Genetic underpinnings of affective temperaments: a pilot GWAS investigation identifies a new genome-wide significant SNP for anxious temperament in ADGRB3 gene

Although recently a large-sample GWASs identified significant loci in the background of depression, the heterogeneity of the depressive phenotype and the lack of accurate phenotyping hinders applicability of findings. We carried out a pilot GWAS with in-depth phenotyping of affective temperaments, considered as subclinical manifestations and high-risk states for affective disorders, in a general population sample of European origin. Affective temperaments were measured by TEMPS-A. SNP-level association was assessed by linear regression models, assuming an additive genetic effect, using PLINK1.9. Gender, age, the first ten principal components (PCs) and the other four temperaments were included in the regression models as covariates. SNP-level relevances (p-values) were aggregated to gene level using the PEGASUS method1. In SNP-based tests, a Bonferroni-corrected significance threshold of p ≤ 5.0 × 10−8 and a suggestive significance threshold of p ≤ 1.0 × 10−5, whereas in gene-based tests a Bonferroni-corrected significance of 2.0 × 10−6 and a suggestive significance of p ≤ 4.0 × 10−4 was established. To explore known functional effects of the most significant SNPs, FUMA v1.3.5 was used. We identified 1 significant and 21 suggestively significant SNPs in ADGRB3, expressed in the brain, for anxious temperament. Several other brain-relevant SNPs and genes emerged at suggestive significance for the other temperaments. Functional analyses reflecting effect on gene expression and participation in chromatin interactions also pointed to several genes expressed in the brain with potentially relevant phenotypes regulated by our top SNPs. Our findings need to be tested in larger GWA studies and candidate gene analyses in well-phenotyped samples in relation to affective disorders and related phenotypes.


Introduction
Depression is a severe illness causing significant dysfunction and suffering. Unlike other illnesses where there is significant new progress and paradigm shifts in therapy also due to understanding and exploiting genetic variation associated with the illness, there is, with the exception of the promising introduction of glutamatergic approaches, a void of new approaches to understanding and treatment of affective disorders contributing to depression predicted to be the illness associated with the highest disease burden in the next very few years 2 .
One reason for our lack of sufficient insight into the etiopathology and neurobiology of depression is the failure to identify replicable genetic variation associated with the emergence and clinical characteristics of this illness. Although candidate gene studies targeted a large number of possible variants, only a few of these were investigated in at least three studies and even less were replicated 3 .
Genome-wide analysis approaches similarly yielded less results in case of depression compared to other psychiatric illnesses and, where suggestive significant results were retrieved, these did not confirm the role of previous candidate genes, nor could they be replicated in subsequent genome-wide association studies (GWASs). However, the most recent large-sample GWAS attempts involving joint databases and megasamples at the sacrifice of accurate phenotyping managed to identify a few significant variants with 102 significant hits in the latest study 4 .
However, the lack of in-depth and precise phenotyping may be a crucial weakness of such studies, as depression as a disease category is a highly heterogeneous phenomenon, where divergent clinical manifestations may equally be labelled as depressive disorder without a single overlapping symptom 5 , and in the background of such heterogeneous clinical symptoms divergent neurobiological pathways and distinct genetic variation are likely to play a role 6 . Subtypes of depression with clinically highly distinct features, such as unipolar or bipolar depression 7 , alexythmic depression 8 , melancholic and atypical depression 9,10 , or depression associated with increased suicidal risk 10 to name only a few, require focusing on different psychosocial or neurobiological targets, and for this, understanding their distinctive genetic determinants would provide the missing background and first step. Given the lack of proper efficacy of current, monoaminebased treatment approaches to depression, not only new pharmacological targets would be needed but novel agents need to be matched to more clear-cut illness phenotypes within the heterogeneous depression group 6 .
One possible approach to understanding depression via a more homogeneous categorization is the use of endophenotypes related to mood disorders, which are, by definition, better characterized and can be more closely mapped to singular neurobiological alterations 11 . Affective temperaments, thought to possess a strong biological background, manifesting in an early age and persisting through the life span, and being closely linked to affective disorders constituting in their more dominant manifestation a high-risk state or the subclinical form of affective disorders, and possessing a strong pathoplastic role, offer themselves as possible endophenotypes 12,13 . Previous research indicated their genetic associations, strong heritability, more marked manifestation not only in affective disorder patients but also in their first-degree relatives, dimensional distribution in the population, and a strong association with several outcomes of affective illness and general illness course 13 , which argue for using them in GWASs to bring us closer to the identification of genes in depression with an in-depth and precise endophenotyping. Yet, previously, no wholegenome-wide analysis was performed for affective temperaments in a general population sample and there has been only one GWAS in a bipolar patient sample 14 .
The aim of our present study was to perform a pilot GWAS on affective temperaments in a European general population sample.

