited from several psychiatric care centers in mitted vs non-transmitted parental alleles. We Toronto and across central Canada. The study was searched for evidence for biased transmission of the approved by the University of Toronto and The Centre alleles using the allele-wise and genotype-wise analy- for Addiction and Mental Health Research Ethics sis and thereafter we analyzed the transmission for Board. After a complete description of the study to the each allele individually. The alleles with a frequency

Molecular Psychiatry DRD4 and TH in bipolar disorder P Muglia et al 862 lower than 3% (alleles 179 and 199 for HUMTH01 and the HUMTH01 and DRD4 VNTR alleles are indicated the 3-, 5- and 6 repeat DRD4 alleles) were not con- in Table 1. The analysis for HUMTH01 did not reveal sidered in the analysis for individual alleles because preferential transmission for any of the alleles nor for of the inadequate power. Furthermore, a parent-sex- maternal or paternal preferential transmission. TDT speciﬁc analysis of the transmission of the DRD4 and analysis for the DRD4 VNTR revealed biased trans- HUMTH01 common alleles was conducted using the mission of the 4- and 2-repeat alleles. Despite the fact command for parental sex-speciﬁc analysis in the that 40 parents had the 2-repeat allele, it was trans- ETDT package. In addition to the TDT analysis, the mitted only 12 times to affected offspring (␹2 for geno- Haplotype Relative Risk (HRR) statistic42 was applied. type-wise = 14.84; df = 8; P = 0.062; ␹2 for allele-wise This is a standard chi-squared statistic that, unlike the = 10.32; df = 5; P = 0.066). The results from the trans- TDT, includes all parental genotypes, both homo- and missions for individual alleles of DRD4 showed an heterozygous. For the homozygous parents the same excess of 2-repeat allele non-transmission to the affec- allele is scored both in the transmitted as well as the ted offspring (12 transmitted vs 28 non-transmitted, ␹2 non-transmitted category. Because the variance esti- = 6.40; df = 1; P = 0.0114). On the other hand the analy- mate for HRR uses more information than the sis of the 4-repeat alleles revealed an excess of trans- TDT/McNemar statistic, HRR can lead to a more mission for this allele (77 transmitted vs 52 non-trans- powerful statistical test for association; although, mitted, ␹2 = 4.845 df = 1; P = 0.0278). The separate under certain conditions, HRR may be less powerful analysis of the individual alleles from maternal and than the TDT/McNemar test.43 Furthermore, because paternal meioses revealed that the biased transmission HRR uses all the transmitted and non-transmitted for the 2- and 4-repeat alleles were derived mostly from alleles, the HRR contingency two by two tables maternal meioses. In fact, the 2-repeat allele was (obtained computing the transmission and non trans- maternally transmitted only twice while not transmission of the alleles) allows calculation of the odds mitted 16 times (␹2 = 10.889; df = 1; P = 0.0010), and ratios for the ‘risk’ and ‘non-risk’ alleles. The polymor- the 4-repeat was maternally transmitted 40 times and phisms we studied were multiallelic and therefore the non transmitted 20 times (␹2 = 6.667; df = 1; P = 0.009). two by two contingency tables that were used for the The same alleles showed a non biased transmission HRR analyses were obtained considering in one col- when the paternal meioses were considered (see Table umn, the transmissions and non transmissions of the 1). In Tables 3, 4 and 5 the contingency tables with the ‘risk’ or ‘non-risk’ allele (as obtained through the ETDT transmitted and non-transmitted DRD4 alleles used for analysis), and the transmissions and non transmission the HRR analysis are shown and in Table 2 the odds count for all the remaining alleles were grouped into ratios (OR) derived from the HRR analyses are summar- the other column. The alleles that the TDT analyses ized. The odds ratios indicate that the 4-repeat alleles showed to have biased transmission were considered increase the risk for bipolar disorder (OR = 1.7; CI in the HRR tables. The odds ratio (that is approxi- (95%) = 1.18–2.46; P = 0.004), while the non trans- mately equal to the relative risk) provides an esti- mission of the 2-repeat allele is associated with a pro- mation of the risk associated with the transmission of tective effect (OR = 0.44; CI (95%) = 0.22–0.86; P = a given allele. 0.015). The analyses of the maternal and paternal transmissions clearly indicate that the risk associated with the 4-repeat alleles is derived almost exclusively from Results the maternal meiosis (OR = 2.34; CI (95%) = 1.29–4.26; The results of the transmission of the individual alleles P = 0.004) and not from the paternal side (OR = 1.25; from heterozygous parents to the affected offspring of CI (95%) = 0.69–2.27; P = 0.5). In a similar way, the

