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Keywords:

  • genetic variation;
  • polymerase chain reaction;
  • restriction fragment length polymorphism;
  • serotonin 1A receptor;
  • suicide

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Aim:  The association of serotonin 1A receptor (5-HTR1A) gene polymorphisms with suicidal behavior has been reported in several previous studies, but the results have been inconsistent, which might be due to ethnic differences. The aim of the present study was therefore to investigate the association between polymorphisms −1019C>G, 47C>T (Pro16Leu) and 815G>A (Gly272Asp) and suicidal behavior, taking into account age, gender, and the presence of stressful life and loss events in 1 year prior to suicide.

Methods:  A total of 191 suicide victims and 218 healthy control subjects were included in the present study. 5-HT1RA gene polymorphisms were determined on polymerase chain reaction–restriction fragment length polymorphism.

Results:  The distribution of −1019C>G genotypes was significantly different in suicide victims and healthy controls (P = 0.002), and the GG genotype was associated with a significantly higher number of more stressful life and loss events in the suicide victims (P = 0.017, P = 0.037, respectively). The distribution of 47C>T (Pro16Leu) and 815G>A (Gly272Asp) genotypes was not significantly different in the suicide victims and control subjects (P > 0.05). Moreover, these genotypes were not associated with stressful life and loss events (P > 0.05).

Conclusion:  The frequency of the −1019G allele in the 5-HTR1A gene was higher in suicide victims (with stressful life events) as compared with the control group. In contrast, neither 47C>T (Pro16Leu) nor 815G>A (Gly272Asp) polymorphisms were related with suicide and stressful life events.

SUICIDE IS AN important public health issue and despite extensive implementation of different strategies for early detection of possible victims, the worldwide rate of suicide is high. The World Health Organization (WHO) estimates that approximately 1.53 million people will die from suicide in 2020.1

There have been several reports demonstrating the association of suicidal behavior with neurotransmitters or neuroactive peptides, which are connected to human emotional states such as happiness and depression.2,3 There is a large body of evidence suggesting that alteration of serotonergic neurotransmission can affect suicidal behavior. An early report of such findings was published by Asberg et al.,4 who reported a low level of 5-hydroxyindolacetic acid (5-HIAA) in the cerebrospinal fluid of victims of suicide.5 Therefore, an accumulated number of studies have focused on identifying the genes affecting suicidal behavior in the serotonergic system.6

In the human brain, the serotonin 1A receptor (5-HTR1A) is highly abundant,7,8 and is expressed presynaptically on serotonin (5-HT) cell bodies in the raphe (somatodendritic autoreceptors), and post-synaptically in limbic and cortex regions.9 The 5-HTR1A gene, mapped to chromosome 5q11.2-q13, is intronless and codes for a 422-amino-acid protein.10 This gene has a number of single nucleotide polymorphisms (SNP): 47C>T (Pro16Leu) and 815G>A (Gly272Asp) as functional polymorphisms, and −1019 C > G in the promoter region. Several studies have investigated the association between 5-HTR1A polymorphisms, suicide and other mental diseases. In some previous studies that investigated two functional polymorphisms, 47C>T (Pro16Leu) and 815G>A (Gly272Asp), in connection with suicide, there was no statistically significant difference in the distribution of genotypes or allele frequencies between suicide victims and controls.11–13 In contrast, several studies, which investigated the association between the 5-HTR1A −1019 C > G and suicide, have reported conflicting results. Some studies have shown an increased susceptibility to suicide among carriers of the G allele,14 while others have reported a lack of an association.15,16

Because the distribution of certain genes and genotypes differs in different ethnic groups and no studies have been performed on the association of 5-HTR1A polymorphisms and suicide in the Iranian population, the current case–control study was designed to investigate this. Furthermore, because there is strong evidence for the effect of environmental stress on various components of the serotonergic system,17 the aim of the present study was to evaluate the association between 5HTR1A polymorphism and stressful life events in 1 year before suicide completion in suicide victims.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Subjects

The present study was approved by the Ethics Committee at the Department of Iranian Forensic Medical Organization. Before the study, all the subjects (family members of the suicide victims and control subjects) were given written information about the project and written consent was subsequently obtained from them. Venous blood used for DNA extraction was collected from all subjects in the course of autopsy during 2009–2011. The mean age of the suicide victims was 46.6 ± 16.2 years and that of control group was 44.8 ± 18.2 years. The male : female ratio in the control group was 1.7:1 and in the suicide group it was 2.8:1. Family members, who lived with the subjects before the suicide, were invited to provide information about the stressful life events of suicide victims. Of 177 invited family members, 130 participated, of whom 93 were male with a mean age of 49.1 ± 13 years, and 37 were female with a mean age of 47.0 ± 15 years.

