Family-Based Study of HTR2A in Suicide Attempts: Observed Gene, Gene Â Environment and Parent-Of-Origin Associations

ORIGINAL ARTICLE Family-based study of HTR2A in suicide attempts: observed gene, gene Â environment and parent-of-origin associations

YJ Ben-Efraim, D Wasserman, J Wasserman and M Sokolowski

While suicidal behavior is frequently accompanied by serotonergic system alterations, specific associations with genetic variation in the serotonin 2A receptor (HTR2A) gene have been inconsistent. Using a family-based study design of 660 offspring who have made a suicide attempt (SA) and both parents, we conducted an association and linkage analysis using single- nucleotide polymorphisms (SNPs) with extensive gene coverage, and included the study of parent-of-origin (POE) and gene-- environment interaction (G Â E), also using previously unstudied exposures. The main finding was a G Â E between the exon 1 SNP rs6313 and exposure to cumulative types of lifetime stressful life events (SLEs), driven by overtransmission of CT and undertransmission of TT, both in relation to other genotypes. Further exploratory analysis revealed a significant POE in this G Â E in female subjects, which followed a polar overdominant inheritance pattern. In addition, rs6310 and rs6305 were found to significantly associate with SA in the total sample. A G Â E in female subjects (rs7322347 Â physical assault in childhood/ adolescence) confirmed features of a previously observed association with SA. Other potentially interesting nominally significant findings were observed, but like the G Â E of rs7322347 did not pass a false-discovery rate cutoff. Taken together, this study found multiple associations of HTR2A SNPs on SA, with strongest statistical evidence for a G Â E involving rs6313, and further suggested the importance of taking into account different inheritance patterns and G Â Es with regard to HTR2A.

Molecular Psychiatry (2013) 18, 758--766; doi:10.1038/mp.2012.86; published online 3 July 2012 Keywords: family-based association; gene--environment interaction; parent of origin; serotonin receptor; stress; suicidality

INTRODUCTION Gene--environment interactions (G Â Es) with stressful life Approximately 1 million people commit suicide each year,1 while events (SLEs) are of importance in a stress-diathesis model of 19 at least 10 times as many make a suicide attempt (SA).2 Identifying the suicidal process. Exposure to traumatic SLEs during child- genetic variants that underlie complex disorders such as hood/adolescence, in particular childhood sexual and physical suicidal behavior may be aided by studying endophenotypes3 abuse (CSA and CPA), are frequently observed in suicidality.20 and severity measures.4 Serotonergic system alterations have Stress sustained throughout life, however, may act on a back- been found in several suicidal behavior endophenotypes,5 and ground of heightened environmental susceptibility caused by lowered cerebrospinal fluid concentration of a serotonin metabo- allostatic loading of the neuroendocrine response to psychosocial lite is one of the best-known biological predictors of future suicide stress.21 G Â Es between intron 2 SNPs and CSA/CPA on SA have along with dexamethasone non-suppression.6,7 Changes in brain been reported.17 serotonin level8 and receptor expression9 occur in suicidality, HTR2A may also be epigenetically regulated. Studies focusing including binding potential changes of the serotonin 2A receptor on rs6313 show evidence of reduced C-allelic expression in suicide (HTR2A) in the prefrontal cortex in vivo10 and in suicide post-mortem brain tissue and DNA hypermethylation in peripheral post-mortem tissue.11 Further studies of HTR2A in suicidality, leukocytes of schizophrenic SA.22,23 Maternally inherited rs6313 T- however, yielded inconsistent observations with regard to alleles (Tmat) showed nonsignificant (NS) trends for association expression changes in specific brain regions12 and peripheral cell with SA and suicidal behavior severity,22,23 suggesting possible types, for example, platelets.13 parent-of-origin effects (POEs). Such inconsistencies may be caused partly by genetic variation. To complement and possibly extend the previously reported Three single-nucleotide polymorphisms (‘SNPs’, that is, A-1438G HTR2A genetic studies on SA, we wished to investigate the coding (rs6311), T102C (rs6313) and His452Tyr (rs6314)) have been and flanking regions for genetic associations, G Â Es and POE (at widely investigated in expression and association studies on rs6313) using a family-based design. Family-based genetic suicidality; however, also yielding inconsistent observations.13,14 association studies are robust to population stratification, which Meta-analyses of rs6313 showed no association with suicidal may generate spurious associations in population-based studies. behavior,15,16 although promoter SNP rs6311 (often in perfect To our knowledge, few family-based studies of HTR2A with linkage disequilibrium (LD) with rs6313) was found to associate adequate power to detect anticipated modest effects of common with suicidal behavior and/or ideation in fewer studies.16 variants have investigated endophenotype-related outcomes,24--26 More comprehensive investigations of variation have revealed and none have investigated SA as the primary outcome. We also novel associations with SA,17 and a different promoter SNP investigated previously unstudied SNPs with low minor allele affecting transcription.18 frequency (MAF), as they may display larger effects than common

