Objective: Suicide is a significant public health issue and a major cause of death in all ages worldwide. Previous studies have shown the involvement of genetics in suicidal behaviour. This study aimed to assess the role of the genetic variants of the serotonin transporter genes (5HTTLPR, SLC6A4 intron 2) and receptor gene (5HTR2AT102C) in individuals who died of suicide. The study compares the serum levels of serotonin between the cases and controls. Methods: We conducted a case-control study with 120 cases and 126 controls. Socio-economic details of the subjects were collected using a semi-structured proforma, and psychological autopsy was used to collect details of medical and other clinical conditions. Blood was drawn after taking informed consent and serum levels of serotonin were estimated by enzyme-linked immunosorbent assay. Genotyping was performed using appropriate primers followed by polymerase chain reaction and a restriction fragment length polymorphism. Results: Mean age was 32.59 ± 12.58 for cases and 33.64 ± 9.78 for controls. The risk-associated LL genotype of 5HTTLPR was higher among cases. The heterozygous 12/10 genotype of SLC6A4 intron 2 polymorphism was increased among controls. Serum levels of serotonin were lower among cases. Variant genotypes of all the 3 polymorphisms showed significant interaction (OR = 39.26) indicating that this model may increase suicidal tendency. Conclusion: The findings of this study suggest that low serum levels of serotonin and two variants of the serotonin gene may influence suicide behaviour in a south Indian population.

Highlights of the Study

  • Role of serotonin gene polymorphisms and serum levels of serotonin in suicidal behaviour was evaluated.

  • Serotonin transporter 5HTTLPR polymorphism and SLC6A4 intron 2 polymorphisms are associated with suicidal behaviour.

  • The mean levels of serum serotonin are significantly lower in suicidal behaviour compared to the controls.

  • In silico analysis shows a significant BIOGRID interaction for SLC6A4.

Suicidal behaviour (SB) is the major cause of death in all ages worldwide. According to the World Health Organisation (WHO) about 1 million people commit suicide every year. Though it is a major concern all over the world, 78% of the suicides are reported in low and middle-income countries. WHO defines SB as “a range of behaviours that include thinking about suicide (or ideation), planning for suicide, attempting and committing suicide itself” [1]. Attempted suicide is the act of causing injury to oneself with the desire to end one’s life but not resulting in death. On the other hand, when death occurs as a result of the attempted suicide it is known as completed suicide.

Aetiologically suicide is considered as a neurobiological disorder, but genetic and epidemiological studies have indicated that genes play a significant role in the predisposition to SB. Family, twin and adoption studies have shown that genetics has a role to play in SB. It is said to be passed from one generation to another through complex hereditament. Interactions between multiple genes and environmental factors are said to contribute to this inheritance.

Though recent studies such as the one by Harika et al. [2] suggest that psychiatric disorders may interact with serotonin gene polymorphism, results of studies based on such gene-by-stress interactions have been contradictory and inconsistent. One such study found that there is a possibility of genetic predisposition to suicidal behaviour that could be independent of comorbid psychiatric disorders [3]. Hence, considering the genetics and the neurobiological basis of suicidal behaviour, the genes related to the serotonergic system gains special interest.

Studies from the suicide attempters have shown a reduced concentration of 5-hydroxyindoleaceticacid (5-HIAA) in the cerebrospinal fluid [4] and plasma [5]. Post-mortem examination of brains of suicide victims also shows considerable alterations in the binding kinetics of 5HT receptor subtypes. It has also been demonstrated that the number of serotonin transporter binding sites on the brain and on platelets were reduced and the density of 5-HT2A receptors were increased in regions of the brain as well as platelets of depressed suicidal patients [6]. Hence, studies have focused on analysing genetic variations in the serotonergic genes as a cause for suicidal behaviour.