Assessment of stress

The 12 categories in the list of threatening experiences, which was originally developed by Brugha et al.,18 have been used for assessment of stressful life events. The list is widely accepted and used by research groups throughout the world.16,19 The items were modified according to our understanding of Iranian culture. Because there are previous reports of the significance of the contribution of loss events to the risk of suicide,19 the association between polymorphisms and loss events was analyzed separately, and other stressful events were pooled together. The loss events were identified in the interviews using the Life Events and Difficulties Schedule (LEDS) scale. This scale classifies loss events into four major types: loss of (i) health; (ii) person; (iii) a cherished idea; and (iv) employment/material possessions. The number of all stressful life events, including loss events, was recorded separately for each suicide victim during the period of 12 months prior to suicide.

Genotyping

Genomic DNA was extracted from peripheral blood leukocytes using the salting-out method as previously described.20 For 5-HTR1A −1019C>G genotyping, a 182-bp fragment was amplified on polymerase chain reaction (PCR) using the forward primer 5′-TGT CGT CGT TGT TCG TTT GT-3′ and reverse primer 5′-CGT GTC AGC ATC CCA GAG TG-3′.13 Genomic DNA was amplified in a 50-µL mixture containing 100 ng DNA template, 0.4 µmol/L each primers, 200 µmol/L dNTPs, 5 µL of 10 × reaction buffer and 1.25 U Taq DNA polymerase. After DNA denaturation for 10 min at 95°C, the reaction mixture was subjected to 35 cycles of denaturation for 50 s at 94°C, 45 s of annealing at 55°C, and 45 s of extension at 72°C. PCR product was digested with HpyCH4 IV restriction enzyme (New England Biolabs, Mannheim, Germany) and restriction fragment length polymorphism fragments were separated and identified on 3% agarose gel electrophoresis and staining with ethidium bromide. Allele C corresponded to a 182-bp fragment and allele G to 158- and 24-bp fragments. Genotyping for 5-HTR1A 815G>A (Gly272Asp) and 47C>T (Pro16Leu) polymorphisms was performed according to previous reports.11,21 In summary, for 5-HTR1A Gly272Asp genotyping, a 384-bp fragment was amplified on PCR using the forward primer 5′-CCG CAA GAC GGT CAA AAA GG-3′ and reverse primer 5′-AAG GTG CCC ATG ATG CC-3′. PCR products were digested using FokI restriction enzyme (New England Biolabs), and the digested fragments were separated on 4% agarose gels. The restriction pattern was identified as Gly with a 384-bp fragment and as Asp with 232-bp and 152-bp fragments. For 5-HTR1A Pro16Leu genotyping, a 203-bp fragment was amplified on PCR using the forward primer 5′-GGG TCT CTG CAT TCC CTT CC-3′ and the reverse primer 5′-AGT AGT GTT GCC GCC GGT CT-3′. PCR products were digested using the Bsr I restriction enzyme (New England Biolabs), which did not cut the Pro/Pro genotype but cut the Leu/Leu genotype into two fragments: 175 bp and 28 bp. The digested fragments were separated on a 4% agarose gel.

Statistical analysis

Genotype and allele frequencies were compared using the chi-squared and Fisher's exact tests. Allele frequencies were determined by the gene counting method, and Hardy–Weinberg equilibrium was tested using the chi-squared test. The mean number of stressful life events was calculated for each subgroup and compared using one-way anova. P < 0.05 was considered significant and all calculations were performed using spss Statistics 17.00 (SPSS, Chicago, IL, USA).

For comparisons involving genotype frequencies in suicide victims, PGA version 2.0 (Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA; http://dceg.cancer.gov/bb/tools/pga) was used for calculation of power assuming a dominant mode of inheritance for the SNP and a perfect linkage disequilibrium (D' = 1) between the marker and disease alleles.22 The suicide prevalence in Iran has been assumed to be 1.4% according to Mohammadi et al.,23 and this figure was used for calculation of the frequency of the disease allele.