The National Centre for Suicide Research and Prevention of Mental Ill-Health (NASP), Karolinska Institute (KI), Stockholm, Sweden. Correspondence: Dr M Sokolowski, The National Centre for Suicide Research and Prevention of Mental Ill-Health (NASP), Karolinska Institute, Stockholm 171 77, Sweden. E-mail: [email protected] Received 17 October 2011; revised 2 April 2012; accepted 29 May 2012; published online 3 July 2012 Family-based study of HTR2A in suicide attempts YJ Ben-Efraim et al 759 SNPs on complex disorders.27 Overall, this study aimed to map the childhood/adolescence sexual assault (11.8%) and physical assault linkage and association of HTR2A SNPs on SA in a detailed manner. (18.6%); adulthood sexual assault (8.5%) and physical assault (21.9%); and high lifetime SLEs (51.6%).

PATIENTS AND METHODS Outcome measures Research subjects SA main outcome measure and secondary outcome covariates. All Subjects were collected in the Ukraine in 2001--2006, as described X 28 index SA had a score of 2 on the Medical Damage Rating previously, numbering 660 complete and extensively quality- 37 29 Scale, and this was the main outcome measure of the study. To controlled nuclear family trios. The collection of research better characterize index SA and covariation with SA-related subjects followed the code of ethics of the World Medical outcomes, seven SA-concomitant secondary outcomes and twelve Association (Declaration of Helsinki), and a written consent was other covariates with diverse life timing were assessed at the obtained. The study was approved by the Research Ethics interview (Supplementary Methods). Committee at the Karolinska Institute (Dnr 97--188) and by the Ministry of Health in the Ukraine. Selection criteria, sample Statistical analyses demographics, International Classification of Diseases 10th edn. Tests of Hardy--Weinberg equilibrium were performed in Haplo- (ICD-10) psychiatric diagnoses, DNA preparation and genotyping 38 have been previously described for trios.30,31 Briefly, SA offspring view v.4.2 using the default settings, and additionally confirmed were Ukrainian citizens with 94.1% having X3 grandparents of with the threshold P40.05 for any significant associations. Allelic Ukrainian/Russian ethnicity, 51.1% were male and 48.9% were tests of association were performed on all SNPs and genotypic female. The mean (±s.d.) age of offspring was 24.6 (7.3) and 23.8 tests were performed on the set of SNPs with previously reported non-additive effects on SA (rs6313, rs7322347, rs6561333 and (7.1) years for males and females, respectively. Comorbidity has 13,14,17 39 been described previously,31 although some disorders were rs7997012) or central serotonergic activity (rs6314). Allelic aggregated in SA offspring (for power) according to ICD-10, that and genotypic tests of main genetic effects were performed by is, depression or anxiety disorders were prevalent in 41.8%; using an additive model family-based association test in FBAT schizophrenia, schizotypal or delusional disorders were prevalent v.2.0.3 (http://www.biostat.harvard.edu/~fbat/default.html) or genotype relative risks command GTRR40 in software package in 12.9%; and past-year alcohol or substance-use disorders were 41 prevalent in 18.1% (further sample description in Supplementary GENASSOC as implemented in Stata v.11 (StataCorp, College ± Station, TX, USA), respectively. Allelic tests of G Â E were performed Methods). Adult SA had mean ( s.d.) of 12.4 (2.3) years of 42,43 education, 32.3% were unemployed, 29.8% were married or living by using fbat-i in pbatR; P-values are reported for minor alleles. with a partner, while 16.2% were divorced, separated or widowed. G Â E effect size estimates were calculated by using conditional logistic regression using one or three matched pseudosibs per SA offspring44--46 and are displayed as odds ratio with 95% confidence SNPs intervals for the SLE-exposed stratum relative to the SLE- We selected 43 ‘common’ SNPs (MAFX0.05) that tagged32 all the 2 unexposed