Molecular Psychiatry (2007) 12, 842–853 & 2007 Nature Publishing Group All rights reserved 1359-4184/07 $30.00 www.nature.com/mp ORIGINAL ARTICLE A region of 35 kb containing the associate receptor 6 (TAAR6) is associated with in the Irish study of high-density schizophrenia families V Vladimirov1, DL Thiselton1, P-H Kuo1, J McClay1, A Fanous1,2,3, B Wormley1, J Vittum1, R Ribble1, B Moher1, E van den Oord1, FA O’Neill4, D Walsh5, KS Kendler1 and BP Riley1 1Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA, USA; 2Mental Health Service Line, Washington VA Medical Center, Washington, DC, USA; 3Georgetown University School of Medicine, Washington, DC, USA; 4Department of Psychiatry, Queens University, Belfast, Northern Ireland and 5Health Research Board, Dublin, Ireland

The TAAR6 gene has been previously associated with schizophrenia in 192 pedigrees of European and African ancestry. To replicate these findings we performed an association study of TAAR6 in 265 pedigrees of the Irish Study of High-Density Schizophrenia Families (ISHDSF). Of the 24 genotyped single-nucleotide polymorphisms only rs12189813 and rs9389011 provided single-marker evidence for association (0.0094pPp0.03). Two-marker haplotypes (rs7772821 and rs12189813; 0.0071pPp0.0023) and four-marker haplotypes (rs8192622, rs7772821, rs12189813 and rs9389011; 0.0047pPp0.018) gave strongest evidence for association. The associated high-risk (HR) haplotype in the ISHDSF is defined by the major alleles at rs7772821 and rs12189813 (0.00097pPp0.023). The associated HR remains positive in a case only test of association by Operational Criteria score analysis in which significant association was observed only with the highest threshold for delusions (P < 0.009). When analysis was restricted to affected individuals under the core schizophrenia (D2) diagnosis, the observed associations for HR were most significant in the highest threshold for delusions (P < 0.004) and hallucinations (P < 0.0004), supporting the family-based association with schizophrenia. Molecular Psychiatry (2007) 12, 842–853; doi:10.1038/sj.mp.4001984; published online 15 May 2007 Keywords: schizophrenia; association; trace amine receptor; gene; haplotype; expression