Among the serotonergic system, the gene SLC6A4, located on chromosome 17q11.1–q12 codes for the human serotonin transporter (5-HTT) protein [7]. Two common polymorphisms have been identified in this gene. One is a functional polymorphism (5-HTTLPR) which consists of different lengths of the repetitive sequence containing GC-rich, 20 to 23bp-long repeat elements in the upstream regulatory region of the gene. A deletion/insertion in the 5-HTTLPR creates a short (S) allele and a long (L) allele (14 and 16-repeat alleles) which alters the promoter activity. The S variant has been reported to be associated with lower basal and transcriptional efficiency of the SLC6A4, resulting in lower serotonin uptake activity when compared with the L variant that could lead to depression [8]. However, a study by Culverhouse et al. [9] reports that it is difficult to affirm that the S allele of the 5-HTTLPR increases risk of depression only in the stressed individuals, indicating that contrary results have been obtained with respect to association of S allele of the 5-HTTLPR and risk of suicide.

The second functional VNTR polymorphism located in intron 2 known as STin2, consists of 9, 10, or 12 copies of the 17bp repeat sequence (STin2.9, STin2.10, and STin2.12). The repetitive element conformed to the consensus sequence, GGCTGYGACCY(R)GRRTG, where Y = T/C, R = G/A, with loss of the 12th base pair in one of the repeating elements. An individual may be homozygous (9/9 or 10/10 or 12/12 repeat) or heterozygous (9/10 or 9/12 or 10/12 repeat) for the repeat sequence. It has been suggested that the VNTR region may act as a transcriptional regulator of the SLC6A4 gene [8].

Another candidate gene that has been studied in relation to serotonin system dysfunction and suicidal behaviour is the 5HTR2A gene encoding the serotonin 2A receptor. Located on chromosome 13q14-q21, the 5HTR2A gene spans 20 kb and contains 3 exons [10, 11]. The 5HTR2AT102C polymorphism has been widely explored, in various psychiatric disorders [12].

The results obtained from case-control studies on the association of 5HTTLPR, SLC6A4 intron 2, and 5HTR2A T102C polymorphisms are controversial. Considering that there exists a wide difference in the genotype frequencies among different populations and that there is no data on the role of gene polymorphisms in SB in Indian population, we studied the role of the two genetic variants of the serotonin transporter (5HTTLPR, SLC6A4 intron 2), receptor 5HTR2AT102C, and serotonin levels in a sample of South Indian population.

The study was approved by the Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research (CSP-MED/16/JAN/27/31). The study was conducted over a period of 2 years from 2016 to 2018, during which a total of 150 cases of suicide were evaluated; 10 of the 150 cases (6.66%) were excluded as they had diagnosed psychiatric illness and had undergone treatment. In 10 cases, details related to suicide could not be collected and in 10 cases the relatives were not willing to give consent. Finally, 120 cases were included in the study as they met the inclusion criteria. Cases included individuals in the age group of 15–65 years who died of suicide and who were autopsied. Socio-demographic and other clinical data about the deceased were obtained using psychological autopsy (performed by a psychiatrist) from the deceased relatives, friends, investigating officer and the inquest report. Diagnosed major psychiatric disorders such as schizophrenia and other related psychotic disorders, bipolar affective disorders, and major depressive disorders were not included in the study to avoid genetic bias arising due to the psychiatric illness per se. It is a well-known fact that these major psychiatric disorders increase the tendency for suicide. The polymorphisms associated with these disorders can have a bearing on suicide. Hence this exclusion was made in our study. A customized semi-structured proforma, designed and validated was used for the purpose of collecting the socio-demographic details such as the age, sex, education, occupation, and socio-economic status.

The control group consisted of age and sex-matched healthy volunteers without a history of suicide attempt. 126 controls were directly interviewed to get the information. To exclude the presence of the major psychiatric disorders, the MINI International Neuropsychiatric interview – DSM – IV English Version 5.0.0 was administered to the control subjects with the help of a psychiatrist. Control subjects were directly interviewed to obtain relevant information. To exclude major psychiatric disorders among cases, an informant variant of the MINI was validated and used by the mental health expert with the next of kin. Written informed consent was obtained from the relatives/legal heirs in the presence of a witness for suicide completers. For the control subjects informed consent was directly obtained from the individuals.

Blood samples were collected subsequent to taking consent from both cases and controls. Femoral vein blood samples were collected during the autopsy from the cases. Samples were separated for phenotypic and genotypic analyses.

Serum Levels of Serotonin

Estimation of serum levels of serotonin was done by using enzyme-linked immunosorbent assay (Demeditec Diagnostics GmbH, Kiel, Germany). This competitive enzyme-linked immunosorbent assay kit uses the microtiter plate format. Antigen is bound to the solid phase of the microtiter plate and acylated standards, controls, samples, and the solid phase are bound to the analyte compete for a fixed number of antiserum-binding sites. After the system is in equilibrium, free antigen and free antigen-antiserum complexes are removed by washing. The antibody bound to the solid phase is detected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The reaction is monitored at 450 nm. Quantification of unknown samples is achieved by comparing their absorbance with a reference curve prepared with known standard concentrations. All test samples were run in duplicates, and the quantification of test samples was achieved by comparing their absorbance with a reference curve prepared with the five standard concentrations ranging from 0, 15, 50, 150, 500 ng/mL. The intra- and inter-assay coefficients of variation for serotonin were 1.5% and 4.5%, respectively.

Genotyping

Genomic DNA was extracted using the blood DNA isolation kit (QIAGEN, Hilden, Germany). The HTTLPR and SLC6A4 genotyping was performed using the primers forward – GTC​AGT​ATC​ACA​GGC​TGC​GAG, reverse – TGT​TCC​TAG​TCT​TAC​GCC​AGT​G, forward – GGC​GTT​GCC​GCT​CTG​AAT​GC, reverse – GAG​GGA​CTG​AGC​TGG​ACA​ACC​AC, respectively. Polymerase chain reaction (PCR) amplification was performed in total volumes of 20 µL using 50 ng of DNA, 1XTaq buffer, 200 μm of dNTP, 1.5 units of Taq DNA polymerase, and 30pM of primers (forward and reverse). The amplified products were separated on agarose gel electrophoresis, and based on the product size, the genotypes were assigned as follows: for 5-HTTLPR, the homozygous insertion allele abbreviated as L/L = 528 bp, the heterozygous insertion and deletion abbreviated as L/S = 528, 484 bp, the homozygous deletion allele abbreviated as S/S = 484 bp. For SLC6A4 VNTR repeat polymorphism, the abbreviations are 12/12 repeat = 204 bp, 12/10 = 170, 204 bp, 10/10 = 170 bp, 12/9 = 204, 153 bp, 10/9 = 170, 153 bp, 9/9 = 153 bp.

The 5HTR2A T102C polymorphism was genotyped using PCR restriction fragment length polymorphism. PCR was performed using the forward primer TTG​AGC​TCA​ACT​ACG​AAC​TCC and reverse primer ACC​CAT​ACA​GGA​TGG​TTA​ACA​T, and the amplification was confirmed using agarose gel. The amplified products were subjected to restriction fragment length polymorphism using HpaII restriction enzyme and based on the restriction pattern the genotypes were assigned as follows CC = 314, 71 bp, CT = 385, 314, 71 bp, TT = 385 bp.

Computational Prediction Analysis

In silico prediction for the SLC6A4 was performed using the online tools GeneMANIA and STRING databases. GeneMANIA (Gene Function Prediction using a Multiple Association Network Integration Algorithm; www.genemania.org) was used to predict the genes association network and genes functions. GeneMANIA is an integrated interaction network program that predicts gene functions and possible interaction networks using many large publicly available datasets including protein-protein and genetic interaction network.

In silico Analysis of the SLC6A4

To analyse the gene interaction, functional network and protein-protein interaction (PPI) of the SLC6A4 gene we used two computational prediction tools. The prediction of interaction relationship and PPI for the SLC6A4 gene was performed using the STRING database. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) is a database of known and predicted protein interactions. The interactions include direct (physical) and indirect (functional) associations; they are derived from four sources – genomic context, high-throughput experiments, conserved co-expression, and previous knowledge. The functional association is the basic interaction unit in STRING and it establishes the link between two proteins that both contribute together to a specific biological function.

Statistical Analysis

We calculated descriptive measures like mean and standard deviation for characteristics like age. To test whether the study population followed Hardy-Weinberg equilibrium (HWE), the expected genotype and allele frequencies were calculated for both cases and controls for each polymorphism. The significance level between the cases and controls was determined by calculating the odds ratio at 95% confidence interval (CI) and p value <0.05 was considered significant. Stratified analysis based on the age was performed by categorising the patients into less than 30 years and greater than and equal to 30 years. While haplotype analysis was performed using the SNP Stats online tool, the linkage disequilibrium (LD) analysis was performed using the Haploview version 4.2 program. Lewontin’s coefficient D’ and Pearson’s correlation r2 were employed to estimate LD between the two polymorphisms in the 5HTT gene. Interaction between the two gene polymorphisms of the 5HTT and 5HTR2A genes was determined using multifactorial dimensionality reduction (MDR) analysis software (v3.2).

Demographic details of the study subjects are presented in Table 1. The mean age of the study population was 32.59 ± 12.58 for the cases and 33.64 ± 9.78 for the controls. No significant differences were observed in the mean age and gender distribution between the cases and controls, confirming that there was no gender bias in the recruitment of participants. Analysis of gender distribution across two age categories, mean age less than or equal to 30 years and more than 30 years, showed that 76% females committed suicide when they were less than 30 years of age. In contrast, there was a higher frequency of males (63.6%) in the age group above 30 years. Analysis with regard to their family type (nuclear vs. joint) revealed that 80% of cases who committed suicide belonged to nuclear families (p = 0.02). However, parameters of employment, marital status, and education did not show any significant difference between the cases and controls.

Table 1.

Demographic details of the study subjects

DetailsCases (n = 120)Controls (n = 126)p value
Age (mean±SD), years 32.59±12.58 33.64±9.78 0.53 
Gender, n (%) 
 Males 66 (55) 70 (56) 0.15 
 Females 54 (45) 56 (44) 
Family pattern 
 Nuclear 96 77 0.02 
 Joint 24 49 
Employment 
 Employed 60 80 0.10 
 Unemployed 60 46 
Education 
 High school and below 46 45 0.15 
 Post high school and above 74 81 
Marital status 
 Married 61 73 0.44 
 Unmarried 55 47 
 Divorced 
Method of suicide 
 Hanging 73 Not applicable  
 Poison 32 
 Drug overdose 15 
DetailsCases (n = 120)Controls (n = 126)p value
Age (mean±SD), years 32.59±12.58 33.64±9.78 0.53 
Gender, n (%) 
 Males 66 (55) 70 (56) 0.15 
 Females 54 (45) 56 (44) 
Family pattern 
 Nuclear 96 77 0.02 
 Joint 24 49 
Employment 
 Employed 60 80 0.10 
 Unemployed 60 46 
Education 
 High school and below 46 45 0.15 
 Post high school and above 74 81 
Marital status 
 Married 61 73 0.44 
 Unmarried 55 47 
 Divorced 
Method of suicide 
 Hanging 73 Not applicable  
 Poison 32 
 Drug overdose 15 

Distribution of cases based on the reason for suicide was analysed and it was observed that about 40% of the cases committed suicide for personal issues which include any one of the following – a personal health issue such as suffering from chronic pain due to terminal illness or suffering from an irreversible disease. It could be failure or inability to succeed in an event or task undertaken by the individual such as failure to pass an examination. It could also include discord with someone at work place or stress at work place or at home, fall in social reputation, death of a near or dear one, so on and so forth. The other reasons included family issues (31%), financial difficulties (24%), and the cause was unknown in 5% of the cases. A sub-analysis revealed that 62% of women committed suicide for personal issues, and for 90% of the males, suicide was due to financial difficulties. Analysis of gender distribution across two age categories, mean age less than or equal to 30 years and more than 30 years, showed that 76% females committed suicide when they were less than 30 years of age. In contrast, there was a higher frequency of males (63.6%) in the age group above 30 years.

Out of two polymorphisms of SLC6A4 analysed, the genotype distribution of 5-HTTLPR polymorphism revealed a significant difference between the controls and cases. The risk associated LL genotype was significantly higher in cases compared to controls (OR: 3.76; 95% CI: [1.84–7.69]). This remained consistent even when a sub-analysis of male-female distribution was performed. Also, the recessive model revealed a statistically significant association with suicide (OR = 3.82; 95% CI: [2.11–6.90]) (Table 2).

Table 2.

Genotype comparison between controls and cases

GenotypeControl (n = 126), n (%)Case (n = 120), nORCIp value
HTTLPR 
 S/S 38 (30) 25 Ref   
 L/S 67 (53) 43 0.97 0.51–1.83 NS 
 L/L 21 (17) 52 3.76 1.84–7.69 0.0004 
 Dominant model L/S + L/L 88 95 1.64 0.91–2.93 NS 
 Recessive model S/S + L/S 105 68 Ref   
 L/L 21 52 3.82 2.11–6.90 0.00001 
SLC6A4 
 12/12 80 (63) 94 Ref   
 12/10 26 (21) 0.29 0.13–0.66 0.004 
 Others (10/10, 10/9, 9/9) 20 (16) 17 0.72 0.36–1.47 NS 
 Dominant model 12/10 + Others 46 26 0.481 0.27–0.85 0.02 
 Recessive model 12/12 + 12/10 106 103 Ref   
 Others (10/10, 10/9, 9/9) 20 17 0.88 0.43–1.76 NS 
HTR2A 
 TT 53 (42) 41 Ref   
 TC 49 (39) 50 1.32 0.75–2.32 NS 
 CC 24 (19) 29 1.56 0.79–3.07 NS 
 Dominant model TC + CC 73 79 1.39 0.83–2.34 NS 
 Recessive model TT + TC 102 91 Ref   
 CC 24 29 1.35 0.74–2.49 NS 
GenotypeControl (n = 126), n (%)Case (n = 120), nORCIp value
HTTLPR 
 S/S 38 (30) 25 Ref   
 L/S 67 (53) 43 0.97 0.51–1.83 NS 
 L/L 21 (17) 52 3.76 1.84–7.69 0.0004 
 Dominant model L/S + L/L 88 95 1.64 0.91–2.93 NS 
 Recessive model S/S + L/S 105 68 Ref   
 L/L 21 52 3.82 2.11–6.90 0.00001 
SLC6A4 
 12/12 80 (63) 94 Ref   
 12/10 26 (21) 0.29 0.13–0.66 0.004 
 Others (10/10, 10/9, 9/9) 20 (16) 17 0.72 0.36–1.47 NS 
 Dominant model 12/10 + Others 46 26 0.481 0.27–0.85 0.02 
 Recessive model 12/12 + 12/10 106 103 Ref   
 Others (10/10, 10/9, 9/9) 20 17 0.88 0.43–1.76 NS 
HTR2A 
 TT 53 (42) 41 Ref   
 TC 49 (39) 50 1.32 0.75–2.32 NS 
 CC 24 (19) 29 1.56 0.79–3.07 NS 
 Dominant model TC + CC 73 79 1.39 0.83–2.34 NS 
 Recessive model TT + TC 102 91 Ref   
 CC 24 29 1.35 0.74–2.49 NS 

NS, not significant.