Participants
The study was part of the NewMood project (New Molecules in Mood Disorders, Sixth Framework Program of the EU, LSHM-CT-2004-503474) funded by the European Union. NewMood has been a collaboration between 13 clinical and basic science research groups, across 10 European countries aimed at identifying new molecules in the background of mood disorders (www. newmood.co.uk), focusing on genetics with the specific aim of demonstrating allelic variation in association with vulnerability markers of depression, translation from rodent to human models, and specifically using translational endophenotypic markers rather than symptoms or diagnoses [15][16][17] . In the human clinical part of the project over 3000 participants of European White ethnic origin aged 18-60 years, in Greater Manchester and Budapest, were recruited via advertisements, general practices and a website. Participants filled out a questionnaire pack for precise and in-depth phenotyping of multiple endophenotypes related to susceptibility to depression, provided detailed background information on personal and family psychiatric history, as well as demographic and socioeconomic circumstances, and provided DNA using a saliva sampling kit. In the present study, we used phenotypic and genotypic data from 775 participants in the Budapest cohort. All procedures were carried out in accordance with the Declaration of Helsinki and were approved by the Scientific and Research Ethics Committee of the Medical Research Council, Budapest, Hungary. All participants provided a written informed consent prior to participating in the study.

Phenotype
Participants filled out the NewMood questionnaire pack, including the standardized Hungarian version of the 110-item Temperament Evaluation of Memphis, Pisa, Paris and San Diego (TEMPS-A) questionnaire to measure affective temperaments 12,18 and a background questionnaire including questions on age, gender, previous psychiatric illness, family history of psychiatric illness, somatic disorders and relevant demographic information. The TEMPS-A consists of five scales measuring the five affective temperaments described by Akiskal based on a clinical population and their first-degree relatives, including the depressive, cyclothymic, hyperthymic, irritable and anxious temperaments. Each affective temperament score was calculated as a continuous weighted score by dividing the sum of item scores by the number of completed items.

Genotyping, quality control and imputation
Participants provided DNA by a genetic saliva sampling kit. Genomic DNA was extracted from buccal mucosa cells according to established protocols 19 . Genotyping was performed using Illumina's CoreExom PsychChip, yielding a total of 573,141 variants, the genomic positions of which were defined according to the build GRCh37/hg19. Quality control and imputation was based on refs. 20,21 (see Supplementary File 1).

Statistical analyses
Descriptive statistics were done with SPSS25. Primary single-nucleotide polymorphism (SNP)-based association tests for each affective temperament phenotype were calculated using linear regression models in Plink 1.9 (https://www.cog-genomics.org/plink2), assuming an additive genetic effect. All models contained the first ten calculated principal components (PCs), gender, age, and the other four temperaments as covariates.
Gene-based tests for each affective temperament phenotype were calculated using Pegasus 1 . Significance (pvalue) of genes is calculated by a method that aggregates variant-level p-values and takes into account the dependence between them (induced by the linkage between the respective variants) by applying a null model of a multivariate normal distribution with a covariance matrix reflecting the aforementioned linkage.
To explore the known functional effects of our most significant SNPs as reported in public open databases based on expression quantitative trait loci (eQTL) and three-dimensional chromatin interaction, we used FUMA v1.3.5 22 , with a p ≤ 1 × 10 −5 threshold for lead SNPs, an R 2 ≥ 0.5 to define a genomic risk locus around a lead SNP, and a p ≤ 0.05 to involve SNPs into it. Each SNP of the genomic risk loci (referred to as top SNPs or our most significant SNPs) were mapped to a gene if either residing within gene boundaries extended by 10,000 base pairs, or having a false discovery rate q ≤ 0.05 with it in the external eQTL, or a q ≤ 1 × 10 −6 with its promoter region in the external chromatin interaction dataset 22 .

Characteristics of the sample
Imputation and quality-control steps yielded 2,550,710 SNPs and 775 subjects with data on the 5 affective temperaments, gender, age, and quality-controlled genomic data. By convention the threshold for statistical significance is based on a Bonferroni correction for 1 million comparisons, yielding a threshold of p ≤ 5.0 × 10 −8 , whereas at the SNP-level p ≤ 1.0 × 10 −5 was determined as threshold for suggestive significance 23 . For gene-based tests, a Bonferroni-corrected significance threshold of 2.0 × 10 −6 was applied and, at the gene level, p ≤ 4.0 × 10 −4 was determined as threshold for suggestive significance, the former one corresponding to the Bonferroni-corrected nominal significance level of 0.05 assuming 25,000 independent tests (the approximate number of the genes tested) and the latter one corresponding to that number multiplied by 200 (as in the case of variant-level tests).
Descriptive statistics on affective temperament scores, gender, age, self-reported psychiatric history and treatment are provided in Supplementary Table S1. Affective temperament scales were significantly and at least moderately correlated with each other in our sample (Supplementary Table S2); thus, when analysing specific variability of each affective temperament scale, the other four temperament scales were covariates in the model.  Table 1).

Suggestively significant findings in SNP-based tests for cyclothymic temperament
For cyclothymic temperament, λ-value resulting from genome-wide SNP-based tests was λ = 1.00000. For the QQ plot, see Supplementary Fig. S2. No SNP survived Bonferroni correction for multiple testing; however, 12 SNPs showed a suggestive significance, 4 of which reside in ERBB4 (Fig. 1B), whereas the others reside in genes CSMD1, NAP1L4, SNORA54 or are intergenic (Fig. 2 I-B and Table 1).

Suggestively significant findings in SNP-based tests for depressive temperament
In case of depressive temperament, genome-wide SNPbased tests yielded a genomic inflation factor of λ =  Table 1).

Suggestively significant findings in SNP-based tests for irritable temperament
In case of irritable temperament, genome-wide SNPbased tests yielded a genomic inflation factor of λ =   Table 1). Except for one intergenic SNP, the other two SNPs are mapped to the SHISA6 gene (Fig. 1C).

Suggestively significant findings in SNP-based tests for hyperthymic temperament
In case of hyperthymic temperament, genome-wide SNP-based tests yielded a genomic inflation factor of λ =  Table 1).

Suggestively significant findings in gene-based tests for affective temperament phenotypes
Hits with genome-wide suggestive p-values at the gene level for each temperament together with the most significant SNP within each gene are shown in Table 2 and

Genomic location of top SNPs for affective temperament phenotypes
Proportion of intronic SNPs was nominally significant in case of all five affective temperament phenotypes, and with the exception of anxious temperament phenotype, in case of the other four temperament phenotypes proportion of noncoding RNA intronic SNPs was also nominally significant (Fig. 3). In case of all affective temperament phenotypes, proportion of intronic SNPs, and, with the exception of anxious temperament, proportion of noncoding RNA intronic SNPs were significant, thus, the majority of identified SNPs likely influence function of protein-or RNA-coding genes by mechanisms other than amino acid replacement.

Functional effects of the top SNPs identified for affective temperament phenotypes on gene expression regulation in the brain
We carried out functional mapping and annotation concerning genes regulated in all tissues and, as we investigated a psychological phenotype, specifically in the brain, by our top SNPs, based on external chromatin interaction databases and eQTL databases GTEx v6 and v7 24,25 , and BRAINEAC 26 , which comprise several brain regions, as well as xQTLServer 27 and CommonMind Consortium (CMC) 28

Functional effects of top SNPs identified for the anxious temperament phenotype
We found that top SNPs for the anxious temperament phenotype on chromosome 11 were associated with gene expression levels in SLC35F2 in the DLPFC (in CMC samples), in the hypothalamus (in GTEx/v7 samples), in nucleus accumbens basal ganglia (in GTEx/v7 samples) and in the pituitary (in GTEx/v7 samples); in ACAT1 (in GTEx/ v6 samples) and SLN (GTEx/v7 samples) in the cerebellum; and in ELMOD1 in the DLPFC (in CMC samples) (Supplementary Table S1 and Supplementary Fig. S13). Top SNPs for the anxious temperament took part in chromatin interactions in the fetal cortex with Lnc-NPAT-2:ACAT; NPAT:   Table S5 and Supplementary Fig. S16).

Functional effects of top SNPs identified for the depressive temperament phenotype
In case of the depressive temperament phenotype, the identified top SNPs did not affect expression levels of genes in the brain (Supplementary Table S7) and did not take part in chromatin interactions with known genes (Supplementary Table S7).

Functional effects of top SNPs identified for the irritable temperament phenotype
Top SNPs associated with the irritable temperament did not influence expression levels of any genes according to FUMA (Supplementary Table S9); however, they took part in chromatin interactions in the adult and fetal cortex with NBEA 29 , and in the fetal cortex with RFC3 29 (Supplementary Table S10).  Table S11 and Supplementary Fig. S22). Top SNPs for the hyperthymic temperament were also   Table S12).

Discussion
Our present study is the first to analyse the genomewide association of affective temperaments in a general European population sample with precise in-depth phenotyping for these endophenotypes. As affective temperaments can be considered sub-affective manifestations of, and when present in a dominant form, high-risk states for the development of different types of affective illnesses 12 , they may provide a complex approach to understand the genetic background of different affective disorders and mood syndromes yielding clinical relevance for our present results. Although several suggestively significant SNPs and genes with potential relevance for affective disorders and other psychiatric and psychological phenotypes emerged, we most notably identified one SNP in case of the anxious temperament, which survived correction for multiple testing in ADGRB3 where several other suggestively significant SNPs also underline the potential importance of this gene.
Previously, only one study investigated the genetic background of affective temperaments in a whole-genome analysis approach, in 1263 bipolar patients in the Bipolar Genome Study 14 identifying 3 genome-wide significant SNPs for the hyperthymic temperament near MDMI on chromosome 12 and FBLNI on chromosome 22, and 2 significant SNPs on chromosome 1 within neighbouring INTS7 and DTL genes, which led the authors to conclude that temperamental aspects may define clinically and genetically homogeneous bipolar disorder subtypes. Although that study differed from our present one in that they applied a bipolar patient sample as opposed to our general sample, none of the above SNPs or regions have been significant or suggestively significant in our present analysis.
In the present study, we analysed genome-wide associations for each of the five affective temperaments separately and given the significant moderate intercorrelations between them, all models were corrected for the effect of all other temperaments at SNP-based levels.
In the next step, as assigning SNPs to genes purely based on their proximity yields only a limited insight into the complexity of the genetic background and little information on the whole spectrum of possible functional effects, and as the majority of identified SNPs are intronic or located in noncoding RNA introns and thus likely influence function of protein-or RNA-coding genes by mechanisms other than amino acid replacement, we carried out functional analysis using FUMA focusing on chromatin interactions and effects on gene expression in the brain. Here we discuss findings related to genes expressed in the brain or implicated in relevant psychiatric or psychological phenotypes in previous analyses.

Genetic underpinnings of the anxious affective temperament
The most robust finding of the study was identification of a novel intronic SNP in the ADGRB3 gene on chromosome 6, which survived correction for multiple testing in addition to 21 other intronic variants with suggestive pvalues within this gene supporting its involvement in anxious temperament. The ADGRB3 gene is expressed almost exclusively in the brain and encodes the Adhesion G protein-coupled receptor B3, or brain-specific angiogenesis inhibitor 3 (BAI3), a protein with a pivotal role in the central nervous system development, maintenance and plasticity, including regulating synaptic development, axon guidance, synapse formation, myelination, with high expression in the postsynaptic density, especially in the cerebellum, and crucial roles in Purkinje cell synaptogenesis, both during development and adulthood [30][31][32] . Specifically, the encoded protein controls synaptic connectivity of excitatory inputs, and several SNPs and copy number variations within this gene have previously been associated with different psychiatric disorders including bipolar disorder 33 , schizophrenia 34 and addiction 35 , as well as with traits such as impulsivity/negative urgency/ behavioural disinhibition 36 and educational attainment 37 . ADGRB3 shows high expression in the cerebellum and in hippocampal neurons where it acts as a regulator of synapse density and where its knockdown leads to significant impairment in dendrite morphogenesis in mouse models, whereas in a human study the clinical spectrum in patients with biallelic intragenic duplication in ADGRB3 also included, among other symptoms, anxiety and mood instability, which are associated with hippocampal dysfunction 32,38,39 and which is specifically in line with our findings that variation in this gene shows association of anxious affective temperament. Further research, focusing on the manipulation of this gene in knockout rodent paradigms, as well as human studies focusing on its association with other anxiety-related endophenotypes and neuropsychiatric symptoms and illnesses should further validate the potential role of this gene and its variation in association with anxiety-related traits and disorders, and eventually the receptor encoded by this gene should be investigated as a potential treatment target.
In gene-based analyses for the anxious temperament, several genes reached suggestive significance after correction for multiple testing, some of which play a role in the central nervous system processes and thus may have potential relevance. Genes with a suggestive significance for the anxious temperament include FYN, encoding the proto-oncogene tyrosine protein kinase fyn, which, among several other functions, plays a role in neuronal development and axon guidance, and is implicated in various neural functions and processes. Fyn kinase has, in previous studies, been implicated to play a role in the background of anger/aggression 40 and in the pathophysiology of major depression 41,42 , bipolar disorder 43 , schizophrenia 44,45 and Parkinson's disease 46,47 , and has been suggested as a potential drug target in Alzheimer's disease 48 . PIP encoding prolactin-induced protein has also showed suggestive significance in association with anxious temperament. An SNP in the PIP gene in a previous GWAS has been found to be associated with unipolar depression (rs28672333) 49 . HECTD1, another gene suggestively associated with the anxious temperament in our present analysis, regulates the expression of SNAIL 50 , which in turn regulates neural crest differentiation and neurogenesis 51 . Finally, ROGDI also emerged as a suggestive gene in association with anxious temperament. ROGDI encodes a typical leucine zipper protein with an unknown function, with highest expression in the adult brain 52 and lower expression in the fetal brain and other tissues.
Furthermore, functional prediction analyses implicated top SNPs for the anxious temperament to be associated with the expression of various genes in the central nervous system in areas relevant both for emotional reactivity and affective illnesses, such as SLC35F2 in the DLPFC, hypothalamus and nucleus accumbens, and implicated in GWASs for unipolar depression, bipolar disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder 53 . Top SNPs for the anxious temperament also influence expression of ACAT1 encoding the acetyl-CoA Acetyltransferase1, the major isoform in the brain, which has been implicated in several neurodegenerative disorders and has recently emerged as a promising target in the treatment of Alzheimer's disease 54 and glioblastoma 55 . Top SNPs for the anxious temperament also regulate the expression of ELMOD1, which has previously been associated with personality 56 and delayed discounting measures 57 .

Genetic underpinnings of the cyclothymic affective temperament
In SNP-based tests in case of the cyclothymic temperament, suggestive SNPs were located either on chromosome 2 within the ERBB4 gene or on chromosome 11, with 1 out of 6 of these latter SNPs in the NAP1L4 or SNORA genes. One more suggestive SNP was located in the CSMD1 gene.
ERBB4 is a member of the Tyr kinase family and encodes a neuregulin surface receptor regulating development of the central nervous system including gene transcription, cell proliferation, differentiation, migration and apoptosis, expressed in interneurons in the frontal cortex and involved in postsynaptic modulation of GABAergic function 58 .
There were several suggestive hits for cyclothymic temperament at the gene level as well, including NAP1L4 where a functional effect was also observed and which has been mentioned at a suggestive significance level in association with major depression in a transcriptomewide analysis 95 . Top SNPs for cyclothymic temperament influenced brain expression of a few potentially relevant genes and participated in chromatin interactions, most notably with ERBB4 and SLC36A4, which latter encodes a sodium-independent electroneutral transporter for tryptophan, proline and alanine, and is also associated with chronotype 71 .

Genetic underpinnings of the depressive affective temperament
At the SNP level, several suggestively significant SNPs for the depressive temperament were identified in the SGCZ gene, which is expressed in several brain regions including highest expression in the cerebellum, basal ganglia and cortex, and has been implicated in the sarcoglycan complex and muscular dystrophy. SGCZ has, in previous studies, been associated with some potentially relevant phenotypes including anxiety 61,62 , neuroticism 61,62,96 , cognitive function 65 , mathematical ability 37 , intelligence 64,97 , self-reported educational attainment 37 , depressive symptom and response to antidepressant 98 , schizophrenia 99 and response to paliperidone 84 .
At the gene level, UGT2B7 encoding UDP-Glucuronosyltransferase-2B7, a phase II metabolic isoenzyme, also found in the brain showed a suggestive-level association with depressive temperament. For top SNPs in association with depressive temperament, we could not identify relevant effects on gene expression in the brain or chromatin interactions.

Genetic underpinnings of the irritable temperament
At the SNP level, suggestive SNPs for the irritable temperament were identified on chromosome 17 in the SHISA6 gene, which encodes a protein involved in the maintenance of high-frequency synaptic transmission in hippocampal CA3-CA1 synapses as an auxiliary α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) subunit, and regulates postsynaptic AMPAR glutamate receptor immobilization keeping the channel in an activated state in the presence of glutamate, preventing synaptic depression and desensitization 100 . Variation in the SHISA6 gene in GWASs has been implicated in risk-taking behaviour/adventurousness 101 , insomnia 70 , sleep duration 102 and schizophrenia 88 . In gene-level analyses, suggestive hits included ENTPD6, which has been found to be associated with smoking status/smoking initiation 103 , and brain volume of the left pallidum 104 . In functional analyses, no effect of our top SNPs for irritable temperament emerged for influencing gene expression, but chromatin interactions were found with NBEA and RFC3 in the adult and fetal cortices. NBEA encodes neurobeachin, lack of which protein in the BEACH domain in neurons was associated with a sharp reduction in synaptic responses as a consequence of reduced surface GABA-A and glutamate receptors, accumulation of immature AMPA receptors, and NMDA, kainite and GABA-A receptors not reaching the synapse, suggesting that Nbea plays a role in regulating basal neurotransmission and targeting of neurotransmitter receptors to synapses 105 . NBEA variations in GWASs have been reported in antipsychotic response in schizophrenia 106 , nicotine dependence 78 , self-reported educational attainment 37 , PTSD 76 , and cognitive function 37 .

Genetic underpinnings of the hyperthymic temperament
At the SNP level, suggestive hits for the hyperthymic temperament were identified on chromosome 4, 8 and 20, and on chromosome 6 in the region of CDSN and PSORSC1 genes. PSORSC1 is expressed in the brain and has been associated in previous GWASs with neuroticism 63 , risk-taking behaviour 101 , wellbeing/positive affect and wellbeing/life satisfaction, and subjective wellbeing 63,107 , depressive symptom measurement 63 , schizophrenia 44,88,108 , a broad depression or schizophrenia phenotype 109 and autism spectrum disorder 108 . On the gene level, suggestively significant hits for hyperthymic temperament included SNX6 and CANT1 with high expression in the brain, but no relevant phenotypic associations in previous studies. Top SNPs associated with hyperthymic temperament participated in chromatin interactions with a few brain-expressed genes but without previous association with relevant phenotypes.

Potential transdiagnostic and pathoplastic nature of affective temperaments
Our results indicate that the five distinct affective temperaments have non-overlapping genetic backgrounds; however, several suggestively significant hits were observable for genes which have previously been implicated in association with relevant psychological traits related to the development of mood disorders such as neuroticism, impulsiveness, aggression or wellbeing, as well as in other psychiatric disorders. These two findings, namely distinct genetic underpinnings of the five distinct temperaments and the same disorders these distinct genes confer a risk for, suggest a potentially transdiagnostic endophenotypic nature of the TEMPS affective temperaments. If considering psychiatric disorders other than mood disorders, schizophrenia has been found to have common genetic risk factors with all five temperaments. Addiction may share some genetics with anxious, cyclothymic and irritable temperaments. Autism spectrum disorder has been implicated in relation to risk genes for anxious and hyperthymic temperaments, and PTSD in genes for cyclothymic and irritable temperaments. Distinct temperaments with their distinct genetic underpinnings may represent distinct subgroups within these aetiologically heterogeneous disorders. Stratification of patients or at-risk individuals according to these welldefined endophenotypic pathways, from certain genes through certain affective temperaments to the equifinal endpoint of the disorder, may help in the personalization of preventive and therapeutic strategies for these disorders. However, further longitudinal studies are needed to clarify the mediatory role of affective temperaments between genes and the emergence of these various disorders.
A major weakness of our study is the small sample size highlighted by terming the present analysis a pilot study and which is in part balanced by the careful sample characterization and precise attainment of the investigated phenotype, and multiple analytical approaches focusing on functional analyses besides SNP and gene levels. Furthermore, although precise deep phenotyping of affective temperaments is a strength of the present analysis, affective temperaments are measured based on self-report and are ascertained cross-sectionally, and thus may be influenced by state-like factors such as mood or possible mood disorder episode.
In conclusion, our GWAS focusing on affective temperaments, besides several suggestively significant hits at SNP and gene levels, have most notably identified a new genome-wide significant variant in association with anxious temperament in AGDRB3 gene. Furthermore, although the five affective temperament phenotypes have non-overlapping genetic backgrounds, we identified several suggestively significant variants previously associated with psychological traits implicated in the development of both mood and other types of psychiatric disorders, implicating the potential transdiagnostic endophenotypic nature of affective temperaments. In the future, our findings may help pave the way for personalized and precision approaches for both prevention and intervention strategies across various psychiatric disorders. Our results should further be tested in larger samples in genome-wide analytical approaches for related phenotypes and candidate gene studies in well-characterized and phenotyped samples, to more precisely establish the role of our identified genes and variants in mood disorders or high-risk states for the development of affective illness, as well as its role as a potential target of therapeutic interventions. Longitudinal studies should also test our findings to understand the potential mediative role of affective temperaments between genes and emergence of various psychiatric disorders.