Introduction of European and 48 families of African descent).4 After correction for multiple testing, rs4305745 Schizophrenia (MIM 181500) is a common and debi- (located in the 30UTR of TAAR6) was the only marker litating psychiatric disorder with a lifetime preva- to remain significantly associated with the disease. lence of 0.5–1% worldwide. Symptoms of the disease Multiple-marker haplotype analyses gave less signifi- include delusions and hallucinations (psychotic cant results than the analysis of rs4305745 alone. The symptoms), disordered thought, blunted emotion authors suggest that rs4305745 is thus the polymorph- and impairment of memory, attention, cognition and ism most likely to account for the association of executive function. Family, twin and adoption stu- TAAR6 with schizophrenia. dies show consistent evidence of a substantial genetic Trace amines (TAs) are endogenous compounds component in schizophrenia, in conjunction with structurally related to classical biogenic amines such environmental and developmental factors.1–3 as p-, b-phenylethylamine, and Using single-nucleotide polymorphism (SNP)-based octapamine. TAs have been implicated in highly association fine mapping, the trace amine-associated prevalent psychiatric disorders like depression and receptor 6 (TAAR6/TRAR4) gene on schizophrenia.5–8 They are present in the central 6q23.2 was implicated as a schizophrenia suscepti- nervous system (CNS) at low levels and they share bility gene in a sample of 192 pedigrees (144 families significant overlap with classical biogenic amines in their chemical properties, biosynthesis and break- Correspondence: Dr V Vladimirov, Virginia Institute for Psychia- down. The absence of specialized TA receptor(s) tric and Behavioral Genetics, Virginia Commonwealth University, fostered the assumption that TAs would function Biotech1/Suite110, 800 East Leigh Street, Richmond, VA 23298- via classical receptors.9 This view 0424, USA. changed with the identification of a novel family E-mail: [email protected] Received 5 January 2006; revised 4 February 2007; accepted 27 of TA-sensitive receptors belonging to G- February 2007; published online 15 May 2007 coupled receptors gene superfamily.10,11 TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 843 Six functional and three pseudo TA receptor sion criteria for controls were (1) age less than 30 and have been identified in humans, all localized on (2) substance abuse within 1 year of death.15 chromosome 6q23.2. With the exception of TAAR2, which is encoded by two exons, the coding sequence SNP selection, genotyping and error checking of TAAR genes is contained in one exon of approxi- At the beginning of this study, few SNPs in the mately 1 kb, with very high homology between the TAAR6 region had been deposited in HAPMAP. We mouse, chimpanzee and rat TAAR orthologues.5 The therefore chose the most significant SNPs reported in expression of the best studied TAAR1 gene has been the original study (rs8192625, rs4305745, rs6903874 detected in different tissues at very low levels. In and rs6937506) and additionally selected rs8192622, brain the overall expression of TAAR1 was also low, rs8192624 and rs7772821, which were the only although some regions showed higher mRNA abun- additional TAAR6 polymorphisms available in HAP- dance compared with others. The highest TAAR6 MAP to extend coverage of the gene. rs8192623 expression was detected in , whereas was monomorphic and was excluded. TAAR6 belongs the lowest expression was seen in basal ganglia. Other to a gene family of highly homologous members brain regions such as frontal cortex, substantia nigra all located in a small genomic region of 108 kb. To or amygdala showed comparable levels of TAAR6 exclude the possibility of association signal from expression and these regions have been implicated in another member of this gene cluster, we included the pathophysiology of schizophrenia.4,12 17 additional SNPs from the region either within or Here we report the results of a family-based flanking additional TAAR loci, based on the HapMap association study performed in the Irish Study of data (http://www.hapmap.org) available at the time. High-Density Schizophrenia Families (ISHDSF) sam- Since the TAGGER option was not implemented in ple and present evidence for association between the earlier versions of Haploview, haplotype tagging schizophrenia and a haplotype of 35 kb overlapping SNPs were selected within each LD block to dis- TAAR6 in the ISHDSF. criminate haplotypes with frequency X1% using the default HAPLOVIEW settings. Materials and methods The DNA samples were genotyped by fluorescence polarization template-directed incorporation of dye- Subjects and phenotypes terminator using the relevant AcycloPrime FP SNP The ISHDSF sample consists of 265 high-density detection kit (Perkin-Elmer Life Sciences, Boston, schizophrenia families with 1408 people available for MA, USA), according to manufacturer’s instructions. genotyping.13 All participating individuals gave ap- The FP data were converted to genotypes by an auto- propriate informed consent for the study. The sample mated allele scoring platform.16 Polymerase chain was assessed using multiple concentric diagnostic reaction (PCR) primers were designed using PRI- categories, ranging from narrow to very broad. Briefly, MER3.17 PCR and single-base extension primer se- D2 (core schizophrenia, 625 affected individuals) quences for markers in this study are given in the includes schizophrenia, poor-outcome schizoaffective Supplementary Table. The average completion rate of disorder and simple schizophrenia. D5 (narrow the genotype experiments was 94%. psychosis spectrum, 804 affected individuals) adds Mendelian inconsistencies within families were to these schizotypal personality disorder and all other identified with PEDCHECK v1.1 (20) and unlikely nonaffective psychotic disorders. D8 (broad psychosis recombinants were identified with MERLIN.18 For all spectrum, 888 affected individuals) additionally in- Mendelian inconsistencies and unlikely recombi- cludes mood-incongruent and mood-congruent psy- nants, marker genotypes were zeroed for the entire chotic affective illness, and paranoid, avoidant and pedigree. Hardy–Weinberg equilibrium was assessed schizoid personality disorders. D9 (very broad, 1172 for all markers using HAPLOVIEW v3.2 and no signi- affected individuals) includes all psychiatric disorder ficant deviations were observed.19 The schizophrenic (i.e. all psychosis spectrum disorders plus non- (n = 35) cases and controls (n = 35) from the SBS series psychotic affective disorders, anxiety, alcoholism, were genotyped for four markers in the same methods etc.). All patients with a history of any psychotic as for the ISHSDF. episode were assessed for lifetime psychotic symp- toms using the Operational Criteria (OPCRIT) check- Intermarker linkage disequilibrium analysis list of psychotic illness.14 Linkage disequilibrium (LD) between SNPs was esti- The Stanley Brain Series (SBS) post-mortem collec- mated with Haploview from the genotypes of unrela- tion donated by the Stanley Medical Research ted founders. Haplotype frequencies were estimated Institute consists of DNA and mRNA samples extrac- from genotype data using the expectation-maximiza- ted from dorsolateral prefrontal cortex (Brodmann’s tion algorithm.20 The LD blocks based on confidence area 46) from 35 individuals each with schizophrenia intervals (D0) were determined by the default settings and bipolar affective disorder and 35 controls. The of Haploview.21 exclusion criteria for collection of the samples included (1) significant structural brain pathology, Association analysis (2) history of pre-existing CNS disease, (3) poor RNA We analyzed genotypic data using the Family-Based quality or (4) documented IQ < 70. Additional exclu- Association Test as implemented in the program

