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 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, 3, 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 5′ and 3′ 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 >0.05, synonymous SNPs with minor allele frequencies >0.10). 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-disequilibrium-test (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=0.014) to the affected child, and a trend toward association for rs8192625 (P=0.063) (Table 1). The results of the haplotype analysis did not strengthen our findings (data not shown). In the replication step, we analyzed the three TRAR4 variants in the case–control sample. Allele G of SNP rs8192624 again showed association with BPAD disease status (P=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=0.004) (Table 1). The combined analysis of rs8192624 for the two samples resulted in a significant association (P=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=0.05 (P=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 3′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.
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