Dopamine transporter haplotype and attention- deficit ... - Nature

Molecular Psychiatry (2005) 10, 617–621 & 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00 www.nature.com/mp

SCIENTIFIC CORRESPONDENCE

Dopamine transporter haplotype and attentiondeficit hyperactivity disorder Molecular Psychiatry (2005) 10, 617–618. doi:10.1038/sj.mp.4001655 Published online 10 May 2005 SIR—The dopamine transporter gene (DAT1 SCL6A3) is an important candidate for mediating susceptibility to attention-deficit hyperactivity disorder (ADHD), given its relevance for related behaviors, and its blockade by effective stimulant drugs. To date, most studies examined a 40 bp variable number tandem repeat (VNTR) in the 30 -untranslated region of the gene. Along with recurrent reports of preferential transmission of the 10-repeat (480 bp) allele, there are some negative reports, and a meta-analysis of 11 studies with a total of 824 informative meioses yielded a nonsignificant pooled odds ratio estimate of 1.27 (95% CI 0.99–1.63, 0.06).1A previous study that did not detect significant association with the VNTR found a haplotype consisting of the 10-repeat allele and two 30 biallelic sites significantly associated with ADHD.2 A recent study replicated association of the VNTR with ADHD, with stronger associations observed for 30 microsatellite marker haplotypes that include the 10-repeat allele.3 If the VNTR is a marker for an adjacent functional DAT1 variant, a linkage disequilibrium (LD) association approach may increase the sensitivity for locating association with the phenotype, and may help explain part of the variance in previous reports that examined the VNTR as a single marker. In the present study, we examined an exon 15 haplotype located in the 30 -untranslated region, consisting of the VNTR and an upstream G2319A substitution in LD,4 in a sample of ADHD triads, confirming association with the VNTR, and stronger association with the haplotype. The sample was previously described and consisted of 76 nuclear ADHD families.5 Of these, 68 trios were informative Table 1

for the VNTR site, 50 trios were informative for the A2319G site, and a total of 64 trios were informative for the haplotype analysis. The extended transmission disequilibrium test6 was used to determine allelic association with individual markers, and HAPMAX (http://www.uwcm.ac.uk/uwcm/mg/ download) was used for the multimarker haplotype relative risk (HRR) analysis. The DAT1 VNTR alleles showed marginally significant biased transmission to affected children (w2 for genotype-wise TDT ¼ 9.08, df ¼ 3, P ¼ 0.028); preferential transmission was also observed for the 2319 G allele (w2 ¼ 3.97, df ¼ 1, P ¼ 0.046) (Table 1). The two sites demonstrated strong LD (D0 coefficient ¼ 1.000). Multimarker HRR analysis showed a globally significant biased transmission of haplotypes (w2 ¼ 14.56, df ¼ 5, P ¼ 0.0124). Among the individual haplotypes that contributed to the global significance of the haplotype analysis, relative risk calculation for the haplotype 11-G vs the others provided a significant protective RR of 0.69 (CI 1.09–84.48; w2 ¼ 4.097, df ¼ 1, P ¼ 0.043), although with a very small effect size (w ¼ 0.179), and haplotype 10-G vs the others provided a modest RR of 1.56 (CI 0.84–2.86). The rest of the haplotype combinations did not show any transmission differences among affected and unaffected children (Table 2). All subjects were Jewish, of Ahskenazi, nonAskenazi or mixed (Ashkenazi and non-Ashkenazi) background. Both markers did not show differences in allele or genotype frequencies among subjects of different Jewish backgrounds in our sample. The observation of stronger haplotype associations in the present report, as well as in two previous studies,2,3 suggests that the inconsistently reported association of the VNTR with ADHD may reflect variable LD with a functional site lying in proximity. Current in vitro and in vivo data provide conflicting results regarding a possible functional role for VNTR alleles in affecting gene expression. This may be explained in part by reports showing that VNTR alleles within different haplotype combinations correlate with different expression indices in vitro.7,8

Association tests between DAT markers and ADHD (TDT)

Allele freq.

Number of transmitted alleles

Number of nontransmitted alleles

w2

df

P-value

9 repeats 10 repeats 11 repeats

0.46 0.48 0.07

34 33 1

28 32 8

0.58 0.01 5.44 9.08

1 1 1 3

0.45 0.90 0.02 0.028

A G

0.50 0.50

18 32

32 18

3.92 3.92

1 1

0.048 0.048

Marker

Alleles

VNTR Locus-wise A2319G Locus-wise

Scientific Correspondence

618

Table 2

Family-based association test between DAT markers and ADHD (haplotypes)

Haplotype

9-G 10-G 11-G 9-A 10-A

Transmitted Number of freq.

