Identiﬁcation of genomic variants in regulatory elements associated with risk of suicide

Suicide attempt is the consequence of a complex interaction between social traumatizing relations, environmental and genetics factors that might induce mood disorders. The genetic framework of schizophrenia, depression, bipolarity and anxiety disorders are very similar, with important genes involved in neurotransmission, circadian rhythm, serotonin pathways and more. We perform a non-coding scanning analysis using GWAS datasets of four previous suicidal attempts articles. P-value signiﬁcant genetic variants (p ≤ 0.05 ) were processed in a novel inferring software (http://inferno.lisanwanglab.org/ index.php). We map up to 40 regulatory elements near to those genetic variants, that might be interfering in the expression of genes related to excitability of neurons, period and neuronal development, essential for a good mental health. This investigation allows the identiﬁcation of new loci of interest related with suicidality and emphasizes the importance of including biological context enrichment to post GWAS analysis.


INTRODUCTION
Suicide is the intention of taking one's life. Every year, this public health issue account for approximately 703,000 deaths (WHO, 2021) mostly attributed to men who have a higher reported rate of completed suicide, whereas women have a higher rate of attempted suicide [1]. Unfortunately, suicidality has risen in young people, and it was the third leading cause of death worldwide in 2014 [2]. Temperamental traits of impulsive aggression, neuroticism, childhood abuse and introversion have also been implicated in suicidal risk. However, it is unlikely that these factors, themselves, can explain suicide [1].
Components of suicidal thoughts and behaviors, such as feeling that life is not worth living or contemplating self-harm, are relatively common in the general population, as well as in patients affected by a variety of separate clinical diagnoses [3].
Therefore, diagnosis of suicide tendencies still biases and encompasses a broad spectrum of phenotype and even, genotype traits. In fact, those individuals who do not progress from the preceding steps towards death may be distinct biologically from those that do [4].
Although suicide results from a complex interaction of multiple social and psychological factors, predisposition to suicidality is at least partly genetic [3]. Over 90% of the suicidals had recognizable psychiatric illness at the time of their death [5] and major depressive disorder (MDD), neuroticism, mood instability and schizophrenia have the strongest correlations with suicidality [3].
Genetic framework of mental illness and suicide attempt might be similar; however, the genetic architecture of suicidal behaviors remains poorly understood [6].
First single-nucleotide polymorphism (SNP) studies found statistics supporting relations between some variants and a specific phenotype, but the highly polygenic nature of complex Selected genetic variants in serotonergic system genes have been the major historic focus of suicide related investigations during the past decades, but the polygenetic perspective is nowadays also being addressed by the study of candidate genes in other neurosystems [10].
Recent advances in this area include a GWAS of suicide attempts in approximate 50,000 individuals with and without psychiatric disorders which identified some suggestive loci [3]. These studies have examined individuals with depression or bipolar disorder, comparing attempters with no attempters and testing for genetic variants that might contribute independently to suicide attempt that were underpowered to detect with typical SNP stu- Although, large-scale genetic studies remains difficult due to the necessity of achieve a collection of enough samples to have the power to identify replicable genetic associations, or to directly estimate the proportion of heritability contributed from common genetic variation [6].
Due to the majority of GWAS hits mapped within non-coding regions (such as intergenic or intronic regions), new functional RNA species (such as lncRNAs or circRNAs) have started to emerge. In recent years, in parallel to an increased knowledge of the non-coding genome, new studies started to characterize disease-associated variants located within non-coding RNA regions [9].
Considering this, we reanalyze four previous genome-wide association studies (GWAS) of suicide attempts, using a novel software to infer the molecular mechanisms of noncoding genetic variants. To our knowledge, this investigation is one of the few that uncover new insights about downstream biological processes related to suicidality.

Genome-wide association studies (GWAS) data collection
Study subjects were drawn from previous GWAS investigations.
We made contact with the correspondence author of each study and they enabled us to use the datasets. Genetics and Clinical Genomics DOI: 10.37980/im.journal.ggcl.20232167

P-value quality filter
We obtain 31,233,308 variants from the GWAS summary statistics. Based on p-value criteria, we selected 6,492 variants with a significant value between 1x10x -5 and 1x10x -8 .

Statistical based-on filter
After LD analysis was performed, the blocks of variants with TAG-MAF ≤ 0.1 and r 2 ≥ 0.9 were selected. We searched the regulatory elements and the genes linked to those blocks, using two public databases (UniProt https://www.uniprot.org) and NCBI (https://www.ncbi.nlm.nih.gov)

RESULTS
It was found a major proportion of TFBS (52%) above Enhancers (36%) and miRNA regulatory elements (12%) near to p -value significant variants, eQTL could not be found ( Table 2). We set up 23 TFBS, 18 Enhancer and 6 mi-RNA regulatory elements. After this, we reorganized these regulatory elements in nine groups, based on the similarity of the biological functions of the target gene ( Figure 1).

Voltage-dependent calcium channels
Calcium ions are involved in several human cellular processes including transcription, corticogenesis and synaptogenesis  [12]. These channels mediate the entry of calcium ions into excitable cells and they are essential in the neurotransmitter release and in a variety of processes including muscle contraction). Dysregulation of these processes and associated alterations in calcium channel activity have been linked to various types of neurological disorders, including epilepsy, migraine, and chronic pain [13].
We identify CACNA1E gene on enhancer, it maintains synaptic plasticity, neuronal survival and fear conditioning and it is expressed on dendrites [12]. The role of calcium channels in mental health is further supported by findings that show that SNPs located in CACNA1C are linked to development of other psychiatric disorders such as bipolar disorder, schizophrenia, and depression [13]. Some schizophrenic patients have an intergenic deletion between classical CDH12 and CDH18 genes [14], suggesting the important role of cadherin genes in neural function. Finally, CLSTN2 gene is predominantly expressed in the brain, and it is involved in learning and memory process. to create specific morphologies and synaptic connections [18].

Period genes (circadian clock)
Sleep and circadian rhythm disruption (SCRD) are a common feature of schizophrenia and is associated with symptom severity and patient quality of life [19]. Genetic association studies have demonstrated that mutations in several circadian clock-related genes are associated with a higher risk of schizophrenia as well as alterations in dopamine signaling. Thereby, dopamine is closely linked to sleep and the circadian system [19].
Previous GWAS studies have associated mutations in circadian clock genes with an increased risk of schizophrenia.
Among them are the single nucleotide polymorphisms (SNPs) in CLOCK, PER2, PER3, RORB, and TIMELESS. Respecting HDAC1, HDAC2 and PML genes, may be involved in the transcriptional repression of circadian target genes, such as PER1 and PER2, mediated by CRY1 through histone deacetylation [20]. Moreover, BHLHE40, a transcriptional repressor is involved in the regulation of the circadian rhythm by negatively regulating the activity of the circadian clock genes and circadian clock-controlled genes [21]. TFBS elements are involved in neuronal differentiation (SIN3A), catalysis of important circadian complex (EZH2), and recruitment of the methyltransferase SUV39H1 to the E-box elements of the circadian target genes (like CBX3).

Differentiation and neuronal development/plasticity
This biological process already has an elevated genetic burden.
Due to this, we wanted to focus this finding on a few facts. The MEF2-TFBS complex reduces the expression of MEF2 gene in hippocampal neurons, suggesting that activation of this factor restricts synapse number [22].
According to Fatjó-Vilas, M., 2016, Neuritin 1 gene (NRN1) is involved in neurodevelopment processes and synaptic plasticity and its expression is regulated by brain-derived neurotrophic factor (BDNF). Results suggest that NRN1 variability is a shared risk factor for both schizophrenia-spectrum disorder and bipolar disorders and that NRN1 may have selective impact on age at onset and intelligence in Somatic Sympton disorder SSD.

CONCLUSION
Of all the psychiatric phenotypes, suicidality remains especially challenging to assess due to the highly polygenic condition. This research is the first approach to understanding the functionality and potential of INFERNO. The volume of the output data was significantly high, approximately 100 MB or more for each of the four GWAS data sets. We have processing tools limitation, so we had to prioritize the information that would give us better results, and it was the block of variants with a minor MAF and good linear correlation value that was expressed in Brain tissue. However, we recognize that other variants of a medium-high frequency could be potentially associated with suicidal behaviors and they must be examined in future studies. We obtained GWAS from Americans and United Kingdom people, whereas Latin American, Asian and other European countries were sub-represented.

Data availability
Derived data supporting the findings of this study are available from the corresponding author on request.
of novel genome-wide associations for suicidality in UK Biobank, genetic correlation with psychiatric disorders and polygenic association with completed suicide.