For comparisons involving genotype frequencies in different genders, ages, and life events, power was calculated using G*Power 3.1.0 (University of Kiel, Kiel, Germany).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Table 1 lists the genotype distribution and allele frequencies of the 5-HTR1A gene polymorphisms −1019C>G, 815G>A (Gly272Asp), and 47C>T (Pro16Leu) in suicide victims and control subjects. Genotype distribution was in Hardy–Weinberg equilibrium (χ2 = 0.03, P = 0.97). As shown in Table 1, the possibility of having a −1019G allele in the genotype of suicide victims was significantly higher than for control subjects, with an odds ratio of 4.78 (P = 0.001). In respect to 47C>G (Pro16Leu) and 815G>A (Gly272Asp) polymorphisms, however, we found no significant differences in the genotype frequencies of suicide victims and control subjects (P > 0.05), but the low powers of the latter tests prevent the drawing of any conclusions. Table 2 lists stressful life events, loss events, age, and gender of suicide victims according to the genotypes of 5-HTR1A gene polymorphisms. As shown in Table 2, stressful life events, including loss events, were reported to have a significant association with the GG genotype of −1019C>G (P = 0.017, P = 0.037 respectively). But there was no association between these events and the genotypes of 815G>A (Gly272Asp) and 47C>T (Pro16Leu) polymorphisms (P > 0.05). The power of the latter analysis, however, was very low. To check for lack of sampling bias for age and gender, we tested the present cases and controls for genotype frequencies across different ages and genders and did not find any significant differences.

Table 1.  5-HTR1A polymorphisms
GenotypeSuicide victims n (%)Control subjects n (%)Pχ2Power (%)OR (95%CI)
  • Calculated using PGA (version 2.0).

  • CI, confidence interval; 5-HTR1A, serotonin 1A receptor; OR, odds ratio.

C-1019G
 CC82 (42.9)150 (68.8)    
 CG53 (27.7)42 (19.2)    
 GG56 (29.3)26 (12)0.00212.6291.28 
 C165 (43.2)342 (78.4)    
 G217 (56.8)94 (21.6)0.001107.3491.284.78 (3.53–6.49)
Gly272ASP
 Gly/Gly155 (81.2)181 (83)    
 Gly/Asp29 (15.7)28 (12.8)    
 Asp/Asp6 (3.1)9 (4.1)0.7010.7115.27 
 Gly340 (89)390 (89.4)    
 Asp42 (11)46 (10.6)0.8380.0425.271.05 (0.67–1.63)
Pro16Leu
 Pro/Pro151 (79.1)183 (83.9)    
 Pro/Leo30 (15.7)28 (12.8)    
 Leu/Leu10 (5.2)7 (3.2)0.3891.89021.59 
 Pro332 (86.9)394 (90.4)    
 Leu50 (13.1)42 (9.6)0.1192.43621.591.41 (0.91–2.18)
Table 2.  Characteristic and life events in suicide victims vs 5-HTR1A polymorphism
GenotypesCharacteristicsLife events
Gender, n (%)Age, n (%)No. occurrences (mean ± SD)
Male (n = 141)Female (n = 50)≤30 years (n = 63)>30 years (n = 128)Stressful life eventsLoss events
  1. χ2 or Fisher's exact test where applicable; one-way anova. §Calculations done using G*Power (version 3.1.0).

  2. 5-HTR1A, serotonin 1A receptor.

C-1019G      
  CC60 (42.55)22 (44)26 (41.26)56 (43.75)1.50 ± 1.391.58 ± 1.42
  CG40 (28.36)13 (26)18 (28.57)35 (27.34)1.61 ± 1.311.60 ± 1.45
  GG41 (29.07)15 (30)19 (30.15)37 (28.90)3.01 ± 1.622.91 ± 1.53
 P0.9500.9480.0170.037
 χ20.1030.106  
 Power (%)§17179999
Gly272Asp      
  Gly/Gly114 (80.85)41 (82)50 (79.36)105 (82.03)1.61 ± 1.321.81 ± 1.25
  Gly/Asp22 (15.60)7 (14)11 (17.46)19 (14.84)1.59 ± 1.541.80 ± 1.34
  Asp/Asp5 (3.54)2 (4)2 (3.17)4 (3.12)1.63 ± 1.581.82 ± 1.54
 P1.000.8910.420.62
 χ20.2160.377  
 Power (%)§162855
Pro16Leu      
  Pro/Pro112 (79.43)39 (78)49 (77.77)102 (79.68)1.78 ± 1.231.67 ± 1.54
  Pro/Leu22 (15.60)8 (16)8 (12.69)22 (17.18)1.75 ± 1.341.66 ± 1.45
  Leu/Leu7 (4.96)3 (6)6 (9.52)4 (3.12)1.77 ± 1.451.68 ± 1.42
 P0.9510.1480.290.53
 Fisher's exact test0.2593.661  
 Power (%)§169255