stratum. Genotypes were coded as indicator vari- other known common SNPs by LD (r X0.80), using the HapMap ables.44,47 Likelihood ratio test P-values are reported for genotypic database (release 24) reference panel of Utah residents with 33 tests and for post-hoc data fitting of genetic models (Supplemen- ancestry from northern and western Europe, and seven ‘low- tary Methods). A transmission disequilibrium test in Haploview frequency’ SNPs (0.01oMAFo0.05) for genotyping (SNP&SEQ v.4.238 or command GTRR in Stata v.11 were used to determine Technology Platform, Uppsala, Sweden). After imputation of allelic or genotypic counts, respectively, and transmission distor- sporadic missing genotypes, untyped SNPs and processing tions (Po0.05) within exposure strata. Minimum counts were set to (Supplementary Methods), 49 SNPs were included in the analysis: nX20 transmissions of each allele or nX5 informative SA offspring 38 genotyped and 4 imputed common SNPs that tag 98% of per genotype (no pooled genotypes) for each exposure stratum. currently known common variation in the coding region (HapMap Analysis of low-frequency SNPs was performed with command database, release 24) and 100% of flanking regions 11.9 kb GTRR using aggregated cells for heterozygotes and minor 0 0 upstream (5 ) and 6.5 kb downstream (3 ), and 7 low-frequency allele homozygotes.48 A threshold of P ¼ 0.25 was used to decide genotyped SNPs (Figure 1 and Supplementary Table 1). whether to follow-up tests in the total sample with sex-specific testing. SLEs The false discovery rate (FDR)49 was controlled by calculating Environmental exposures were assessed with questions originally positive FDR analogs (Q-values) of P-values50 (Table 1) by using obtained from the Life Events section of the European Parasuicide the software fdrtool.51 An FDR cutoff of 0.10 was used, as Study Interview Schedule v.5.134 and the post-traumatic stress recommended for genetic discovery52 (conservative control), as disorder section of the Composite International Diagnostic Inter- well as a cutoff of fewer than 1 false positive among declared view v.2.1.35 Three questions from the post-traumatic stress ‘significant’ findings (liberal control). Uncorrected (nominal) P- disorder section of Composite International Diagnostic Interview values are shown, which are referred to as NS (P40.10), NS covering exposure to rape, molestation and serious physical tendencies (0.05oPo0.10) or nominally significant (uncorrected assault or attack were assessed in relation to the age of exposure Po0.05). All P-values reported in this study are two-tailed. to distinguish different life timing: 18 years or younger (childhood/ Statistical power was calculated by using the software QUANTO adolescence) and 18 years or older (adulthood). Rape and v.1.2.3 (http://hydra.usc.edu/gxe/).53 Power was calculated at molestation were combined into a single exposure (sexual a ¼ 0.01 (in anticipation of presumed FDR control at 0.10)52 for a assault). In addition, a broad lifetime exposure checklist assessed range of MAF specific for each test according to the previously SLE types selected according to Kendler et al.36 and all of the post- mentioned inclusion criteria of minimum transmissions (Supple- traumatic stress disorder section of Composite International mentary Table 2). Diagnostic Interview, as described previously.29 An SLE scale was constructed from the checklist by summing the number of different types of lifetime exposures for each individual (lifetime RESULTS SLEs). Exposure to high lifetime SLEs was operationally defined as Association and linkage of HTR2A SNPs on SA outcome a sum of X5 (median score in either sex) and low lifetime SLEs as SNPs did not deviate from Hardy--Weinberg equilibrium (P40.001 a sum of o5. Exposure prevalence in SA offspring was as follows: for all SNPs and P40.05 for 45 out of 49 SNPs). We first tested 49

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 758 -- 766 Family-based study of HTR2A in suicide attempts YJ Ben-Efraim et al 760

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Figure 1. Schematic of the gene and linkage disequilibrium map. Above: Schematic diagram of the serotonin 2A receptor gene (HTR2A) and its reference position on the complementary strand of chromosome 13. Ensembl protein coding sequences show alternative 5’ untranslated regions (clear boxes on the right), 3 exons (dark boxes) and 2 introns. Alternative splice variants are not shown. The arrow marks the direction of transcription. The distance of LD coverage by tag SNPs1 used in this study (81.1 kb) is marked by the ruler, and the range of base pair positions covered is displayed at the ﬂank edges. Below: LD (r2) map showing 45 genotyped and 4 imputed SNPs included in the study relative to their position. Solid circles mark the position of imputed SNPs, and dotted circles mark the position of genotyped SNPs with minor allele frequency o0.05. LD, linkage disequilibrium; SNP, single-nucleotide polymorphism.

common and low-frequency SNPs (Supplementary Table 1) for G Â E no. 3. A genotypic test of rs7322347 Â childhood/adoles- association and linkage in the total sample, and observed cence physical assault was nominally significant in females only nominally significant associations for two common SNPs in the (Table 1). A synergistic G Â E pattern was observed with a TT- 50 flanking region, rs17289304 and rs6310, and for a low- homozygote protective effect among the exposed (Figure 2c). frequency synonymous SNP in exon 2: rs6305 (Table 1). Minor Accordingly, a recessive model was observed to fit best in post-hoc alleles were overtransmitted (that is, had a ‘risk effect’) for all three analysis (Supplementary Table 3). SNPs, and associations were best fit by an additive genetic model (Supplementary Table 3). Subsequent sex-stratified analysis did FDR not reveal increased significance for these SNP associations Q-values were calculated for all study-wide genetic and G Â E relative to the total sample; however, another low-frequency effect P-values generated (n ¼ 316 tests). The genotypic test of intron 1 SNP rs2070036 was nominally significant in females only rs6313 Â lifetime SLEs in the total sample passed our conservative (Table 1). threshold for significance (FDRo0.10), whereas the top four findings (ranked according to P-value, that is, rs6313 G Â Es, G Â Es on SA outcome rs6310 and rs6305; Table 1) passed a more liberal threshold for G Â E no. 1. A genotypic test of rs6313 Â lifetime SLEs was significance, that is, 4 Â 0.219o1 false positive estimated. We also nominally significant in both the total sample and in females present all findings with FDRo0.25 (Table 1), but at least two false only (Table 1). An antagonistic G Â E pattern was observed, that is, positives are then expected at this level of FDR control among all with CT-heterozygote risk effect and TT-homozygote under- 10 associations. transmission (‘protective effect’) among the unexposed (Figure 2a, Table 1 and Supplementary Table 3). An overdominant Exploratory analyses model best fit the total and females-only data in post-hoc analysis POE. Maternal genetic effects were NS for all allelic tests of POE (Supplementary Table 3). (data not shown). When not accounting for G Â E, POE of rs6313 was NS (data not shown). We next took G Â E no. 1 into account, G Â E no. 2. An allelic test of rs1928042 Â lifetime SLEs was investigating POE in the low lifetime SLEs stratum only and nominally significant in males only (Table 1). A synergistic G Â E the allelic POE test was NS in the total sample (Table 2). In pattern was observed with a minor C-allele protective effect females, an NS tendency was, however, observed for Tmat risk and among the exposed (Figure 2b). Both additive and recessive paternal T-allele (Tpat) protective effects (Table 2). As several models fit the data to a similar degree in post-hoc analysis imprinting patterns are possible,54 we next investigated POE in a (Supplementary Table 3). genotypic test, and this rendered POE nominally significant with

Molecular Psychiatry (2013), 758 -- 766 & 2013 Macmillan Publishers Limited Family-based study of HTR2A in suicide attempts YJ Ben-Efraim et al 761 Table 1. Linkage and association of SNPs and interaction effects on SAs

Effect size Allelic: MiA GT: MiA/MiA No. of informative Position relative Allelic or -/MiA transmissions SNP ( Â SLE) to coding region MiA GT test Sex CLR OR (95% CI) P-valuea Q-value GT: SA offspring

Main effects rs17289304 50-Upstream C Allelic MF 1.46 (1.12--1.90) 0.0052 0.222 134 C: 92 A rs6310 50-UTR G Allelic MF 1.61 (1.18--2.20) 0.0025 0.194 103 G: 64 A M 1.77 (1.14--2.76) 0.0097 0.238 55 G: 31 A rs2070036b Intron 1 C Allelic F 0.41 (0.20--0.83) 0.0095 0.237 26 A: 11 C rs6305b Exon 2 T Allelic MF 2.03 (1.22--3.38) 0.0047 0.219 47 T: 22 C F 2.44 (1.21--4.91) 0.0087 0.236 28 T: 11 C G Â E effects rs6313 ( Â Exon 1 T GT MF 0.51 (0.25--1.04) 0.0002 0.049 90 CC: 113 CT: 46 TTc lifetime SLEs) 1.64 (1.06--2.50) 64 CC: 140 CT: 20 TTd F 0.61 (0.23--1.64) 0.0033 0.206 49 CC: 58 CT: 25 TTc 2.00 (1.08--3.70) 26 CC: 69 CT: 11 TTd rs1928042 Intron 2 C Allelic M 0.55 (0.35--0.87) 0.0078 0.233 92 A: 62 Cc ( Â lifetime SLEs) 81 C: 66 Ad rs7322347 Intron 2 T GT F 0.07 (0.01--0.64) 0.0087 0.236 17 AA: 18 AT: 1 TTc ( Â PA-U18) 0.74 (0.34--1.62) 63 AA: 87 AT: 46 TTd Abbreviations: CI, conﬁdence interval; CLR, conditional logistic regression with matched pseudosibs; F, female; GT, genotype or genotypic; G Â E, gene-- environment interaction; lifetime SLEs, exposure to median-dichotomized ‘high’ vs ‘low’ cumulative lifetime SLE types (median ¼ 5 in either sex); M, male; MiA, minor allele; OR, odds ratio; PA-U18, exposure to serious physical assault or attack at or under age 18 years; SA, suicide attempt; SLE, stressful life event; SNP, single-nucleotide polymorphism; UTR, untranslated region. aApproximates but may not exactly match the effect size P-value (see Patients and methods). bLow-frequency SNP (0.01ominor allele frequency o0.05). cExposed. dUnexposed.

Tmat overtransmission in the form of TmatCpat heterozygotes P ¼ 0.0247), and best fit by additive and recessive models, (Table 2). The transmission distortion (Figure 3) was best fit by an respectively (data not shown). imprinting pattern called polar overdominance (Table 2).55 A POE in the context of G Â E no. 1 was subsequently confirmed in females (P ¼ 0.0340), with TmatCpat heterozygotes showing the DISCUSSION only significant interaction parameter (odds ratio (95% confidence Primary findings intervals) ¼ 3.98 (1.51--10.49)), and the G Â E data were similarly A previous study examined G Â E on SA between HTR2A best fit by polar overdominance (genotypic test P ¼ 0.0034). variants and CSA and CPA exposures, but did not detect G Â E at rs6313.17 We found that G Â E no. 1 was independent of Secondary outcome covariates. We next adjusted the findings with lifetime physical or sexual assault exposures. The heterozygote risk common SNPs that had FDRo0.25 (Table 1) by using 19 covariates and TT-homozygote protective effects observed here in G Â E (Supplementary Methods). Significant covariate interactions agree with certain previous genetic findings on SA,56,57 although (Po0.01) were not observed; however, we observed a number no genetic effects have been observed consistently.13,14 Our of interaction trends in a covariate-stratified analysis of SNPs and observation of POE with increased TmatCpat inheritance in SA may G Â Es (Supplementary Table 4). imply imprinting. To our knowledge, this is the first observation that a neuropsychiatric outcome associates with (1) POE in HTR2A Exposure specificity. The relative contributions of specific SLEs in (although the maternal copy of nearby chromosomal region the observed G Â E nos. 1--3 was further probed by removing or 13q12 has been linked with bipolar disorder)58 and (2) a polar combining them before G Â E testing. G Â E no. 1 (in the total overdominant inheritance pattern (however, this was observed for sample and females) and no. 2 remained nominally significant DLK1 on obesity).59 after removing those trios with SA offspring exposed to lifetime Imprinting and methylation have previously been studied for physical or sexual assault, with maintenance of transmission HTR2A in association with suicidality. Analysis of rs6313 in post- pattern and best-fit genetic model (data not shown), further mortem dorsolateral prefrontal cortex of heterozygous suicides supporting that these G Â Es were driven by the broader panel of showed reduced C-allele expression22 and NS differences in lifetime SLEs rather than specific physical or sexual assault methylation vs controls.23 No suicides exhibited pure monoallelic exposures. The significance of G Â E no. 3 recessive model was expression, indicating that it might operate only in a proportion of abolished after removing trios with SA offspring exposed to cells from each individual,22,23 or partial C-allele silencing. With childhood/adolescence sexual assault and physical or sexual regard to peripheral tissue, increased methylation of rs6313 was assault in adulthood (data not shown), that is, we were therefore observed in leukocytes of schizophrenic but not bipolar SA,23 but unable to demonstrate additional support that G Â E no. 3 was other studies show that monoallelic expression in certain blood driven specifically by childhood/adolescence physical assault. cell populations is frequent and random.60,61 Apart from Finally, we also explored a G Â E shown by others between CSA suicidality, monoallelic (T-allele) expression or reduced C-allele and SNP rs7997012 on SA,17 as it was potentially related to expression was observed in post-mortem brain tissue,60,62,63 but G Â E no. 3 (see Discussion). Only after combining adulthood not always,64 implying that cell- and/or region-specific imprinting physical or sexual assault exposures, a nominally significant may occur only in certain individuals. Considering POE was G Â E was observed with rs7997012 minor T-allele protective observed only in a subgroup of SA, we hypothesize that a complex effects in the total sample (allelic P ¼ 0.0111 and genotypic pattern of cell- and/or tissue-specific paternal imprinting of HTR2A

& 2013 Macmillan Publishers Limited Molecular Psychiatry (2013), 758 -- 766 Family-based study of HTR2A in suicide attempts YJ Ben-Efraim et al 762 may be widespread,65 with some loci showing polar overdominance.66 POE may confound genetic association and other types of epigenetic association; thus, further association, expression, and methylation studies on HTR2A that consider POE are important. Selecting relevant tissue and/or cell types for these studies will be challenging. For example, methylation of rs6313 correlated with methylation levels of perfect LD promoter SNP rs6311 in the brain.67,68 However, the correlation of methylation in the core promoter or near neighboring transcription factor binding sites was observed to co-vary with tissue and other factors, for example, medication use.68--70 Other mechanisms may be involved. Overdominance could reflect several distinct molecular events including epistasis,71 and TT-homozygote effects may also be involved: TT-homozygote moderated the effect of COMT on impulsiveness72 and more complex three-locus genotype interactions were observed with COMT and TPH1 on antisocial personality disorder.73 Various patterns of genomic imprinting have also been observed to depend on genetic background.74 In addition, rs6313 has predicted functionality, for example, in splice variation,75 implying that post- transcriptional mechanisms may also be of importance. Other SNPs with predicted or reported functionality were tagged by rs6313 (Supplementary Figure 1), that is, rs6311, rs732821 and rs731245.76 Previous studies on SA showed that rs6313 T-allele or TT-genotype (or rs6311 A-allele carriers by LD) had lower impulsivity, anger, aggression and/or had reduced risk for non-violent SA.77--79 In comparison, our results on G Â E no. 1 showed that it was CT-heterozygotes that tended to have increased risk for planned (less impulsive) and non-violent SA, marked by high anger and aggression (Supplementary Table 4). Using the more liberal FDR threshold (Q ¼ 0.219), three more associations were found to be significant (Table 1). The proportion of false positives is controlled similarly to FDR by Q-values when tests are weakly dependent,50,80 which we assume since none of the tested SNPs were in high LD (r2o0.80). Thus, although the FDR threshold here is more liberal (0.219), no false positives were expected. Rs6310 has not previously associated directly with suicidality or endophenotype-related outcomes, but several SNPs were tagged by rs6310 (Supplementary Figure 1). Of these SNPs, only rs6312 has been shown to be functional, that is, it interacted with rs6311 on transcription.18 We observed that the minor G- allele of rs6310 was always in phase with the major C-allele of rs6313 in our sample (data not shown). Thus, the direction of our G-allele (rs6310) risk effects were congruent (by high LD) with a reduction of promoter activity in vitro using an unmodified version of the regulatory region.18 Our CT-heterozygote (rs6313) risk effects were also congruent with a reduction of mRNA expression in ex vivo fibroblasts in the same study. An interaction was further observed between rs6311 and rs6312 on mRNA expression, while Figure 2. Gene--environment interactions (G Es) on suicide Â our associations were observed separately as genetic and G E attempt (SA) outcome. (a) SNP rs6313 Â lifetime SLEs in the overall Â sample, (b) SNP rs1928042 Â lifetime SLEs in male subjects and (c) associations. Thus, extrapolating to SA association is not SNP rs7322347 Â PA-U18 in female subjects. Minor alleles are dis- straightforward, particularly since in vivo expression may be played in black and major alleles and heterozygotes in gray or affected by additional factors, for example, stress hormones, hatching pattern. Exposure strata are labeled on the X axis for low drugs, and so on. Finally, the risk effect observed for rs6305 T- lifetime SLEs/unexposed (À) and high lifetime SLEs/exposed ( þ ). allele was previously shown on obsessive-compulsive disorder,81 Significant transmission distortions are marked within the strata (1 and this SNP has predicted splicing function.75,76 degrees of freedom (d.f.) tests unadjusted for the other genotypes). Significant interactions are also marked across the strata (either 1 d.f. unadjusted tests, or 2 d.f. full genotypic tests as shown in Table 1). Secondary findings Lifetime SLEs, exposure to median-dichotomized ‘high’ vs ‘low’ cumulative lifetime SLE types (median ¼ 5 in either sex); PA-U18, Secondary finding associations (0.222pQp0.238) are discussed exposure to serious physical assault or attack at or under age 18 years; here with the caveat that false positives are expected using these SLE, stressful life event; SNP, single-nucleotide polymorphism. FDR cutoffs. Some of these secondary findings are supported by #0.05oPo0.10, *Po0.05, **Po0.01, ***Po0.001. previous research, which may aid in SNP prioritization for future studies, but we cannot predict which are false based on this study alone. Rs17289304 has not previously been associated with either occurs in female SA that have increased vulnerability to SLEs. suicidality, endophenotype-related outcomes or gene function. Although the mechanism is unclear, recent studies in mice have SNPs tagged by rs17289304 (Supplementary Figure 1) have shown that sex- and environment-dependent genomic imprinting predicted functionality: rs1328685,75 rs2760345 and rs2760346.76

Molecular Psychiatry (2013), 758 -- 766 & 2013 Macmillan Publishers Limited Family-based study of HTR2A in suicide attempts YJ Ben-Efraim et al 763 Table 2. Parent-of-origin effects for single-nucleotide polymorphism rs6313 among SA offspring exposed to low lifetime SLEs

Parent-of-origin

Effect size Allelic: MiAmat GT: MiAmat/MiApat No. of informative Effect size GT: -/MiAmat transmissions Allelic or MiA/MiA -/MiA CLR--LRTa -/MiApat POE (CLR--LRT)a GT: SA offspring: GT test Sex GT OR (95% CI) P-value MiA or GT OR (95% CI) P-value pseudosibs

Allelic MF NT NT NT Tmat 1.60 (0.82--3.12) 0.1635b 50 Tmat:50Cmat 37 Tpat:55Cpat M 0.93 (0.37--2.32) 0.8781b 23 Tmat:30Cmat 20 Tpat:26Cpat F 2.48 (0.86--7.13) 0.0874b 27 Tmat:20Cmat 17 Tpat:29Cpat GT MF TT 0.55 (0.31--0.97) 0.0152 TmatTpat 0.54 (0.30--0.97) 0.1652 (CLR--LRT, 0.0162) 20: 79 TmatTpat TC 1.20 (0.84--1.71) TmatCpat 1.49 (0.93--2.40) 50: 108 TmatCpat CmatTpat 0.93 (0.56--1.54) 35: 99 CmatTpat M TT 0.50 (0.22--1.16) 0.2167 TmatTpat 0.50 (0.22--1.16) 0.8753 (CLR--LRT, 0.3790) 9: 33 TmatTpat TC 1.00 (0.62--1.62) TmatCpat 0.97 (0.52--1.82) 22: 62 TmatCpat CmatTpat 1.04 (0.51--2.12) 18: 48 CmatTpat F TT 0.62 (0.28--1.39) 0.0386 TmatTpat 0.61 (0.27--1.39) 0.0233 (CLR--LRT, 0.0087) 11: 46 TmatTpat TC 1.50 (0.87--2.57) TmatCpat 2.69 (1.23--5.90) 28: 46 TmatCpat CmatTpat 0.85 (0.41--1.76) 17: 51 CmatTpat F TC 1.73 (1.07--2.80) 0.0233 TmatCpat 3.11 (1.47--6.59) 0.0240 (CLR--LRT, 0.0060) 28: 46 TmatCpat CmatTpat 1 (0.51--1.96) 17: 51 CmatTpat

Abbreviations: CI, conﬁdence interval; CLR, conditional logistic regression with matched pseudosibs; F, female; GT, genotype or genotypic; G Â E, gene-- environment interaction; low lifetime SLEs, exposure to o5 cumulative lifetime SLE types in either sex; LRT, likelihood ratio test; M, male; mat, maternal copy; MiA, minor allele on negative reference strand; NT, not tested; OR, odds ratio; pat, paternal copy; POE, parent-of-origin effect; SA, suicide attempt. aCLR was performed as in Table 1 but with a reduced set of trios as for POE tests, that is, trios with two heterozygous parents were dropped. bAllelic POE P-values and no. of informative transmissions were assessed with non-equivalent methods.

Rs7322347 lies downstream of the previously discussed SNPs, and is not in LD with any of them. In previous genetic studies, rs7322347 did not associate with SA in Han Chinese psychiatric patients,83 although it associated with childhood-onset attention- deficit hyperactivity disorder.84 AGÂ E between rs7997012 and CSA on SA was reported (TT-homozygote ‘protective’) in mostly female French Canadians,17 and we could also observe a nominally significant G Â E in exploratory post-hoc analyses; however, with combined physical or sexual assault exposures in adulthood. SNPs rs7322347 and rs7997012 were in moderate LD in French Canadians;17 however, they were not in LD in our sample or in the HapMap reference panel. Nevertheless, the previously reported G Â E17 was congruent to G Â E no. 3 with regard to sex, life timing of exposure, allelic direction and recessive model. As neither SNP has predicted functionality, perhaps they are both in LD with another functional variant in the Figure 3. Parent-of-origin effects for single-nucleotide polymorph- 0 ism rs6313 among female offspring exposed to low lifetime 3 coding or flanking region. SLEs. Genotype transmission ratios maintaining parent-of-origin Associations of low-frequency SNPs had larger effect sizes than 27 are displayed for SA offspring vs matched pseudosibs. Significant common SNPs, in line with predictions, but also had wider transmission distortions are marked (1 degrees of freedom (d.f.) tests confidence intervals. To our knowledge, we are first to report unadjusted for the other genotypes). Low lifetime SLEs, exposure to association of low-frequency SNP rs2070036 with a neuropsychia- o5 cumulative lifetime SLE types in either sex; mat, maternal copy; tric outcome, but its potential function is currently unpredicted. pat, paternal copy; SLE, stressful life event; SNP, single-nucleotide polymorphism. *Po0.05, **Po0.01. Methodological limitations Rs1928042 has neither been previously associated with Associations using imputed SNPs typically account for uncertainty suicidality or potential endophenotypes, nor does it have by, for example, using posterior genotype probabilities. However, predicted functionality. However, nominally significant imputed our main findings were with genotyped SNPs (Table 1), which SNP rs594242 (r2 ¼ 0.77 with rs1928042 here) has predicted splice were not affected by imputation. G Â E testing was limited by variant functionality,75 and has previously shown association (in a exposure prevalence and our inclusion criteria for minimum haplotype with rs6311) with impulsivity in SA in the same allelic transmissions (Supplementary Table 2). Therefore, while our direction.77 Rs594242 was also nominally significant on antide- significant genetic and G Â E findings were well-powered to pressant outcome.82 detect modest effects, potentially missed effects were possible,

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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

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