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 844 FBAT version 1.1.5 and the Pedigree Disequilibrium Statistical analysis of gene expression Test as implemented in the program PDTPHASE Differences in TAAR gene expression level between version 4.0.22–25 Both are able to deal with the cases and controls were evaluated by the non- transmission of multilocus haplotypes, even when parametric Kruskal–Wallis test implemented in Prism the phase is unknown and parental genotypes may v4.0 (GraphPad v4.03 for Windows, San Diego, CA, be missing. Although the approaches are broadly USA; www.graphpad.com). Effects of potential con- similar, we include both for several reasons. First, founder variables (age, post-mortem interval (PMI), FBAT was the only analytic tool used in the original refrigeration interval, brain pH and smoking) were study, and second PDTPHASE reports the compo- assessed by multiple regression analysis implemen- nents of the association from vertical parental ted in analysis of variance (ANOVA) using NCSS 2004 transmissions and horizontal sibling genotype con- (Number Crunching Statistical System, Kaysville, UT, cordance or discordance separately. Since the direc- USA; www.ncss.com). tions of these two components of the test are not always the same, it is important to be able to assess Haplotype reconstruction them separately. In FBAT analyses, we did not use Haplotypes were reconstructed in the SBS samples the empirical-variance estimator (-e flag) since we had using the PHASE v2.1.30,31 The program implements a no prior evidence of linkage to this region in our Bayesian statistical method and for haplotype recon- sample and for the analysis we assumed the additive struction PHASE uses an iterative approach. The model. number of iterations required to obtain accurate Analyses of OPCRIT factor scores were performed answer depends on the complexity and size of the using TRANSMIT. The robust variance estimate was data. For the haplotype reconstruction in the SBS we used to allow for multiple-affected offspring per have used 100 iterations. pedigree.26,27 Bioinformatics tools We used VISTA (http://genome.lbl.gov/vista/index. Quantitative real-time PCR shtml) to define conserved regions in genomic Expression of the TAAR genes was analyzed by sequences from different species.32 To predict secon- quantitative real-time PCR using SYBR Green I on a dary structure of TAAR6 mRNA we used RNAstructure Bio-Rad (Hercules, CA, USA) iCycler platform and version 4.2, available through Isis Pharmaceuticals normalized against two reference genes TBP and Inc., Carlsbad, CA, USA (http://rna.urmc.rochester. GAPDH (used as an internal controls) by the 2ÀDDCT edu/rnastructure.html). These predictions are based algorithm implemented in the Relative Gene Expres- on the algorithm for free energy minimization using sion Macro software (Bio-Rad Inc.). The inclusion of the nearest neighbor parameters.33,34 Default values set multiple housekeeping genes as internal controls has (1) the lowest free energy structure at À100 kcal/mol been reported to give more accurate and reliable (DG137) and (2) the maximum energy difference at normalization of gene-expression data.28,29 Reverse 10%. The graphical visualization of the RNA second- transcription of mRNA samples into cDNA was ary structures was performed by XRNA software performed using the ready-to-go kit from Amersham version 1.1.12Beta available through University of Bioscience (Piscataway, NJ, USA) following the California, Santa Cruz, CA, USA (http://rna.ucsc.edu/ manufacturer’s instructions. The primers for TAAR6 rnacenter/). TBP and GAPDH were designed using the Beacon To find potential target sites for miRNAs in the Designer software version 4.02. Primer sequences TAAR6 30UTR sequence we used the miRanda used in this experiment are shown in the Supple- package (http://www.microrna.org/miranda.html).35 mentary Table. TAAR2, TAAR5 and TAAR6 primer Potential target sites are identified using a two-step sets optimized for SYBR Green I-based real-time strategy. First a dynamic programing local alignment PCR were obtained from SuperArray (Frederick, MD, is carried out between the query miRNA sequence USA). To generate the standard curves, the cDNAs and the reference sequence. The miRNAs sequences for these TAAR genes, TBP and GAPDH were cloned were retrieved from the miRNA registry.36 This in the pcDNA3.1 expression vector. The concen- alignment procedure scores is based on sequence tration of such prepared vectors was determined by complementarity rather than sequence identity. The measuring their absorbance at 260 nm. The quality second phase of the algorithm takes high-scoring

of the DNA sample was verified by the A260/A280 alignments (those above a user-defined score thresh- ratio, which was between 1.7 and 1.9. The standard old) detected in phase 1 and estimates the thermo- points were made using five 1:5 serial dilutions dynamic stability of RNA duplexes based on these and the quality of the standard curve was judged alignments. Optionally some rudimentary statistics from the slope and the correlation coefficient (r). about each target site can be generated by performing A correlation coefficient r > 0.99 (showing that a a number of alignments using shuffled reference linear relationship is observed) was obtained for all sequences generated from the input sequence. A loci. The PCR efficiency ranged from 98 to 102%. distribution is built from these data and statistical Each point on the standard curve was assayed in parameters from this distribution are used to produce triplicate. a Z score for a detected target site. A Z score of X5

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 845 was used as cutoff value to screen against false which also identifies these LD blocks interspersed positive matches. with regions of very low LD.

Single-marker association results Results Results of single-marker analyses are presented in Table 1. The single-marker association study was LD structure of TAAR region and SNP selection performed across D2, D5, D8 and D9 diagnostic In our sample of pedigrees from Ireland, the haplo- categories. Of the 24 SNPs screened, only two showed typic structure of the region is defined by four LD significant evidence for association, rs12189813 blocks (using the default confidence interval setting (0.0094pPp0.02), and rs9389011 (0.03pPp0.1597) in HAPLOVIEW). Haplotype and LD structure with in the narrowest D2 category. These two markers are r2 values of the region in our sample are shown located 35 and 42 kb telomeric from TAAR6, respec- in Figure 1. TAAR9 and TAAR8 are located in the tively, and fall between LD block 2 and 3. The observed first (most centromeric) LD block, TAAR6 between associations were due to excess transmission and/or LD blocks 1 and 2, TAAR5 and TAAR2 between LD sharing of the common allele at both markers. Of the blocks 2 and 3 and TAAR1 in LD block 4. These four SNP variants in the TAAR6 gene, none gave results are similar to the regional data from HapMap, significant evidence of association individually in any

Figure 1 In ISHDSF sample, the LD map represents a region of 108 kb covering the TRAR genes on 6q23.2. Four LD blocks (D0 confidence interval (CI)) are defined, with block 2 showing extended LD with markers rs12189813 and rs9389011. Markers rs8192622, rs8192624, rs8192625 and rs7772821 localized in the TAAR6 gene fall between LD block 1 and 2. The LD map color intensity is based on D0 with darker shades representing higher LD. Values in boxes represent the pair-wise r2 measure between markers.

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 846 Table 1 Single-marker analysis provided association evidence for two markers, rs12189813 and rs9389011 (significant P-values below nominal 0.05 are given in bold), using a narrow diagnostic model

Marker dbSNP Position Distance SNP MAF FBAT PDT (bp) (bp) nucleotide Transmissions

Observed Expected P-value Trio-T Trio-NT Affsib Unafsib P-value

1 rs2842899 132 840 179 0 T > A 0.280 202 201.53 0.94 722 724.6 896 933 0.2852 2 rs2153174 132 842 919 2740 G > T 0.482 184 186.12 0.75 498 488.1 632 608 0.5723 3 rs1933988 132 852 223 9304 A > C 0.401 214 209.57 0.51 595 575.2 758 728 0.2953 4 rs2255071 132 861 218 8995 C > T 0.340 201 204.77 0.56 648 633.2 800 794 0.5342 5 rs1361280 132 865 906 4688 T > C 0.406 182 182.1 0.99 506 513.8 609 604 0.9480 6 rs8192622 132 872 108 6202 C > T 0.052 82 79.92 0.52 985 976.9 1328 1323 0.4072 7 rs8192624 132 872 823 715 G > A 0.073 118 113.48 0.24 954 936.4 1270 1256 0.2503 8 rs8192625 132 872 902 79 G > A 0.068 95 96.82 0.61 939 928.6 1199 1215 0.9210 9 rs7772821 132 873 076 174 T > G 0.122 158 152.05 0.22 834 825.9 1053 1015 0.0751 10 rs4305745 132 874 282 1206 A > G 0.455 164 171.33 0.22 465 474.8 480 514 0.1562 11 rs6903874 132 877 480 3198 T > C 0.167 122 129.51 0.12 787 783.1 939 963 0.7121 12 rs7765655 132 878 428 948 G > A 0.267 173 178.53 0.32 612 638.7 678 724 0.1350 13 rs6937506 132 879 969 1541 G > A 0.177 113 119.04 0.16 763 758.6 914 929 0.3771 14 rs7744878 132 882 090 2121 G > A 0.183 146 156.32 0.06 783 782.8 948 963 0.8617 15 rs7759367 132 884 315 2225 G > A 0.445 164 159.97 0.50 459 476.5 537 537 0.2175 16 rs17195227 132 889 521 5206 T > C 0.355 228 217.65 0.13 662 623.7 866 844 0.1253 17 rs3813354 132 891 204 1683 G > A 0.098 77 73.65 0.31 895 888.1 1148 1135 0.7337 18 rs17061477 132 891 840 636 A > C 0.100 82 79.42 0.45 926 921.1 1203 1186 0.8290 19 rs4320395 132 900 518 8678 G > A 0.460 187 178.56 0.21 514 488.5 723 686 0.1566 20 rs12189813 132 908 072 7554 G > C 0.408 184 168.88 0.02 532 495.4 645 592 0.0094 21 rs9389011 132 915 149 7077 T > C 0.229 247 233.16 0.03 803 789.4 1021 996 0.1597 22 rs9389015 132 924 006 8857 C > T 0.466 201 207.53 0.35 520 525.4 716 729 0.8803 23 rs9389020 132 943 623 19 617 T > C 0.280 247 242.41 0.47 760 730 985 969 0.05437 24 rs9402439 132 948 172 4549 C > G 0.406 175 176.98 0.78 557 572.6 725 727 0.2773

Both markers are located 35 and 42 kb telomeric from the TAAR6 gene. Of the four TAAR6 SNP variants (M6, M7, M8 and M9) only rs7772821 showed trend toward association.

diagnostic category. SNP rs7772821 showed a trend the single-marker level, we used either one or both as toward association only in D2 (0.075pPp0.22). anchor markers in combination with other specific SNPs (Table 2). All SNPs within TAAR6 in two-marker Haplotype analyses combinations with rs12189813 (M20) gave evidence of We initially analyzed haplotypes composed of SNPs association. The most significant of these, and the within the four haplotype blocks (Block 1: M1-M5; most significant two-marker haplotype experiment- Block 2: M11-M16; Block 3 and 4: M21-M24), those wide was for rs7772821 (M9) and rs12189813 (M20) defining specific genes not located within blocks (0.0023pGlobal Pp0.0071). Again, the specific over- (TAAR6: M6-M9; TAAR5: M16-M18; TAAR2: M21- represented haplotype was composed of the common M22) and those defining one small unit of high LD not alleles at both markers (0.00097pPp0.023). A similar identified as a block by default settings in HAPLO- pattern was observed in a three-marker haplotype test VIEW (M19-M20). These analyses were conducted in of rs8192622 (M6), rs7772821 (M9) and rs12189813 sliding windows using 2–5 contiguous markers across (M20), and a four-marker haplotype test of rs8192622 all categories. No contiguous-marker haplotype com- (M6), rs7772821 (M9), rs12189813 (M20) and bination was significantly associated with schizo- rs9389011 (M21), which gave the second most phrenia in any diagnostic category (data not shown), significant association experiment-wide at the global with the exception of the two-marker haplotype (0.0047pGlobal Pp0.018) level with the overrepre- composed of the two individually associated markers, sented haplotype composed of the common alleles rs12189813 (M20) and rs9389011 (M21) (0.0094 (0.0016pPp0.0045). The associated haplotypes were pGlobal Pp0.01, Table 2). Consistent with the always composed of the major allele at each marker. single-marker analysis, the specific haplotype over- These associations were all observed in the core represented in affected family members was com- schizophrenia (D2) diagnostic group. When these posed of the common alleles at both markers haplotypes were analyzed for the broader D5, D8 and (0.012pPp0.049). D9 categories, there was no evidence for association. Since rs12189813 and rs9389011 were the only These results are consistent with the observed LD polymorphisms to show evidence for association at structure of the region (Figure 1), showing moderate

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 847 Table 2 Haplotype analysis of the core markers with rs12189813 and rs9389011 revealed positive association with schizophrenia in the ISHDSF

FBAT v1.5.5 PDTPHASE v5.1

D2

Window Marker HT HT Freq HT Global Trio-T Trio-NT AffSib UnafSib HT Global size P-value P P-value P

SM M20 1 0.58 0.016 0.016 532 495 645 592 0.0094 0.0094 M21 1 0.80 0.028 0.028 803 789 1021 966 0.1597 0.1597 2 M6/M20 1–1 0.54 0.022 0.058 591 548 843 790 0.0052 0.010 M7/M20 1–1 0.52 0.018 0.032 574 530 805 760 0.010 0.0080 M8/M20 1–1 0.54 0.1 0.061 584 545 805 770 0.020 0.024 M9/M20 1–1 0.46 0.023 0.0071 509 467 721 645 0.00097 0.0023 M9/M21 1–1 0.68 0.013 0.021 722 705 967 908 0.02735 0.05722 M20/M21 1–1 0.46 0.049 0.010 496 465 667 637 0.012 0.0084 3 M6/M9/M20 1–1–1 0.45 0.014 0.064 509 465 743 671 0.00090 0.0054 M6/M9/M21 1–1–1 0.67 0.021 0.008 712 696 963 904 0.04513 0.163 M7/M9/M20 1–1–1 0.44 0.013 0.060 491 446 706 644 0.0023 0.032 M8/M9/M20 1–1–1 0.45 0.017 0.034 504 461 730 669 0.0016 0.010 M9/M20/M21 1–1–1 0.35 0.011 0.006 388 358 549 493 0.0020 0.0023 4 M6/M7/M8/M9 1–1–1–1 0.81 0.242 0.660 869 847 1197 1173 0.2331 0.5023 M6/M7/M9/M20 1–1–1–1 0.44 0.016 0.049 493 450 710 648 0.0020 0.014 M7/M8/M9/M20 1–1–1–1 0.43 0.017 0.044 491 442 711 647 0.0012 0.0091 M6/M9/M20/M21 1–1–1–1 0.35 0.0016 0.018 380 354 547 490 0.0045 0.0047 5 M6/M7/M8/ 1–1–1–1–1 0.44 0.023 0.061 492 445 711 649 0.0013 0.0070 M9/M20 6 M6/M7/M8/M9/ 1–1–1–1–1–1 0.37 0.006 0.031 M20/21

M6 rs8192622 TAAR6 M7 rs8192624 TAAR6 M8 rs8192625 TAAR6 M9 rs7772821 TAAR6 30UTR M20 rs12189813 35 kb from the TAAR6 gene M21 rs9389011 42 kb from the TAAR6 gene

Abbreviations: ISHDSF, Irish study of High–Density Schizophrenia Families. The strongest association is represented by the most abundant 1–1 alleles for two-marker haplotype defined by rs7772821 and rs12189813. The second most significant association was found for the 1–1–1–1 four-marker haplotype, defined by rs8192622 and rs8192624 in the TAAR6 gene and rs12189813 and rs9389011 markers. The P-values for two and four marker haplotypes are in typed bold. levels of LD between rs7772821 (M9) and either were also quantified in each sample to control for rs12189813 (M20) or rs9389011 (M21). The sequential variation in mRNA extraction and reverse transcrip- addition of further markers to the sets yielding the tion. Expression of TAAR6, TAAR5 and TAAR2 were results described above did not improve the evidence normalized against these two endogenous controls. for association (data not shown). This region also When analyzed by one-way ANOVA, no significant contains the gene for a second-trace amine receptor, difference in the mRNA levels between diagnostic TAAR5, and is just centromeric to a third, TAAR2. groups was observed for TAAR6 (P = 0.4673), TAAR5 None of the single-marker or haplotype analyses (P = 0.2831) or TAAR2 (P = 0.9731; Figure 2). The anchored to either TAAR5 or TAAR2, showed any ANOVA analysis of potential confounder variables evidence for association (data not shown), suggesting did not show any significant effect of age, pH, PMI, that effects in these genes are unlikely to account for refrigerator interval, or smoking on the expression the observed association with schizophrenia. of TAAR6 (F = 1.919; d.f. = 5, P = 0.1008), TAAR2 (F = 0.700; d.f. = 5, P = 0.6249) or TAAR5 (F = 0.589; Gene expression analysis d.f. = 5, P = 0.7084). We compared the mRNA levels for TAAR6, TAAR5 and TAAR2 between subjects with schizophrenia, Haplotype expression analyses bipolar disorder and unaffected controls by perform- We stratified TAAR6 expression based on the high- ing a quantitative real-time PCR. GAPDH and TBP risk (HR) haplotype identified in ISDFSH sample.