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

In the present study we evaluated the distribution of −1019C>G, 47C>T (Pro16Leu), and 815G>A (Gly272Asp) polymorphisms of 5-HTR1A genotypes in suicide victims and control subjects, and found that only the −1019C>G polymorphism was significantly higher in suicide victims. Also, a higher number of more stressful life events was observed for the subgroup of suicide victims with the GG genotype of −1019C>G polymorphism.24 5-HTR1A knock-out mice exhibit lack of the autoinhibitory control exerted by 5-HTR1A.25 Moreover, recent studies suggest that 5-HTR1A affect other 5-HT (serotonin) transmitter systems in the brain. For example, animal studies have shown that 5-HTR1A in the cingulate cortex are involved in controlling the release of glutamate in the subcortical structures,26 or that both 5-HTR1A and 5-HTR2A increase brain cyclic guanosine monophosphate production in rat frontal cortex.27 It is not clear, however, how these models can explain the pathophysiology of depressed and anxious behaviors in humans. So far, only a very limited number of studies in humans are available, which explore the effects of 5-HTR1A polymorphism on and its association with depression and anxiety disorders.

It has been shown in a series of experiments that the part of the 5-HTR1A gene promoter that contains −1019C>G SNP binds to the transcription factor nuclear deformed epidermal autoregulatory factor DEAF-1 (NUDR), which subsequently represses transcription of 5-HTR1A. It has been shown that this inhibitory function is impaired in the presence of the −1019G allele, which is the same allele associated with suicide and major depression.16 The repression can enhance the feedback inhibition of serotonergic raphe neurons and lead to a lower serotonergic neurotransmission, causing a predisposition to depression and suicide.28 This finding may explain why in the present study the 5-HTR1A GG genotype was found to be present at an increased frequency in suicide victims.

Some previous studies have investigated the association between 5-HTR1A polymorphisms and suicidal behavior.12,17 The first study for assessment of association of suicide with 47C>G (Pro16Leu) and 815G>A (Gly272Asp) polymorphisms was reported by Nishiguchi et al.11 In accordance with this and some other studies,14 we did not find any association between these two functional polymorphisms and suicidal behavior. Due to the low power of the present study, however, the possibility of involvement of these polymorphisms in suicide cannot be ruled out. The alterations imposed by these genetic variations on 5-HTR1A function may contribute to suicidal behavior. A study with a large sample size and adequate power is needed to properly address this issue. In regards to −1019C>G polymorphism and suicide, some conflicting results have been reported by different groups so far. For example, Videtic et al. found no association between −1019C>G polymorphism and suicidal behavior,16 while in the study by Lemonde et al. the association was present in French-Canadian suicide victims.14 These discrepancies may be explained by the inter-ethnic differences of the frequency distribution of HTR1A polymorphisms or variability of the sample size.

Because there have been several reports of the association of multiple negative life events, and particularly loss events, with suicide attempts and completions, we addressed this issue as well. As a result, there are some interesting findings concerning the influence of gene–environment interaction on suicidal behavior, which has been indirectly confirmed by previous studies on twins, and adopted and own family members. It has been estimated that genetics may account for 43% of variability in suicidal behavior, and the environmental factors may account for the remaining 57%.29 In the present group of suicide victims we observed a tendency for those with the GG genotype to have a higher number of more stressful life events including loss events. The association between −1019C>G polymorphism and stressful life events has been reported in a limited Slovenian sample of suicide victims as well, but the reported associated genotype in that study was CC.17 This discrepancy warrants a large multi-ethnic project to re-assess the effects of genotypic variation on stressful life events.

In conclusion, the present study provides evidence of the effects of genetic factors on suicidal behavior. We found that the −1019C>G polymorphism is more likely to be found in suicide victims and those with stressful life and loss events. But we did not find any association of 815G>A (Gly272Asp) and 47C>T (Pro16Leu) with suicide. To our knowledge, this is the first study on the association between suicide and 815G>A (Gly272Asp) and 815G>A (Pro16Leu) 5-HTR1A genetic variations, taking into account stressful life and loss events. Although we found no association between 815G>A (Gly272Asp) and 47 C > T (Pro16Leu) genotypes and stressful life and loss events, the low power of the present study does not allow the drawing of a definite conclusion. Further studies with large sample sizes are needed for assessment of this association.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The authors thank the Research Deputy of Forensic Medical Organization of Tabriz for funding this project, and have no conflict of interest to declare.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES