EXPERIENCE OF USING THE MOLECULAR GENETIC STUDIES TO ASSESS THE RISK OF SUICIDE

In the recent decades, there has been widespread the opinion that genetic markers of the suicidal behavior (suicide, suicidal attempts, suicidal thoughts) can be used to predict the suicidal behavior. The purpose of the study was to determine the possibility of using the method of molecular genetic research to assess the risk of suicide in men of 18‒27 years. The study used the case-control method. The control group included 100 men of 18‒27 years who never had mental disorders. The suicide group included the persons who committed highly traumatic methods of self-harm and were motivated to commit suicide (30 persons). DNA isolation was performed using a NucleoSpin Blood kit (Macherey‒Nagel, Germany) according to the manufacturer’s protocol. Each DNA sample was analyzed for polymorphism by allelic discrimination using the real-time polymerase chain reaction (PCR). The  frequencies of occurrence of genotypes and alleles of  the  following genes were analyzed: HTR1A,  rs6295  (G/C); BDNF, rs6265 (G/A); COMT, rs4680 (G/A); SKA2, rs7208505 (C/T); SLC6A4 (5HTT), rs25531 (T/C); 5HTR2A, rs6313 (G/A); TPH2, rs4570625 (G/T); TPH1, rs1800532 (G/T). A statistically significant difference was found for the frequency of occurrence of genotypes and alleles of the rs25531 polymorphism of the SLC6A4 (5HTT) gene. The chance of being in the suicide group with a heterozygous genotype (T/C) carriage was 2.346 times higher. The significance of the rs25531 polymorphism of the SLC6A4 (5HTT) gene for the formation of the suicidal behavior was confirmed.

All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional and/or national research ethics committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants involved in the study.
Materials and research methods. The study used the case-control method. The control group included 100 young men (18-27 years old), whose mental disorders were excluded. The control group included 30 people who committed highly traumatic methods of self-harm, motivated to commit suicide.
DNA was isolated using a NucleoSpin Blood kit (Macherey-Nagel, Germany) according to the protocol. Specific polymorphic sites of 9 genes were used as molecular targets. Information on target genes and polymorphic sites is presented in Tab. 1.
T a b l e 1. Molecular targets and polymorphic sites selected for research No. Target gene Specific polymorphic site Genotyping was performed using the allelic discrimination method based on real-time PCR data. The CFX 96 connect and CFX 96 Touch instruments (Bio-Rad, USA) were used for real-time PCR. Primary analysis of PCR data was performed using the software supplied with the instruments (Bio-Rad CFX Maestro 1. Real-time PCR was performed using reagents produced by Primetech (Belarus). The final volume of the reaction mixture was 25 μL and contained all the necessary reagents at the following concentrations: 0.1 mM dNTP mixture, 2 mM MgCl 2 solution, 500 nM oligonucleotide primers, 250 nM fluorescently labeled probes, 1.25 units of thermostable Taq DNA polymerase with the appropriate buffer solution from the kit provided by the enzyme manufacturer. Thermal cycling: 95 °C -3 minutes, then 45 cycles 95 °C -10 seconds, 60 °C -59 seconds.
The obtained results were processed by variational methods of mathematical statistics using the Mic rosoft Office 7.0 application package, the Statistic for Windows software package.
Research results. In genetic studies of serotonin biosynthesis, the main attention is paid to tryptophan hydroxylase (hereinafter TPH). TPH is an enzyme that is involved exclusively in the process of serotonin biosynthesis [2]. To date, two TPH isoforms have been identified, TPH1 and TPH2, which have different tissue organization. The TPH1 isoform is synthesized mainly in the walls of the duodenum, as well as in the liver, heart, lungs, kidneys and adrenal glands. Among the most intensively studied polymorphic markers of the TPH1 gene, A218C and A779C are distinguished (both are localized within intron 7 and are in complete linkage disequilibrium). D. Nielsen et al. [3] report an association between these polymorphisms, the concentration of 5-hydroxyindoloacetic acid (hereinafter referred to as 5-OIAA) in CSF and suicidal attempts in violent offenders with alcohol dependence. J. Mann et al. [4] studied the possible association of the 218A allele with suicidal behavior in depression. They found that the A218C polymorphism was associated with impulsive aggression and anger traits that may predispose to suicide. However, the differences found are relatively small and statistically significant only for large samples.
In our study analysis of genotypes and alleles of the rs1800532 polymorphism of the TPH1 gene revealed a slight increase in mutant type homozygotes (T/T) in the control group compared to suicide group, but this difference was statistically insignificant (Tab. 3).
T a b l e 3. Frequency of occurrence of genotypes and alleles of rs1800532 polymorphism of TPH1 gene The second isoform, TPH2, is characteristic exclusively for brain neurons. The TPH2 gene is localized in the 12q21.1 region and consists of 12 exons and 11 introns. TPH2 is expressed exclusively in the brain and plays an important role in the synthesis of serotonin in the brain. Z. Zhou et al. [5] performed an analysis of 10 single nucleotide polymorphisms (hereinafter referred to as SNPs) and analysis of the TPH2 gene haplotypes, which showed a statistically significant association of this gene with completed suicide in a sample of persons of German descent. When comparing the frequencies of the genotypes and alleles of the rs4570625 polymorphism of the TPH2 gene during our study, no statistically significant difference was observed in the control group and the suicide group (Tab. 4).  The BDNF gene encodes a protein of the same name, which is a neurotrophic factor in the brain. This protein is involved in the growth, maturation (differentiation) and maintenance of neurons. Currently about 30 pathogenic polymorphisms of the BDNF gene are known. Most of them are located in non-coding intron regions. The most common functional mutation of this gene is the missense variant rs6265 (p.196G-A, p.V66M). The 196G-A mutation in the BDNF gene shows connection to effects on general cognitive abilities, eating disorders, bipolar disorder, anxiety disorder, Alzheimer's and Parkinson's disease [4,6]. The data for determining the frequency of occurrence of genotypes and alleles of the rs6265 polymorphism of the BDNF gene are presented in Tab. 5.  The frequency of occurrence of the homozygous mutant genotype A/A is almost 2 times higher in the control group than in suicide group (difference are not statistically significant), however the frequencies of the alleles in both groups are approximately the same.
The COMT gene encodes catechol-O-methyltransferase (COMT), an enzyme involved in the breakdown of catecholamines, including dopamine, which is one of the most important neurotransmitters in the brain. COMT gene polymorphisms can disrupt the work of the dopaminergic system of the brain, especially in the prefrontal cortex, which leads to cognitive dysfunction and is considered one of the rea-sons for the development of schizophrenia, bipolar disorder, and obsessive-compulsive disorder [7]. The data for determining the frequency of occurrence of genotypes and alleles of the rs4680 polymorphism of the COMT gene are presented in Tab. 6. T a b l e 6. Frequency of occurrence of genotypes and alleles polymorphism rs4680 of the COMT gene Genotypes There were no significant differences in the frequency of occurrence of genotypes and alleles in both groups.
The SKA2 gene is active in the prefrontal cortex where it reduces the brain's response to cortisol (a stress hormone), thus neutralizing the effects of negative emotions. Currently the SKA2 gene is considered as one of the biological markers of suicidal behavior [8]. The data for determining the frequency of occurrence of genotypes and alleles of the rs7208505 polymorphism of the SKA2 gene are presented in Tab. 7.  Тhere were no significant differences in the frequency of occurrence of genotypes and alleles between the control groups and suicide groups.
The HTR1A gene is located on the chromosomal region 5q11.2-q13. It was shown that the functional polymorphism -1019G/C (rs6295) in the promoter region controls the transcriptional activity of the gene and is associated with suicidal behavior and unipolar depression [9,10]. Data on determining the frequency of genotypes and alleles of the rs6295 polymorphism of the HTR1A gene are presented in Tab. 8.  As shown in Tab. 8, the frequency of occurrence of the G/G genotype in the control group was 2 times higher than in the suiside group (difference are not statistically significant). When comparing the frequency of occurrence of the G/G genotype in both groups, an odds ratio was 2.284 (the chance of committing suicide with the G/G genotype in the rs6295 polymorphism is 2 times higher than in individuals with a different genotype).
The 5HTR2A gene encodes the serotonin receptor type 2A. The mammalian 5-HT2A receptor is one of the subtypes of 5-HT2 receptors, a subfamily of serotonin receptors. It is a metabotropic G-protein coupled receptor. The receptor of this subtype (5-HT2A) is the main excitatory receptor subtype among all G-protein-coupled receptor subtypes for serotonin (5-HT). However receptors of the 5-HT2A subtype can on the contrary exert an inhibitory effect in some areas of the brain such as the optic cortex and the orbitofrontal cortex. Receptor activation triggers intracellular processes that affect the activity of other mediator systems -glutamate, dopamine, and GABA. The data for determining the frequency of occurrence of genotypes and alleles of the rs6313 polymorphism of the 5HTR2A gene are presented in Tab. 9.
T a b l e 9. Frequency of occurrence of genotypes and alleles of rs6313 polymorphism of the 5HTR2A gene When comparing the frequencies of occurrence of genotypes and alleles in the control group and suicide group, no significant difference was observed.
The SLC6A4 gene, abbreviated as SERT (Serotonin Transporter) or 5-HTT (5-HydroxyTriptamine Transporter), encodes an intracellular protein, a serotonin transporter or sodium-dependent serotonin transporter. The serotonin transporter belongs to the monoamine transporter family. Its physiological function is the reuptake and transport of serotonin from the synaptic cleft back to the presynaptic terminal that secreted it for reuse. This protein is a target of psychomotor stimulants such as amphetamines and cocaine and is a member of the sodium neurotransmitter symporter family. It has been shown that repeat length polymorphism in the promoter of this gene affects the rate of serotonin uptake. There are conflicting results in the literature on the possible effect that this polymorphism may have on behavior and depression [11,12]. The data on determining the frequency of occurrence of genotypes and alleles of the rs25531 polymorphism of the SLC6A4 (5HTT) gene are presented in Tab. 10.  When comparing the frequencies of occurrence of genotypes and alleles in the control group and suicide group, significant differences were noted. In the control group, the percentage of occurrence of the wild type genotype (T/T) is less than in the control group, and the frequency of occurrence of heterozygotes is higher respectively. The numerical value of OR (odds ratio) equal to 2.346 (confidence interval 95 % CI = 1.019-5.4) indicates that the chance of being in the suicide group with the carriage of a heterozygous genotype (T/C) is 2 times higher. That is, the risk factor is the heterozygous genotype (T/C).
We also studied the frequency of occurrence of genotypes and alleles of another polymorphism -rs4795541 of the SLC6A4 (5HTT) gene (Tab. 11).
T a b l e 11. Frequency of occurrence of genotypes and alleles of rs4795541 polymorphism of the SLC6A4 (5HTT) gene No differences in the frequencies of all 3 genotypes and 2 alleles were found. Discussion. As a result of the study, the frequencies of occurrence of genotypes and alleles of the following genes were analyzed: HTR1A, rs6295 (G/C); BDNF, (rs6265 (G/A); COMT, rs4680 (G/A); SKA2, rs7208505 (C/T); 5HTT (SLC6A4), rs25531 (T/C); 5HTR2A, rs6313 (G/A); TPH2, rs4570625 (G/T); TPH1, rs1800532 (G/T). Statistically significant differences in the frequencies of genotypes and alleles of the rs25531 polymorphism of the SLC6A4 (5HTT) gene were found. In the control group, the percentage of the occurrence of the wild-type genotype (T/T) was less than in the suicide group, and the frequency of occurrence of heterozygotes was correspondingly higher. The numerical value of OR (odds ratio) equal to 2.346 (confidence interval 95 % CI) = 1.019-5.4) suggests that the likelihood of falling into the suicide group in the presence of heterozygous genotype (T/C) is 2.346 times higher. Thus, it was found that the frequency of occurrence of the heterozygous genotype (T/C) corresponds to the frequency of occurrence of this genotype in the group of persons prone to true suicide, which is consistent with the data of previous studies [1,13]. The significance of the allele polymorphisms of the genes: HTR1A, rs6295 (G/C); BDNF, (rs6265 (G/A); COMT, rs4680 (G/A); SKA2, rs7208505 (C/T); 5HTT (SLC6A4), rs25531 (T/C); 5HTR2A, rs6313 (G/A); TPH2, rs4570625 (G/T); TPH1, rs1800532 (G/T), which are regarded as possible genetic factors of suicidal behavior however it has not been confirmed. This emphasizes the complexity of this problem, despite a long period (more than 10 years) of research, has not yet found its solution.
Conclusion. This study has confirmed the possibility of using molecular genetic studies to identify individuals prone to suicidal behavior.
Conflict of interests. The authors declare no conflict of interests.