Number of freq.

26 31 0 0 7

0.41 0.48 0 0 0.11

95% CI

0.30–0.52 0.36–0.60 — — 0.05–0.17

Nontransmitted Number of freq.

Number freq.

25 26 4 1 8

0.37 0.38 0.05 0.01 0.12

Global

95% CI

w2

df

P-value

0.26–0.48 0.27–0.49 0.01–0.09 — 0.04–0.20

0.02 0.44 4 — 0.07

1 1 1 — 1

0.89 0.51 0.046 — 0.79

14.56

5

0.0124

Log(likelihood)

278.31

VNTR allele frequencies show substantial ethnic variability,9 suggesting that its observed LD with a putative 30 functional site may show similar variability among different ethnic populations from which ADHD triads were sampled. Three independent reports of systematic mutation screens of the DAT gene did not locate any additional common coding variations that result in amino-acid substitutions, or promoter region variations modulating mRNA expression levels.10–12 The gene exhibits a segmental pattern of LD, with a high but variable LD within 30 polymorphisms (exons 9–15), where two haplotype clades have been described, one of which segregates with the 10-repeat allele of the 30 VNTR and the other with the 9-repeat allele.10,13 Greenwood et al13 suggested the presence of a functional element in the 30 region of the gene, alternative variants of which may segregate with each of the clades and may contribute to various neuropsychiatric disorders. Similar to the above findings in ADHD, Greenwood et al10 reported a trend for biased transmission of VNTR alleles among bipolar disorder triads, with a highly significant association for a haplotype composed of the 10-repeat allele and four 30 SNPs in the same sample. Taken together, these findings suggest that the use of haplotype mapping of the DAT1 30 region in association studies with ADHD as well as other neuropsychiatric disorders may be more informative, and may resolve some of the previous discrepancies in studies using the VNTR as a single marker. Another recently suggested source of variation may result from a gene environmental effect.14 Further sequencing of this region and investigation of the functional significance of its variations are warranted.

Zedek Medical Center, Jerusalem, Israel; 4Department of Psychiatry, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 5Department of Psychiatry, Vita-Salute University, San Raffaele Institute, Milan Italy; 6Life Sciences Institute, The Hebrew University, Jerusalem, Israel

E Galili-Weisstub1, S Levy1, A Frisch2, V Gross-Tsur3, E Michaelovsky2, A Kosov2, A Meltzer1, T Goltser4, A Serretti5, C Cusin5, A Darvasi6, E Inbar6, A Weizman2 and RH Segman4 1 Child & Adolescent Psychiatry Unit, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 2Geha Psychiatric Hospital, Petah Tikva and Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3Neuropediatric Unit, Shaare

Molecular Psychiatry (2005) 10, 618–620. doi:10.1038/sj.mp.4001665 Published online 26 April 2005

Molecular Psychiatry

Correspondence should be addressed to Dr RH Segman, Department of Psychiatry, Hadassah Hebrew University Medical Center, Jerusalem 24035, Israel. E-mail: [email protected]

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Maher BS et al. Psychiatr Genet 2002; 12: 207–215. Barr CL et al. Biol Psychiatry 2001; 49: 333–339. Hawi Z et al. Mol Psychiatry 2003; 8: 299–308. Ueno S et al. Mol Psychiatry 1999; 4: 552–557. Segman RH et al. Mol Psychiatry 2002; 7: 72–74. Sham P, Curtis D. Ann Hum Genet 1995; 59: 323–336. Greenwood TA, Kelsoe JR. Genomics 2003; 82: 511–520. Miller GM, Madras BK. Mol Psychiatry 2002; 7: 44–55. Kang AM et al. Biol Psychiatry 1999; 46: 151–160. Greenwood TA et al. Am J Med Genet 2001; 105: 145–151. Vandenbergh DJ et al. Mol Psychiatry 2000; 5: 283–292. Grunehage F et al. Mol Psychiatry 2000; 5: 275–282. Greenwood TA et al. Mol Psychiatry 2002; 7: 165–173. Khan RS et al. J Pediatr 2003; 143: 104.

A family-based and case– control association study of trace amine receptor genes on chromosome 6q23 in bipolar affective disorder

SIR—Trace amines are of interest in the study of neuropsychiatric disorders as they are found predominantly in the CNS, and altered levels have been observed in disorders such as schizophrenia, bipolar affective disorder, and anxiety disorders.1 The genes

0.13 0.87 0.13 0.86 0.26 0.72 0.00 0.01 0.02

1-1

1-2

2-2

0.024 (1.63, 1.03–2.55) 0.250 (1.36, 0.87–2.15) 0.604 (1.06, 0.75–1.52)

ARMITAGES TREND TEST P-values (odds ratio, 95% CI)

619

0.80 0.82 0.71 0.19 0.17 0.27 Combined analysis (TDT and case-control) for SNP rs8192624: P ¼ 0.004. a 1 is the rare allele, 2 is the frequent allele. b Genotype frequencies were in HWE for cases and controls.

0.01 0.01 0.02 0.93 (G) 0.93 (G) 0.85 (T) 0.89 (G) 0.91 (G) 0.84 (T)

Controls (n ¼ 430) BPAD (n ¼ 263) Replication sample

45 37 Transmitted Not transmitted P-values

0.380 A Allele

rs8192624 rs8192625 rs7772821

1-1

Controls (n ¼ 430)a,b

0.014

1-2

2-2

0.063

BPAD (n ¼ 263)a,b

0.760

5 6 6 5 34 31 31 34 0.710 12 23 23 12 13 29 29 13 37 45

A (aspartic acid) C (alanine) G T

rs8192620

G

G (valine) A (isoleucine) G (cysteine) A (tyrosine)

rs8192627 rs8192624

rs8192625

rs7772821

TRAR5 TRAR4 TRAR1 Initial sample (n ¼ 118 BPAD triads)

Table 1

of the three trace amine receptors (TRARs) 1, 4, and 5 cluster on chromosomal region 6q23.2, a region for which we and others have previously obtained evidence for linkage to bipolar affective disorder.2–4 TRARs also share a high degree of sequence homology, and together form a subfamily of G protein-coupled receptors (GPCRs) that are related to serotonin- (5–HT-), dopamine- (DA-), and norepinephrine- (NE-) receptors.1 To explore genetic variability at this locus, we established a PCR-based strategy to amplify the coding regions, and the 50 and 30 flanking regions of the TRARs, namely TRAR1, TRAR4, and TRAR5. We used this to sequence DNA from a representative sample of 96 individuals from the European population.5 A total of 4467 bp of genomic DNA was screened, 3087 bp of which were exonic. Direct sequencing was performed as described elsewhere.5 We identified 12 SNPs in the three TRARs which were submitted to dbSNP (http://www.ncbi.nlm.nih. gov/) under accession nos. ss125870000–ss12587012 (see Supplementary information). Five SNPs were selected from the identified variants for genotyping in 118 parent –offspring triads with BPAD. These were selected on the basis of their functional aspects, and allele frequencies (nonsynonymous SNPs with minor allele frequencies 40.05, synonymous SNPs with minor allele frequencies 40.10). Positive association was observed in TRAR4, and we therefore analyzed the TRAR4 variants in an independent case–control replication sample consisting of 263 BPAD patients and 430 controls. All patients had been interviewed by experienced psychiatrists and psychologists using the Structured Clinical Interview for DSM-IV Disorders.6 Lifetime ‘best estimate’ diagnoses according to DSM-IV criteria were established, based on multiple sources of information including personal structured interview (SCID I) and medical records. All participants were of German descent. Written informed consent was obtained from all patients and controls. Genotyping was carried out using RFLP assays (see Supplementary information). The transmission-disequilibriumtest (TDT)7 was applied for the triad analysis. We used the Armitages trend test8 for the case–control study and the FAMHAP9 program for the haplotype analysis. Fisher’s method10 of the ’combination of probabilities from tests of significance’ was used to determine the common significance of the case– control sample and the trio sample. We analyzed rs8192620 in TRAR1, rs8192624 (V265I), rs8192625 (C291Y), and rs7772821 in TRAR4 and rs8192627 (D328A) in TRAR5 (Table 1). In the initial step, we genotyped all five SNPs in the triad sample and observed no significant TDT results in TRAR1 and TRAR5. In TRAR4, we observed a preferential transmission of the allele G of rs8192624 (P ¼ 0.014) to the affected child, and a trend toward association for rs8192625 (P ¼ 0.063) (Table 1). The results of the haplotype analysis did not strengthen our findings (data not shown). In the replication step,

TDT and Armitage trend test: association analyses with variants at the trace amine receptor genes TRAR1, TRAR4 and TRAR5 in BPAD samples and controls




620

we analyzed the three TRAR4 variants in the case– control sample. Allele G of SNP rs8192624 again showed association with BPAD disease status (P ¼ 0.024), while the two other SNPs showed no association (Table 1). The combined analysis of both samples with rs8192624 resulted in a significant association (P ¼ 0.004) (Table 1). The combined analysis of rs8192624 for the two samples resulted in a significant association (P ¼ 0.004) (Table 1). After application of the Bonferroni correction for multiple testing, applied since five markers had been tested, the significance persists at alpha ¼ 0.05 (P ¼ 0.02). The result remains significant even when applying a further Bonferroni correction of 2 to account for the fact that a haplotype analysis was also conducted. rs8192624 is responsible for an amino-acid exchange (V265I) in the sixth transmembrane domain of the trace amine receptor 4 (predicted on the basis of seven transmembrane GPCR structures as depicted in the GPCR database (http://www.gpcr.org/)). However, no obvious functional sequelae are associated with this variant; valine and isoleucine are nonpolar neutral amino acids and valine in position 265 is not conserved in other species. Our study provides evidence for the involvement of the TRAR4 locus in the etiology of BPAD. It is of interest to note that a recent study11 has reported an association with SNPs at the 30 UTR of TRAR4 and schizophrenia. It could be speculated that genetic variation at this locus contributes to both disorders. However, given the differing locations of the associated SNPs within the gene, and the fact that Duan et al11 did not observe association with rs8192624 and schizophrenia, there is currently no support for the hypothesis that a single variant contributes to both disorders. Independent replication studies in BPAD and schizophrenia using denser SNP maps are required to further define the role of TRAR4 in the etiology of psychiatric disorders. R Abou Jamra1, I Sircar1, T Becker2, Y Freudenberg-Hua1, S Ohlraun3, J Freudenberg1, F Brockschmidt1, TG Schulze3, M Gross4, F Spira5, M Deschner3, C Schma¨l3, W Maier4, P Propping1, M Rietschel3, S Cichon6, MM No¨then6 and J Schumacher1 1 Institute of Human Genetics, University of Bonn, Bonn, Germany; 2Institute of Medical Biometry, Informatics, and Epidemiology, University of Bonn, Bonn, Germany; 3 Central Institute of Mental Health, Mannheim, Germany; 4 Department of Psychiatry, University of Bonn, Bonn, Germany; 5Mental State Hospital, Psychiatrisches Zentrum Nordbaden, Wiesloch, Germany; 6Life & Brain Center, University of Bonn, Bonn, Germany Correspondence should be addressed to Dr R Abou Jamra, Institute of Human Genetics, University of Bonn, Wilhelmstr. 31, D-5311 Bonn, Germany. E-mail: [email protected]

1 Borowski BAN et al. Proc Natl Acad Sci USA 2001; 98: 8966–8971. 2 Cichon S et al. Hum Mol Genet 2001; 10: 2933–2944. Molecular Psychiatry

3 Ewald H, Flint T, Kruse TA, Mors O. Mol Psychiatry 2002; 7: 734–744. 4 Rice JP et al. Am J Med Genet 1997; 74: 247–253. 5 Freudenberg-Hua Y, Freudenberg J, Kluck N, Cichon S, Propping P, Nothen MM. Genome Res 2003; 13: 2271–2276. 6 First M. Structured Clinical Interview for DSM-IV Axis I Disorders. Patient (ed). New York: Biometrics Research Department, 1997. 7 Spielman RS, McGinnis RE, Ewens WJ. Am J Hum Genet 1993; 52: 506–516. 8 Armitage P. Biometrics 1955; 11: 375–386. 9 Becker T, Knapp M. Genet Epidemiol 2004; 27: 21–32. 10 Fisher R. Statistical Methods for Research Workers. 13th ed. London: Oliver & Loyd, 1925, 99p. 11 Duan J et al. Am J Hum Genet 2004; 75: 624–638. Supplementary Information accompanies the paper Molecular Psychiatry website (http://www.nature.com/mp).

Microarray results suggest altered transport and lowered synthesis of retinoic acid in schizophrenia Molecular Psychiatry (2005) 10, 620–621. doi:10.1038/sj.mp.4001668 Published online 10 April 2005 SIR—A microarray study of schizophrenia in human brains reports that, among others, two proteins affecting vitamin A (retinoid) transport and function are altered. The direction of the alteration of these proteins, aldehyde dehydrogenase 1A1 and albumin, suggest that retinoic acid (RA), the final metabolic product of the retinoid cascade, is insufficient in schizophrenia. Unavailability of RA either directly or indirectly impairs transcriptional regulation of retinoid target genes, for example, DRD21 and ERBB4,2 which are candidates in schizophrenia. The identification of retinoid dysregulation as a common underlying pathway in schizophrenia suggests therapeutic interventions directed toward the development of new RA analogs. A recent series of microarray expression analyses implicate entire pathways involving oxidative stress, energy metabolism and mitochondrial function in schizophrenia prefrontal cortex.3 Dysregulation of the synthesis of RA, which mediates oxidative stress, regulates energy expenditure and influences mitochondrial functioning and apoptosis,4 may exert a combined, cumulative influence on these pathways. Support for this suggestion can be deduced from examination of the microarray analyses referenced above: expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1A1 (ALDH1A1), a protein that is strongly expressed in dopaminergic neurons,5 is significantly decreased; albumin, a ubiquitous serum transporter of RA6 and other free fatty acids, for example, docosahexaenoic acid and arachidonic acid, which ligand the retinoid X receptors7 is highly