Table 1 Transmitted (T) and non-transmitted (N-T) alleles

TH tetranucleotide VNTR alleles DRD4 48-bp repeat VNTR alleles

Maternal Paternal meiosis meiosis

A2 A3 A4 A5 A6 RA A2a A4b A7 RA A2c A4d A2 A4

T 38 27 21 21 39 1 12 77 40 11 2 40 8 26 N-T 32 30 17 28 42 0 28 52 49 11 16 20 10 21

The symbols for TH: A2, A3, A4, A5, A6 represent Alleles 183, 187, 191, 195 and 198 and RA includes the rare allele 179 and 199. For the DRD4 the symbols A2, A4, and A7 represent 48-bp repeat polymorphism of 2, 4, and 7 repeats respectively. The Rare Alleles (RA) group consists of 3, 5 and 6 repeats for DRD4. ETDT for individual alleles: a␹2 = 6.4, df = 1; P = 0.0114; b␹2 = 4.845, df = 1; P = 0.0278; c␹2 = 10.889; df = 1; P = 0.0010; d␹2 = 6.667; df = 1; P = 0.0099.

Molecular Psychiatry DRD4 and TH in bipolar disorder P Muglia et al 863 Table 2 The odds ratios as determined by the Haplotype Table 5 Haplotype Relative Risk of DRD4 paternal alleles Relative Risk analyses for DRD4 48-bp 2- and 4-repeat alleles from the total sample and as derived by the maternal and 4-R Non-4R paternal sides, are illustrated Transmitted 56 35 Alleles Odds 95% Conﬁdence Two-sided Non-Transmitted 51 40 ratio interval P ␹2 = 0.57; 1 df; P = 0.45. 4R vs Non 4R 1.70 1.18–2.46 0.004 Odds ratio = 1.25; CI (95%) = 0.69–2.27; P = 0.54. 2R vs Non 2R 0.44 0.22–0.86 0.015

Maternal meioses 2-R Non-2R 4R vs Non 4R 2.34 1.29–4.26 0.004 2R vs Non 2R 0.15 0.04–0.52 0.0001 Transmitted 8 83 Paternal meioses Non-Transmitted 10 81 4R vs Non 4R 1.25 0.69–2.27 0.54 2R vs Non 2R 0.78 0.29–2.08 0.45 ␹2 = 0.25; 1 df; P = 0.61. Odds ratio = 0.78; CI (95%) = 0.29–2.08; P = 0.45.

reduced transmission for the 2-repeat allele, that may Table 3 Haplotype Relative Risk of the DRD4 2- and 4- play a protective role, is exclusively of maternal origin repeat alleles (OR = 0.15; CI (95%) = 0.04–0.52, P = 0.0001) and not of paternal origin (OR = 0.78, CI (95%) = 0.29–2.08, P 4-R Non-4R = 0.4). Because multiple testing was performed, an Transmitted 190 71 increased number of type I errors should be con- Non-Transmitted 165 105 sidered. However, because the methods that appropri- ately correct for non-independent multiple testing are ␹2 = 8.18; 1 df; P = 0.0042. Odds ratio = 1.7; CI (95%) = 1.18–2.46; P = 0.004. controversial for genetic studies of complex diseases, we presented our data with non-corrected P values. Nevertheless, it is notable that the results from the par- 2-R Non-2R ent-sex-specific analysis of the DRD4 2-repeat allele maternal transmission continued to be significant (P = Transmitted 13 248 0.012) after Bonferroni correction for twelve multiple Non-Transmitted 29 241 tests (ie eight tests to individually analyze the transmission of the TH and DRD4 VNTR alleles plus four ␹2 = = 6.04; 1 df; P 0.013. tests conducted to explore the maternal and paternal = = = Odds ratio 0.44; CI (95%) 0.22–0.86; P 0.015. meiosis independently for each of the two loci). Because of the occurrence of multiple sequences within the 48-bp repeat of the DRD4 exon III28 we Table 4 Haplotype Relative Risk of DRD4 maternal alleles tested for a sequence-specific effect by DNA sequencing the DRD4 2-repeat alleles that were informative 4-R Non-4R from the maternal meioses. No sequence variants beyond the one published were detected. Transmitted 65 29 The Family Interview for Genetic Studies revealed Non-Transmitted 45 47 the presence of family history for bipolar disorder in 54 of the 145 families. In 25 families the history for ␹2 = 7.88; 1 df; P = 0.005. bipolar disorder was from the maternal side, in 23 from Odds ratio = 2.34; CI (95%) = 1.29–4.26; P = 0.004. the paternal, while in six families the disorder was present on both the maternal and paternal sides. The transmissions of the DRD4 VNTR alleles in the families 2-R Non-2R with paternal and maternal lineage for bipolar disorder revealed a similar trend to that observed in the overall Transmitted 3 91 Non-Transmitted 17 75 sample. The maternal meiosis of the families with a maternal history for bipolar disorder showed an effect, ␹2 = 11.32; 1 df; P = 0.0007. with the 4-repeat allele transmitted 10 times vs two ␹2 = = = Odds ratio = 0.15; CI (95%) = 0.04–0.52; P = 0.0001. non-transmissions ( 5.333; df 1; P 0.02). The OR indicate to what extent the 2-repeat allele is protective. analysis of the transmission of DRD4 alleles stratified If we consider the risk given by the transmission of the Non- according to bipolar disorder subtypes continues also 2-repeat alleles: OR = 6.88; CI (95%) = 1.94–24.36; P = 0.0001. to show the same trend of transmissions as in the prin- cipal analysis with the same POE. For example in the

Molecular Psychiatry DRD4 and TH in bipolar disorder P Muglia et al 864 bipolar type I families for the DRD4 4-repeat allele we kers on the Xq26–28 are still showing inconsistent observed 25 transmissions vs 13 non transmissions, results, a gene with a small contribution for bipolar dis- and in the bipolar type II we observed 15 transmissions order cannot be ruled out. vs 7 non transmissions. Genomic imprinting defines an epigenetic phenomenon where gene expression is regulated depending on the parental origin of the gene.49,50 Genomic imprinting Discussion has been found to be involved in the etiology of neur- A significant departure from the expected 50:50 ratio opsychiatric disorders such as Prader–Willi and Angel- was observed for the transmission of the 2- and 4- man syndromes51 and its contribution has been postu- repeat DRD4 alleles in our sample of bipolar families. lated for several psychiatric diseases including bipolar The biased transmission of the 2- and 4-repeat alleles disorder.37,49,52 Linkage studies in bipolar disorder fam- showed a parent of origin effect (POE). The excess ilies have shown evidence for linkage for chromosome transmission of the 4-repeat alleles as well as the 18 markers and some of these markers have shown a increased number of non-transmissions of the 2-repeat POE.53,54 These studies have indicated a paternal spe- allele were derived almost exclusively from the cific effect for two markers (D18S41 and D18S541) on maternal meioses. This result suggests that the 4-repeat 18q2153,54 and a maternal specific effect for another allele may increase the risk for bipolar disorder while marker (D18S464) on 18p.53 However, other studies the 2-repeat may confer a protective role when non were unable to replicate these findings making con- transmitted from the maternal side. The tyrosine clusions elusive.55,56 hydroxylase HUMTH01 alleles conversely did not Interestingly, DRD4 and TH are located on the telo- show biased transmission from the heterozygous par- meric region of the short arm of chromosome 11 close ents to the affected offspring and did not show a POE. to a cluster of imprinted genes. DRD4 is approximately The majority of the published association studies17 that 200 kb telomeric to H19, an imprinted gene located have investigated HUMTH01 alleles in bipolar famil- close to DRD4.57 TH is 9 kb centromeric to the ies, similarly to our results, have failed to detect a imprinted cluster on 11p15.5 that contains the major effect of the HUMTH01 alleles. imprinted insulin-like growth factor 2 (IGF2) gene and Several linkage analyses,6–13 unrelated case-con- the insulin gene (INS).58 The expression of DRD4 was trol18–22,25 and a family-based association study16 have tested in the temporal cortex of two brains from investigated the role of DRD4 exon III VNTR in bipolar patients who underwent a neurosurgery intervention.59 disorder and none of them has shown an effect similar The study showed expression of both maternal and to that we observed in the present study. The discrep- paternal alleles of DRD4. The expression of both DRD4 ancy of the results between previous studies and our alleles in this study however does not exclude the pres- finding may be due to the presence of genetic hetero- ence of imprinting for DRD4. In fact, imprinting can be geneity and/or to the different genetic structure of the a developmental stage- and tissue-specific phenom- populations studied. To our knowledge, the only fam- enon60 as well as polymorphic, ie its presence can vary ily-based association study of DRD4 and bipolar dis- from subject to subject as described for brain expressed order present in the literature was conducted in a small imprinted genes.61 Because of these complexities, gen- sample in a partially isolated population from Sardi- omic imprinting is a multifaceted phenomenon nia16 and POE was not investigated. described only recently in mammals,62,63 with function Several lines of evidence from clinical,44,45 and mol- and mechanism not fully understood.64 ecular genetic studies46 have suggested a POE in the The POE that we observed for DRD4 alleles, together transmission of bipolar disorder. However, the clinical with its chromosomal location close to an imprinted studies that investigated for POE in bipolar disorder gene cluster, suggests that DRD4 may be imprinted in were not consistent. Grigoroiu-Serbanescu and col- humans. Replication in independent samples of leagues44 showed that bipolar patients with an affected bipolar families, that will analyze separately paternal father had earlier age of onset when compared with the and maternal transmission of DRD4 alleles, is war- onset of individuals who had a mother affected. Other ranted. However, further studies will have to consider studies have suggested a prevalent maternal trans- the difficulties encountered in the investigation of mission.45,46 The presence of POE in the transmission imprinted risk alleles for other complex diseases. An of bipolar disorder may be determined by the localiz- example is represented in the difficulties found in the ation of the risk locus in mitochondrial DNA, or on the analyses of the imprinted locus containing the insulin X chromosome, or by the presence of genomic gene (INS) VNTR in diabetes. The INS VNTR consists imprinting. The sequencing of the mitochondrial DNA of numerous alleles that have been grouped into three by McMahon and colleagues,47 in bipolar patients who distinct classes according to the number of repeats inherited the disorder from the mother, excluded a (Classes I, II and III). Class I alleles confer risk to dia- major contribution from the mitochondrial DNA. The betes while Class III alleles have a protective effect. For presence of X-linked risk alleles with a major effect in the Class III alleles, a paternal POE has been described bipolar disorder can be excluded from the results of for its protective role in a data set from US families linkage studies.48 However, since few association stud- and a maternal effect in families from Europe.65 The ies of candidate genes located on the X chromosome population-specific POE has been shown to be deter- have been conducted and because the results from mar- mined by the presence of an allele-specific phenom-

Molecular Psychiatry DRD4 and TH in bipolar disorder P Muglia et al 865 enon associated with one Class III allele, ie a different Tyrosine hydroxylase gene not linked to manic-depression in seven determination of the imprinting status within alleles of of eight pedigrees. Hum Hered 1992; 42:259–263. 9 Sidenberg DG, King N, Kennedy JL. Analysis of new D4 dopamine the same polymorphism. In other words, one allele of receptor (DRD4) coding region variants and TH microsatellite in the INS VNTR is maternally imprinted while another the Old Order Amish family (OOA110). Psychiatr Genet 1994; 4: allele at the same locus appears to be paternally 95–99. imprinted.66 The possibility for the imprinting status 10 Smyth C, Kalsi G, Brynjolfsson J, O’Neill J, Curtis D, Rifkin L et al. Further tests for linkage of bipolar affective disorder to the tyrosine being an allele-speciﬁc phenomenon together with the hydroxylase gene locus on chromosome 11p15 in a new series of relatively high degree of sequence variation of the multiplex British affective disorder pedigrees [published erratum DRD4 48-bp repeat have led us to sequence the 2-repeat appears in Am J Psychiatry 1997; 154: 139]. Am J Psychiatry 1996; alleles. However no variation in the sequence within 153:271–274. the 2-repeat alleles was found. 11 Smyth C, Kalsi G, Curtis D, Brynjolfsson J, O’Neill J, Rifkin L et al. Two-locus admixture linkage analysis of bipolar and unipolar Further molecular studies that will investigate the affective disorder supports the presence of susceptibility loci on expression of maternal and paternal DRD4 alleles are chromosomes 11p15 and 21q22. 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