For the SLC6A4 intron 2 polymorphism, the heterozygous 12/10 genotype was significantly increased in controls when compared to the cases indicating an inverse association with suicide. In contrast, the genotype frequencies of the polymorphism 5HTR2A T102C showed no significant difference between the cases and controls (Table 2). HWE was calculated for all the polymorphism and the analysis revealed that the controls and cases for 5HTR2A T102C and SLC6A4 intron 2 followed HWE. For the 5-HTTLPR polymorphism cases showed deviation in HWE and the controls were in HWE.

Haplotype analysis was performed to increase the diversity of observable patterns of variation in the two polymorphisms 5-HTTLPR and intron 2 located on the SLC6A4 gene. Analysis revealed four combinations for the two polymorphisms in cases and controls, the haplotype of S&12 (OR: −0.49; 95% CI: 0.32–0.74; p < 0.001) and S&10 (OR: −0.35; 95% CI: 0.18–0.67; p < 0.001) were significantly increased in controls when compared to the cases. This indicates that the S allele in any combination has an inverse association with suicide. The LD analysis for the two polymorphisms in 5HTT gene using the Haploview program revealed a D’ of 0.08 and r2 value of 0.002 indicating no LD between the two polymorphisms.

The MDR was performed to understand the epistatic interaction between the SLC6A4 and 5HTR2A T102C polymorphisms. To identify a model that gives the details of genotype combinations and disease risk, we used MDR v3.2 which reduces the dimensionality of multi locus data and improve the ability to detect genetic combinations that will increase the disease risk. The analysis involves a hypothetical mathematical modelling, where the permutations and combinations of the available data are performed. Variant genotypes of all the 3 polymorphisms showed significant interaction (OR = 39.26), indicating that this model may increase the suicidal tendency (Table 3); in the case group, a bivariate analysis of genotypes of all 3 polymorphisms with demographic details showed no significant difference.

Table 3.

MDR analysis

ModelsBalanced accuracy CV trainingBalanced accuracy CV testingCross-validation (CV) consistencyOdds ratiop value
HTTLPR 0.60 0.53 10/10 2.33 <0.001 
SLC6A4, HTR2A 0.58 0.53 4/10 3.69 <0.001 
HTTLPR, SLC6A4, HTR2A 0.53 0.51 10/10 39.26 <0.001 
ModelsBalanced accuracy CV trainingBalanced accuracy CV testingCross-validation (CV) consistencyOdds ratiop value
HTTLPR 0.60 0.53 10/10 2.33 <0.001 
SLC6A4, HTR2A 0.58 0.53 4/10 3.69 <0.001 
HTTLPR, SLC6A4, HTR2A 0.53 0.51 10/10 39.26 <0.001 

The mean levels of serotonin were significantly lower among cases (143.25 ± 60.12 ng/mL) as compared to controls (93.16 ± 40.5 ng/mL) (95% CI: 15.99–68.92; p = 0.002). A comparison of genotypes with serotonin levels revealed that the LL genotype of the 5-HTTLPR polymorphism was associated with lower levels of serum serotonin.

The PPI networks identified for SLC6A4 are presented in Figure 1. STRING analysis revealed 11 nodes and 47 edges and the predicted functional partners include HTR1A, TPH1, HTR7, OMT, HTR2A genes. In STRING analysis nodes represent the proteins and edges represent the protein-protein associations. Gene enrichment analysis revealed significantly enriched BioGRID interactions including serotonin receptor signalling pathway, negative regulation of synaptic transmission, serotonin binding, neurotransmitter receptor activity, and integral component of synaptic membrane.

Fig. 1.

The PPI networks identified for SLC6A4.

Fig. 1.

The PPI networks identified for SLC6A4.

Close modal

Network analysis of single-gene (SLC6A4) entry is presented in Figure 2. The results generated by GeneMANIA showed 21 related genes including one input gene. The analysis revealed that the association network members were linked through these networks: physical interactions 77.64%, co-expression 8.01%, predicted 5.37%, co-localization 3.63%, genetic interactions 2.87%, pathway 1.88%, and shared protein domains 0.60%

Fig. 2.

Network analysis of SLC6A4.

Fig. 2.

Network analysis of SLC6A4.

Close modal

Suicide is a complex behaviour that involves multiple aspects of personality, state of health, and life circumstances. It is predicted to be a significant contributor to the global burden of disease over the coming decades. Reports from the WHO indicate that suicide accounts for the largest share of self-harm in developed countries. However, data from developing countries remain sparse. This study which is possibly one of the few that have been conducted in the South Indian population, attempts to fill the gap.

This study observes the rate of suicide between males and females, among various age categories, nuclear family versus joint family, and according to reasons for suicide. A study by Srivastava and Kumar [13] showed that age less than 30 years is one of the risk factors for suicide. Another study [14] revealed that 91.9% of those who committed suicide were aged 30 years or less. Hence, in our study, we took age of 30 years as cut off to determine the number of suicides of both sexes in the below 30 years and the above 30 years age group. It was observed that gender distribution across two age categories differed; a higher frequency of females committed suicide early in life when compared to the men who committed suicide when they were more than 30 years. This finding is supported by a study done by Shukla et al. [15] in which they documented that woman were significantly younger (≤24.6 years) when they committed suicide compared to men (>28.9 years). The authors also reported more suicides among women (34/100,000) than among men (24/100,000).

In India, the joint family system has been in existence since ancient times. Joint family can be defined as members of a unilineal descent group living together with their spouses and offspring in one homestead and under the authority of one of the members. A nuclear family can be defined as a household consisting of two married, heterosexual parents and their legal children [16]. We observed that 80% of cases who committed suicide belonged to nuclear families. This could be explained based on Durkheim’s principle of social regulation, which states that individuals who are not regulated enough by their family/society tend to commit suicide more. In joint families where work and financial commitments are shared among the various family members, stress tends to be lower and by extension, suicide rates are lower. These findings are in contrast to a study that reported a higher suicide rate (60%) among individuals from a joint family when compared to individuals from nuclear families (24%) which included 16% individuals living alone [17]. These variations in observations with respect to suicide and family pattern could be due to social and cultural differences in different parts of the world.

Discounting psychiatric illnesses, the reason to commit suicide are varied. On analysis, it was seen that the most common reason for females to commit suicide was personal issues, while among the males the most common cause for suicide was financial difficulties. Our observations are in accordance with observations made by Osvath et al. [17].

Further research on the aetiology of suicide has shown that the serotonergic system is linked to suicidal behaviour. Serotonin is a powerful neurotransmitter involved in the pathophysiology of many mood disorders. The serotonergic system forms a diffuse network within the central nervous system and plays a significant role in the regulation of mood and cognition. This study shows that levels of serotonin are significantly decreased among cases as compared to controls. A previous study observed that patients who attempted suicide had significantly lower levels of serum serotonin (10 ng/mL, SD 8.6) compared with the non-suicidal group (35.94 ng/mL, [SD: 23.36], p < 0.001) [18]. Another study reported that the mean serum level of serotonin level in suicidal deaths was 4.85 ± 1.09 pg/mL and 21.30 ± 2.15 pg/mL in controls [19].

Studies have shown that in those who have completed suicide there is reduced binding of serotonin, especially in the ventral or orbital prefrontal cortex suggesting that there is a link between reduced serotonin turnover and the alteration in the genes. This is similar to our observation that the LL genotype of the 5-HTTLPR polymorphism was associated with lower serum levels of serotonin [20].

Stress is said to be one of the major reasons for an individual to commit suicide. However, previous studies have shown biological roots to have a connection to an individual’s susceptibility to stress, especially those related to the serotonin system. Among these, the serotonin transporter gene has captured great attention as it is involved in the reuptake of serotonin at the brain synapses. The functional polymorphism of this gene has two alleles, a short (S) and long (L) allele which influence the transcription rate of serotonin.

Though studies have traced the association between the 5-HTTLPR polymorphism and suicidal behaviour, the results have been conflicting [3, 21]. A meta-analysis by Lin and Tsai [22] reported the association of the 5-HTTLPR S allele with suicidal behaviour in the psychiatric population, the disorder being most studied was depression. Contrary to this, Grabe et al. [23] observed higher active L allele and that it had an interactive effect of traumatic events on anxiety disorders. This could indicate that there is an interplay between stress and genetic and environmental factors as a result of the increased L allele in such individuals leading to possible suicide attempts.

Our study on the single polymorphism and haplotype and epistasis effect of the selected polymorphisms reveals some significant associations. The 5-HTTLPR polymorphism revealed a significant difference between the controls and cases with the risk associated LL genotype was significantly higher in the cases; this is similar to the study of Grabe et al. [23]. A sub-analysis of gender distribution for this polymorphism among males and females did not change the strength of association in the cases.

Such conflicting results in the allele association and suicide could be attributed to differences in the sample population, their ethnicity, and cultural differences. Variations in the age groups studied as well as the differences in the methods, factors used to predict the rate outcomes could also influence the results of such studies.

For the SLC6A4 intron 2 polymorphisms, the heterozygous 12/10 genotype was significantly increased in controls when compared to the cases indicating an inverse association with suicide. This was similar to the finding by de Lopez de Lara et al. [24] who observed that those who had at least one copy of the STin2.10 allele had a significant association with suicide completion. An association of haplotype L10 with completed suicide was observed in a Croatian population [25] which was not observed in our study.

Previous studies have suggested that the 5HTR2A T102C polymorphism may be involved in suicidal behaviour. We did not find such association in our study and this outcome was supported by two meta-analyses [26, 27]. In contrast, Du et al. [28] observed an association between suicide ideation and the 5HTR2A T102C polymorphism. Similar observations were made by Saiz et al. [29] and Tsai et al. [30].

Computational prediction analysis revealed significant BioGRID interactions and functional network for SLC6A4. Interaction analysis showed SLC6A4 with other genes to be involved in the serotonin metabolism. Hence it can be predicted that inherited variations in the SLC6A4 could be associated with alterations in the serotonin metabolism pathway.

To the best of our knowledge, the present study is the first to evaluate the association between the two polymorphisms in SLC6A4 and 5HTR2A T102C polymorphisms with SB among the South Indian population. The findings of this study have demonstrated that 5-HTTLPR and SLC6A4 intron, two variants of the serotonin gene possibly influence suicide behaviour in the South Indian population. Additionally, the study also confirms that low serum levels of serotonin are associated with suicide. This study, unlike other studies, did not find an association of the 5HTR2A T102C gene polymorphism with suicide. Additional analysis with a larger sample size including other gene polymorphisms in the serotonergic pathway will help in better understanding of the molecular basis of this complex trait.

This study was performed 2 years in line with the principles of the Declaration of Helsinki. The study was approved by the Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research (Reference Number - CSP-MED/16/JAN/27/31). A written informed consent was obtained from the relatives/legal heirs in the presence of a witness for suicide completers. For the controls informed consent was directly obtained from the individual.

The authors declare no conflict of interest.

This work was financially supported by Indian Council of Medical Research under the grant (PhD –(INTD)/39/FT/I/2015).

Sneha Sivaramakrishnan conceived the study, performed the sample collection, processing, experimentation, and wrote the initial draft of the manuscript. Vettriselvi Venkatesan designed the primers, supervised the genetic component of experimentation, edited and finalized the manuscript. Sampath Kumar Paranthaman guided data collection, supervised the experiments and edited the manuscript. Sathianathan contributed to subject selection by application of the psychiatric scales and helped in sample collection. Swetha Raghavan designed checklists and helped use the validated scales by using psychological autopsy with the caregivers. Priyadarshee Pradhan gave inputs for data collection and for estimation of serum serotonin levels.

The genotyping data from the findings of this study are not publicly available but are available from the corresponding author upon request.

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