significantly decreased. Both of these findings suggest lowered availability of retinoid receptor ligands in the schizophrenia prefrontal cortex. ALDH1A1 and albumin together with other proteins of the retinoid cascade regulate the transport to and temporal and spatial availability of RA in the brain, and thus control the transcription of retinoid targets.8 That dysregulation of the retinoid cascade may be a contributory mechanism in schizophrenia9 is supported by the observation that the genes coding for many of the proteins showing altered expression in this microarray study of schizophrenia prefrontal cortex are either directly or indirectly regulated by RA and retinoid receptors; for example, pyruvate kinase, muscle (PKM2),10 mitochondrial aconitase 2 (ACO2),11 hexokinase 1 (HK1) and malate dehydrogenase 1 (MDH1),12 gelsolin (GSN),13 neuron-specific enolase (ENO2),14 cardiac muscle actin alpha (ACTC) and actinin, alpha4 (ACTN4),15 and transferrin (TF).16 The reliability of microarray studies reporting small differences in expression can be questionable,17 particularly so in relation to false negatives due to the lack of resolution (hybridization without separation) and false positives due to a lack of sensitivity (multiplicity of probes; complexity of probes). However, in situ hybridization with ALDH1A1 probes finds significant downregulation of ALDH1A1 mRNA in the ventral tegmental area in schizophrenia.18 The hypothesis that dysregulated retinoid transport and function contributes to schizophrenia has recently received substantial additional immunohistochemical support with the discovery that expression of retinoic acid receptor alpha (RARA) protein is more than two-fold greater in the dentate gyrus in post-mortem schizophrenia brains compared to controls.19 RAs and their analogues represent well-studied therapeutic targets in cancer and dermatology. Perusal of the literature from these fields indicates that retinoids modulate cell functioning through diverse

mechanisms, including gene silencing and nuclear translocation, as well as through direct transcriptional activation. Pharmacologic successes with retinoids in cancer and dermatology and the wealth of information gleaned from these successes could inform new directions in therapies for schizophrenia, particularly in relation to mechanisms involved in neuronal programmed cell death and oxidative damage.

621

AB Goodman The Massachusetts Mental Health Center Academic Division of Public Psychiatry in the Department of Psychiatry at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Correspondence should be addressed to AB Goodman, 987 Memorial Drive, Cambridge, MA 02138, USA. E-mail: [email protected]

1 Samad TA et al. Proc Natl Acad Sci USA 1997; 94: 14349–14354. 2 Offterdinger M et al. Biochem Biophys Res Commun 1999; 258: 559–564. 3 Prabakaran S et al. Mol Psychiatry 2004; 9: 684–697. 4 Pfahl M, Piedrafita FJ. Oncogene 2003; 22: 9058–9062. 5 McCaffery P, Drager UC. Proc Natl Acad Sci USA 1994; 91: 7772–7776. 6 Smith JE et al. Biochem J 1973; 132: 821–827. 7 Lengqvist J et al. Mol Cell Proteomics 2004; 3: 692–703. 8 Goodman AB, Pardee AB. Proc Natl Acad Sci USA 2003; 100: 2901–2905. 9 Goodman AB. Proc Natl Acad Sci USA 1998; 95: 7240–7244. 10 Faria TN et al. Mol Cell Endocrinol 1998; 143: 155–166. 11 Bielarczyk H et al. Neurochem Int 2003; 42: 323–331. 12 Feingold K et al. Am J Physiol Endocrinol Metab 2004; 286: E201–E207. 13 Carter CA, Shaw BL. Exp Mol Pathol 2000; 68: 70–86. 14 Jang YK et al. J Neurosci Res 2004; 75: 573–584. 15 Aranega AE et al. Cells Tissues Organs 1999; 164: 82–89. 16 Lecureuil C et al. Hum Reprod 2004; 19: 1300–1307. 17 Liang P, Pardee AB. Nat Rev Cancer 2003; 11: 869–876. 18 Galter D et al. Neurobiol Dis 2003; 14: 637–647. 19 Rioux L, Arnold SE. Psychiatry Res 2005; 133: 13–21.