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 848 HR haplotype stratification regardless of disease status. The stratified analysis did not show any signi- ficant difference between HR þ / þ ,HRþ /À and HRÀ/À (P = 0.1984, Figure 3a). In the second analysis each diagnostic group was analyzed separately. Again, no significant differences were observed for schizophre- nic and bipolar cases and controls (P = 0.4548, 0.2013, 0.6056, respectively; Figure 3b). In both analyses no significant interaction between diagnosis and geno- types for rs7772821 and rs12189813 was detected (F = 0.575; d.f. = 30; P = 0.9506).

Operational criteria checklist for psychotic illness We obtain results similar to the family-based asso- ciation studies (in which association was observed only in the narrow D2 diagnostic group) when analyzing the SNP data in a case-only test of asso- ciation with subgroups defined by a priori thresholds of factor-derived scores derived from symptom factors extracted from the OPCRIT (Table 3).37 In our previous work, we showed that the best fit was obtained with a five factor solution composed of individual factors representing negative symptoms, delusions, halluci- nations, as well as manic and depressive symptoms. For each factor-derived scale, subjects scoring in the upper 20%, 30% and 40% were selected and analyzed against the remaining 80, 70 and 60% for association testing. In the OPCRIT factor score analyses we focused on the two-marker haplotype defined by the overtransmited major alleles at rs7772821 and rs12189813 since this haplotype showed the strongest observed association. In the first analysis we analyzed all cases with OPCRIT data. We observe only one significant association, with the highest (top 20%) threshold for delusions (P < 0.009). There were trends approaching significance for hallucinations and mania. No signi- ficant association was observed in the broader 30 and 40% thresholds for any factor scale. The second analysis was restricted to subjects affected under the core schizophrenia (D2) diagnosis. Again, the observed associations are most significant when using the highest (top 20%) threshold for delusions Figure 2 Expression of TAAR6, TAAR5 and TAAR2 in (P < 0.004) and hallucinations (P < 0.0004). These schizophrenic and bipolar cases and controls. The y-axis values represent the ratio of the normalized TAAR6, TAAR5 associations are also observed at the broader top and TAAR2 gene expression to endogenous controls. The 30% and top 40% thresholds, but are of lesser magni- error bars reflects the SE with 95% CI. tude. We also observe some evidence for association with the depression factor scale, although this is difficult to interpret as it is less significant for the Cases and controls in SBS sample were genotyped top 20% threshold than for the top 30% threshold. for rs7772821 and rs12189813 together with two Nevertheless, these analyses are consistent in show- additional markers to provide sufficient inform- ing evidence for association with schizophrenia only ation for haplotype reconstruction using PHASE. in our narrowest and most severely affected diag- Reconstructed haplotypes with common alleles at nostic group. rs7772821 and rs12189813 were defined as high-risk positive (HR þ ), and all others defined as high-risk Control for multiple testing negative (HRÀ). Both, cases and control samples While unexpected behavior of the test statistic due to were then assigned to HR homozygous (HR þ / þ ), characteristics of our data (patterns of missing data, heterozygous (HR þ /À) and non-HR (HRÀ/À) diplotype etc.) is accounted for by permutation tests within the groups. We performed two analyses. In the first ana- FBAT program, we sought to address the issues of lysis the TAAR6 expression was evaluated based on multiple testing by performing a separate set of Monte

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 849 genotype data are maintained as part of this simula- tion. In each replicate of simulated data, all of the FBAT haplotype analyses performed in the observed data were repeated. These results were used to cons- truct a null distribution of the smallest P-values gene- rated for all global tests from each of the replicates. We believe that the values resulting from Monte Carlo simulations of global tests are more reliable because they exclude P-values generated from very rare haplotypes, which are more likely to generate spuriously small P-values. Since the global tests tend to be more stable, we focused on the question, what is the probability that we would find as the smallest global test P-value one equivalent to or smaller than the one we observed in our data when conducting this number of tests (NB1200 from 24 SNPs in four diagnostic categories with both sliding window and marker-anchored haplotype analyses). We generated 1000 replicate data sets for the purpose of assessing the significance of our most significant global test (P = 0.0071). For each set of permutation tests, significance is calculated by (r þ 1)/ (n þ 1), where n is the total number of Monte Carlo procedure tests conducted and r is the number of those more significant than the most significant observed test from the actual data. As a diagnostic assessment of the performance of the test statistic as a whole, we compared it to the entire null distribution of all global tests conducted over all 1000 replicates. We find an empirical P-value of 0.0073, indicating that P-values less than or equal to our observed P-value of 0.0071 occur with a frequency of 0.0073 in our permutations. This shows that the empirical P-values from the Monte Carlo procedure were similar to those generated from the permutation test in the FBAT package was expected and not the primary focus of the Monte Carlo procedure. In the comparison of our best-observed global P-value to the distribution of per replicate best global test P-values, we generated an empirical P-value of 0.224. Thus, within the assessment of global tests, we have a 22.4% chance of randomly generating a most significant global result of the magnitude of our best-observed global test when performing this total number of tests.

In silico analysis Within the HR defined by rs7772821 and rs12189813 we defined the conserved non-coding sequence by comparative genomic analysis of TAAR6 between Figure 3 (a) TAAR6 expression evaluated based on human, mouse and rat using VISTA. We found that haplotype stratification regardless of disease status. (b) the homology between the coding regions of the The TAAR6 gene expression for three haplotypes (HR þ / þ , þ /À À/À TAAR6 orthologs is about 80%. rs7772821 (M9) is HR and HR ) is evaluated in each diagnostic group. 0 The error bars reflect the s.e. with 95% CI. located in the 3 UTR of TAAR6 eight base pairs from the stop codon. Homology is generally lower in the 30UTR, dropping below 50% at the 30 end, but is Carlo simulations. The simulations were first per- similar to that for the coding sequence in the specific formed in the pedigrees using Merlin to simulate area containing this polymorphism. The closest con- haplotype data and perform gene dropping in the served region (with homology of over 70% between pedigrees.18 Importantly, the LD structure, pheno- human, mouse and rat genomic sequences) is located types, family structures and pattern of missing 0.4 kb downstream from rs7772821 (M9) in the

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 850 Table 3 The two-marker haplotype 1–1 represented by rs7772821 and rs12189813 was further analyzed for an association with the five OPCRIT symptom dimensions

Threshold (%) Negative symptoms Delusions Hallucinations Mania Depression

All individuals with psychosis 20 0.567 (141) 0.009*** (119) 0.075*a (187) 0.089* (110) 0.932 (103) 30 0.221 (181) 0.122 (176) 0.075*a (187) 0.456 (169) 0.995 (144) 40 0.071* (227) 0.285 (208) 0.135 (261) 0.819 (224) 0.960 (190)

Individuals with D2 narrow diagnostic 20 0.491a (139) 0.004*** (113) 0.0004*** (107) 0.733 (99) 0.035** (93) 30 0.491a (139) 0.037** (141) 0.012**a (177) 0.865 (136) 0.007** (131) 40 0.166 (173) 0.047** (166) 0.012**a (177) 0.184 (188) 0.226 (167)

Abbreviation: OPCRIT, operational criteria. The vertical transmissions of the haplotype from parents to affected offspring are given for each psychosis symptom scale. Number of families with transmission to affected offspring is shown in parenthesis. Significant P-values are highlighted in bold using * < 0.1, ** < 0.05 and *** < 0.001. Affected individuals with psychosis (n = 749) and individuals limited to the core schizophrenia diagnosis (n = 614) were included in the analysis. aSame cutoff threshold for each level.

TAAR6 TAAR5 100% intergene intron rs7772821 /Sequence/rn coding - vs - UTR /Sequence/hg repeat 50% 0 9.6kb 19.1kb 28.6kb 38.2kb

TAAR6 TAAR5 100% intergene intron /Sequence/rn coding - vs - UTR rs12189813 /Sequence/hg repeat 50% 0 9.6kb 19.1kb 28.6kb 38.2kb

-100% intergene intron /Sequence/rn coding - vs - UTR /Sequence/hg repeat

50% 38.2kb Figure 4 The conserved non-coding regions are defined by VISTA,32 and rs7772821 and rs12189813 positions are shown by the arrows. The conserved regions are generated by comparing with mouse and rat. The region with sequence similarity reaching 70% is defined as a conserved region, and such peak areas are shaded. The darkest shaded areas represent the TAAR6 and TAAR5 genes.

TAAR6 30UTR. Marker rs12189813 (M20) did not lie sequences that are not folded could be available to in a close proximity to an evolutionarily conserved pair with other regulatory factors (e.g. miRNAs). region (Figure 4). Indeed, after scanning the evolutionarily conserved To evaluate any potential effect rs7772821 could region within the 30UTR, four overlapping target have, we analyzed the secondary structure of sequences for four miRNAs (hsa-miR-92, hsa-miR- the TAAR6 mRNA in silico by the algorithm for 125a, hsa-miR-302b and hsa-miR-483) were pre- free energy minimization. The T and G alleles of dicted. The observation of overlapping sequences rs7772821 are predicted to have a pronounced effect recognized by several miRNAs is not surprising since on the folding of the mRNA affecting its conformation it has been suggested that multiple miRNAs can act in and stability38,39 (Figure 5). The secondary structure combination by binding the same mRNA in concen- of the TAAR6 mRNA 30UTR is also important since tration-dependent manner.40

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 851

Figure 5 The RNA secondary structure was predicted with RNAstructure 4.233 for free energy minimization algorithm using the nearest neighbor parameters. A recursive algorithm is used that generates an optimal structure and a series of structures that are called suboptimal structures (structures with free energy similar to the lowest free energy structure). The number of suboptimal structures generated is controlled by the maximum number of structures (N = 20) and maximum % of energy difference (10 kcal), which is the percent difference from the lowest free energy allowed for the structures output. The arrows indicate the position of the rs7772821 polymorphism within circle.

Discussion replication is differences either in the presence or the frequency of a liability allele between different TAAR genes have been proposed as candidates for populations. It is known that differences in the allele schizophrenia based on the pathophysiological evi- and haplotype frequencies can affect both the LD dence as well as linkage mapping and association structure and strength of association. Thus, the high- data.4,41,42 The original association study reported one risk allele might have a very different pattern of both SNP variant in the 30UTR of the TAAR6 gene to be LD and association across different populations and significantly associated with schizophrenia after marker sets useful in one study may not be as useful correction for multiple testing. in another.48,49 During the course of this work four independent Across various analyses performed, the haplotype studies attempted to replicate the original finding for of rs7772821 (M9) and rs12189813 (M20) gave the association between TAAR6 and schizophrenia, of strongest evidence for association with schizophrenia which three were negative and one positive. The first in the ISHDSF sample. Although separated by 35 kb, two negative replications were performed (in chron- the two markers are in moderate LD with each other ological order of their publications) in 235 family trios (D0 = 0.63). A haplotype of this size is not unusual, as from the Han Chinese population and in two inde- reported the average haplotype size in the European pendent sets of Japanese case–control samples.43,44 population is about 22 kb.21 The third study used 53 Arab Israeli families with The location of rs7772821 in the 30UTR and in the previous highly significant evidence for linkage to vicinity of an evolutionarily conserved region bet- 6q13–q26 in a putative susceptibility region of 3.90 Mb ween mouse and human makes this an interest- containing the TAAR6 gene. This study also failed ing candidate for further study. The predicted RNA to replicate the original association.45 All negative secondary structure for the overtransmited rs7772821T studies claimed to be adequately powered to detect allele differed substantially from the undertransmited association. So far, the only positive report found G allele. Such polymorphisms may have functional was a common two-marker haplotype (rs6907909, effects on mRNA conformation or stability, and hence rs9373026, P < 0.001) in the promoter region of TAAR6 gene expression level.50 However, we observed no to be associated with schizophrenia only in Caucasian differences in mRNA abundance between clinical cases, and an additional study found TAAR6 to be groups or haplotypes. Conversely, miRNAs interfere associated with bipolar disorder in two independently with translation, so their regulatory effect will only be analyzed samples from the German population.46,47 detectable at the protein level. Using the miRanda Since these studies were performed in different package, target sequences for four miRNAs were ethnic groups, a possible explanation for the non- identified in this region. One of these, hsa-miR-125a,

Molecular Psychiatry TAAR6 association with schizophrenia in ISHDSF V Vladimirov et al 852 was shown to be expressed in mouse cortex and bution of the test statistics under the null hypothesis. .51 It is therefore possible that protein-level We observed very good agreement between the expression differences mediated by miRNA might be empirical and actual P-values strengthening the observed. This is an area of growing importance, as robustness of our findings. Additionally, there is a recent reports predict that over one-third of all human higher prior probability of association because we are genes might be regulated by miRNAs.52 studying a region previously associated with the trait The rapid decline in the strength of association as of interest. While we cannot be certain, we would we moved away from the core schizophrenia diag- suggest that these lines of evidence (prior association nostic category suggests that in our sample, TAAR6 and our association) make it unlikely that our results might be implicated as a liability factor only for reflect false positives. As always, replication of these schizophrenia although in a recent study from findings will be necessary. Germany, TAAR6 was implicated as a liability locus In summary, our results show an associated haplo- for bipolar disorder.46 Because of very limited number type overlapping the previously described haplotype of bipolar cases both in our sample and in SBS we had and supporting the original study, and thus providing insufficient power to test meaningfully for association additional evidence of a role for TAAR6 as a between TAAR6 and this disease. susceptibility locus for schizophrenia. Our analyses of clinical subgroups defined by thresholds on OPCRIT factors suggest that genetic variation in TAAR6 predisposes to a form of psychotic Acknowledgments illness marked by high levels of delusions, hallucina- We thank the patients and families for their participa- tions, and possibly depressive symptoms. As such, tion. This work was supported by NIH grants we would classify TAAR6 as a mixed susceptibility- MH041953-13 and MH068881-02. modifier gene, as described previously.53 To date, there have been two reports of susceptibility genes influencing clinical features of illness: DTNBP1 References and negative symptoms of schizophrenia, as well as DAOA and persecutory delusions in bipolar disorder. 1 Jablensky A, Sartorius N, Ernberg G, Anker M, Korten A, Cooper JE et al. 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Molecular Psychiatry