Polymorphisms in the trace amine receptor 4 (TRAR4) gene on chromosome 6q23.2 are associated with susceptibility to schizophrenia

Abstract

Several linkage studies across multiple population groups provide convergent support for a susceptibility locus for schizophrenia--and, more recently, for bipolar disorder--on chromosome 6q13-q26. We genotyped 192 European-ancestry and African American (AA) pedigrees with schizophrenia from samples that previously showed linkage evidence to 6q13-q26, focusing on the MOXD1-STX7-TRARs gene cluster at 6q23.2, which contains a number of prime candidate genes for schizophrenia. Thirty-one screening single-nucleotide polymorphisms (SNPs) were selected, providing a minimum coverage of at least 1 SNP/20 kb. The association observed with rs4305745 (P=.0014) within the TRAR4 (trace amine receptor 4) gene remained significant after correction for multiple testing. Evidence for association was proportionally stronger in the smaller AA sample. We performed database searches and sequenced genomic DNA in a 30-proband subsample to obtain a high-density map of 23 SNPs spanning 21.6 kb of this gene. Single-SNP analyses and also haplotype analyses revealed that rs4305745 and/or two other polymorphisms in perfect linkage disequilibrium (LD) with rs4305745 appear to be the most likely variants underlying the association of the TRAR4 region with schizophrenia. Comparative genomic analyses further revealed that rs4305745 and/or the associated polymorphisms in complete LD with rs4305745 could potentially affect gene expression. Moreover, RT-PCR studies of various human tissues, including brain, confirm that TRAR4 is preferentially expressed in those brain regions that have been implicated in the pathophysiology of schizophrenia. These data provide strong preliminary evidence that TRAR4 is a candidate gene for schizophrenia; replication is currently being attempted in additional clinical samples.

Figure A1

Conserved noncoding regions defined by VISTA (Couronne et al. 2003) and the relative position to associated markers. a, TRAR4 3′-flanking conserved regions, generated by comparing human genome with mouse, rat, and chimpanzee genomes (upper, middle, and lower plots, respectively). The region with sequence similarity reaching 70% is defined as a conserved region, and such peak areas are shaded pink. b, Transformed −log FBAT P values of TRAR4 SNPs versus their relative genomic positions. Panels a and b are aligned according to the genomic position (UCSC Genome Bioinformatics Web site [July 2003 genome draft]). c, The local genomic sequence alignment (human-mouse) around rs4305745 and two other polymorphisms (ss28447873 and rs7452939) in perfect LD with rs4305745.

Figure 1

Genomic structure of the 6q23.2 gene cluster and association mapping of the initial screening. The genomic positions are based on the UCSC July 2003 assembly of the human genome (see the UCSC Genome Bioinformatics Web site). a, The relative position of the 6q23.2 gene cluster to the peak markers from various linkage studies: D6S424 (Cao et al. ; Martinez et al. 1999), D6S416 (Cao et al. 1997), D6S292 (Lerer et al. 2003), and D6S264 (Lindholm et al. 2001). b, Genes in the 6q23.2 gene cluster. c, The −log transformation of the FBAT P value for the 31 SNP markers analyzed in the initial association screening. Each data point of the markers points to its relative position in the gene cluster shown in panel b. The most significant marker is rs4305745, with a P value of .0014 (see table A5 [online only], for FBAT P values and other detailed information for all the initially selected markers, and table 1, for the single-marker association results for all the additional TRAR4 markers examined in the dense mapping effort).

Figure 2

Pairwise LD in 192 founders for the TRAR4 region. a, Relative physical position of 23 markers in the TRAR4 region (shown in table 1). The SNPs ss28447862, ss28447876, ss28447865, rs8192624, and ss28447866 were excluded in the following LD measurements because of their low minor-allele frequencies. b, LD pattern in AA subjects (left panel) and EA subjects (right panel). The graph was generated by GOLD (Abecasis and Cookson 2000). In each LD pattern, the D′ values (upper left diagonal) and the P values (lower right diagonal, converted to log P values) are calculated from the program ldmax of the GOLD package (Abecasis and Cookson 2000).

Figure 3

Expression pattern of TRAR4 in human tissues. a, TRAR4 expression pattern in various human brain regions. Lane 1 is a 100-bp molecular weight standard ladder (Promega). Lanes 2–13 are human brain, human fetal brain, cerebellum, fetal liver, placental, spinal cord, control (no reverse transcriptase added), basal ganglia, frontal cortex, substantia nigra, amygdala, and hippocampus. RT-PCR products from total RNAs are displayed in the photograph of the ethidium bromide–stained agarose gel; β-actin was used as internal control. b, Quantitative real-time PCR determined the relative abundance of the TRAR4 transcript in various human brain regions. c, Comparison of gene expression of TRAR4 with TRAR1. Samples S1–S6 in panels b and c are basal ganglia, frontal cortex, substantia nigra, amygdala, hippocampus, and cerebellum.