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Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population
Accepted Manuscript Title: Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population Authors: Katarina Urˇsiˇc, Tomaˇz Zupanc, Alja Videtiˇc Paska PII: DOI: Reference:
S0304-3940(18)30152-6 https://doi.org/10.1016/j.neulet.2018.02.063 NSL 33455
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
21-12-2017 6-2-2018 27-2-2018
Please cite this article as: Katarina Urˇsiˇc, Tomaˇz Zupanc, Alja Videtiˇc Paska, Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.02.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population
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Running head: MAOA polymorphism in suicide Katarina Uršiča, Tomaž Zupancb, Alja Videtič Paskaa1
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000
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Ljubljana, Slovenia. b
Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Korytkova ulica
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2, SI-1000 Ljubljana, Slovenia.
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Corresponding author: [email protected]
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Highlights
Suicide is a complex phenomenon influenced by a number of different risk factors. Polymorphism in MAOA promoter region affects the activity of transcription.
We genotyped MAOA polymorphism in a population with one of highest suicide
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rates in the world.
We observed a possible effect of MAOA-uVNTR 3R allele on suicide.
3R allele was associated with a choice of non-violent suicide method.
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Abstract
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Suicide is a well-defined public health problem and is a complex phenomenon influenced by a number of different risk factors, including genetic ones. Numerous studies have
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examined serotonin system genes. Monoamine oxidase A (MAO-A) is an outer mitochondrial membrane enzyme which is involved in the metabolic pathway of
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serotonin degradation. Upstream variable number of tandem repeats (uVNTR) in the promoter region of MAOA gene affects the activity of transcription. In the present study
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we genotyped MAOA-uVNTR polymorphism in 266 suicide victims and 191 control subjects of Slovenian population, which ranks among the European and world
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populations with the highest suicide rate. Genotyping was performed with polymerase chain reaction and agarose gel electrophoresis. Using a separate statistical analysis for female and male subjects we determined the differences in genotype distributions of MAOA-uVNTR polymorphism between the studied groups. Statistical analysis showed a trend towards 3R allele and suicide, and associated 3R allele with non-violent suicide
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method on stratified data (20 suicide victims). This is the first study associating highly suicidal Slovenian population with MAOA-uVNTR polymorphism.
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Keywords: suicidal behaviour, polymorphism, monoamine oxidase A, VNTR
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1. Introduction Suicidal behaviour is a complex public health problem of global proportions. Worldwide close to 800,000 people die as a result of suicide, translating to a life lost every 40 seconds. According to the suicide rate (the number of suicides per 100,000 inhabitants), Slovenia
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has long been ranked above European and global average rates [1]. Despite a decrease in number of suicides for the past decade, in 2016 Slovenian suicide rate was 18 (with
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European average being around 11) [2].
Genetic predisposition of an individual plays an important role in development of suicidal
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behaviour, as shown with adoptees studies and studies of identical (monozygotic) and
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fraternal (dizygotic) twins. The impact of genetics on the onset of suicidal thoughts and
[3, 4].
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1.1. Monoamine oxidase A
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behaviour is supposed to be 45 %, while the rest can be attributed to environmental factors
MAO is an external mitochondrial membrane enzyme that degrades biogenic amines,
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mainly catecholamines and serotonin by oxidative deamination. There are two known isoenzymes, MAO-A and MAO-B, located on chromosome X in the region p11.23-11.4.
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The MAOA gene consists of 16 exons, making the gene approximately 70 kb long. MAOA preferentially degrades serotonin and norepinephrine. In the central nervous system it
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is found in catecholaminergic cell groups [5, 6]. Many polymorphisms of the MAOA gene have been identified and studied, indicating a possible association with various behaviour patterns and mental disorders. Single nucleotide polymorphisms (SNPs) C1460T, which causes the formation of the EcoRV restriction site, and T941G, which causes the formation of the Fnu4HI restriction site,
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may affect the activity of MAO-A, but do not alter the amino acid sequence [7]. A family study of five men showed that the absence of MAO-A activity leads to aggression, impulsive behaviour and borderline intellectual functioning. Due to point mutation C936T in exon 8, which causes early stop codon, MAO-A is no longer active [8].
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Approximately 1.2 kb upstream from exon 1 lies a promoter region polymorphism, a variable number of tandem repeats (uVNTR). The individual tandem sequence consists
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of 30 bp, and can be repeated 3 times, 3.5 times, 4 times, or 5 times. The number of
tandem repeats affects the rate of gene transcription. Transcription rate is higher in 3.5repeat (3.5R) and 4-repeat (4R) alleles, and lower in 3-repeat (3R) or 5-repeat (5R) alleles
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[9]. Changes in serotonergic activity of the central nervous system are associated with
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impulsivity, decreased learning ability and increased aggression [10]. Later two more
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alleles were discovered; lowest transcription activity 2-repeat (2R) allele, showing a link
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with antisocial and violent behaviour and a higher possibility of arrest [11, 12], and a 6repeat allele [13].
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MAOA-uVNTR polymorphism has also been studied regarding alcohol dependence.
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Some studies found a weak association of reduced MAO-A activity and alcohol dependence [14], while others failed to draw the same conclusion [15].
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We hypothesized that MAOA-uVNTR polymorphism would be statistically related to suicidal behaviour. The main aim of our study was therefore comparing MAOA-uVNTR polymorphism allele frequencies between suicide victims and control group and see if
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they correlate with choice of method and alcohol abuse.
2. Methods
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2.1. Subjects 272 suicide victims (203 male, 69 female) and 193 control subjects (142 male, 51 female) were included in the presented study. Blood samples were taken during the autopsies, performed at the Institute of Forensic Medicine, Faculty of Medicine University of
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Ljubljana. Genomic DNA was isolated from venous blood, using standard methods and stored at +4 °C. Study samples were collected consecutively through the years 2002–2005
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and 2012–2013. Subjects of both groups were matched for age, sex and ethnicity. Mean age ± SD of the suicide victims group and of the control group were 46.3 years ± 16.5, and 53.0 years ± 17.6 for men and 55.3 years ± 18.6, and 56.3 years ± 18.6 for women.
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Male to female ratio in both groups were 3 to 1, and 2.8 to 1, respectively for suicide
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victims and controls. All subjects were of Slovenian origin.
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Subject selection criteria was sufficient body preservation for blood sample collection.
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Subjects in control group were also selected based on the cause of death which excluded
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possible suicide (predominate cause of death being sudden cardiac arrest). Alcohol abuse was confirmed by autopsy reports and histological liver sections, which showed changes
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of the liver that could be attributed exclusively to excessive alcohol consumption (fatty liver disease with >60% of hepatocytes showing macrovesicular hepatocyte fatty change,
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alcohol hepatitis and hepatic cirrhosis). Simultaneously, causal factors for histologic changes of liver were excluded (abnormalities of hepatic tissue due to obesity, diabetes
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and/or congestive heart failure). The study was approved by the Slovenian National Medical Ethics Committee.
2.2. Genotyping
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We genotyped MAOA-uVNTR polymorphism using polymerase chain reaction (PCR) amplification. Estimated PCR product length was minimum 294 to maximum 384 base pairs, from 2R allele to 5R allele. Products were amplified using a primer pair, based on the work of Sabol [9]. Sequences of primers are as following: sense 5'-
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ACAGCCTGACCGTGGAGAAG-3' (position -1369 to -1350) and antisense primer 5'GAACGGACGCTCCATTCGGA-3' (position -1039 to -1038). Reactions were carried
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out in volume of 15 µl, containing 70 ng of DNA, 7.5 µl of PCR Master Mix (50 U/ml
Taq DNA polymerase, 400 μM dNTPs, 3 mM MgCl2 and reaction buffers) (Promega, Madison, USA), 1.2 µl of each 2.5 µmol primer (Sigma-Aldrich, Hamburg, Germany) and
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water. PCR was performed using GeneAmp PCR system 9700 (Applied Biosystems,
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Foster City, ZDA) with conditions: 30 seconds at 95 °C, 30 seconds at 68 °C and 30
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seconds at 72 °C for 35 cycles. PCR products were separated and visualized using 2.8 %
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agarose gels with ethidium bromide and LAS-4000 (Fujifilm, Tokio, Japan).
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2.3. Sanger sequencing
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The sequence of amplified PCR products was confirmed by Sanger sequencing. For four observed MAOA-uVNTR polymorphism alleles one fragment was send to Macrogen
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(Amsterdam, Netherlands), where they were sequenced. 2.4. Statistics
Data was analysed using R version 3.3.3 (R Core Team, 2017) [16]. Statistical analysis
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was done separated by gender as MAOA-uVNTR polymorphism is located on the X chromosome. For age comparison between groups two-tailed t-test and one-way ANOVA were used. For allele and genotype frequency association between groups two-tailed Fisher’s exact test was used. For allele frequencies odds ratio (OR) with 95% confidence
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interval (CI) was calculated. All female groups where within Hardy-Weinberg equilibrium (supplementary table S1). Power analysis using G*Power 3.1 [17] was conducted to determine a priori sample size (parameters effect size = 0.3, significance level = 0.05, power = 0.80). Total desired sample size was 102. Actual total sample size
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was 341 for male subjects and 116 for female subjects, concluding that sample size is
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appropriate to detect significant differences in the two main studied groups.
3. Results
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Eight samples (four male and four female; three suicide victims and one control for both
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gender) were excluded from the statistical analysis after genotyping. Exclusion criteria
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was presence of a low frequency allele (2R, 3.5R, or 5R allele). Remaining 457 subjects
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were grouped as such: 266 suicide victims (200 male, 66 female) and 191 control subjects (141 male, 50 female). Suicide methods of the 266 suicide victims were distributed as
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follows: hanging, n = 130 (48.8%); shooting, n = 38 (14.3%); jumping from height, n = 31 (11.7%); poisoning, n = 24 (9%); drowning, n = 17 (6.4%); traffic accident, n = 12
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(4.5%); cutting wounds, n = 8 (3%); self-burning and electrocution, n = 4 (1.5%), and
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suffocation, n = 2 (0.8%). Based on previous studies, superficial cutting wounds and poisoning with solid, liquid and gaseous substances were regarded as non-violent suicide methods and remaining methods as violent (hanging, shooting, jumping from height,
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drowning, traffic accident, self-burning, electrocution, suffocation) [18-20] Nucleotide sequence of PCR was determined using Sanger sequencing which confirmed that the correct sequence was amplified (supplementary figure S1 - S4).
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Suicide victims and control group 3R and 4R-allele carriers had equivalent mean age (suicide victims p = 0.3047; control group p = 0.4958 for men and suicide victims F = 1.791 p) = 0.175; control group F = 0.11, p = 0.896 for women). Table 1 shows the distribution of MAOA-uVNTR polymorphism allele frequencies in
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male subjects. Statistically significant differences in allele frequencies were observed between control group and non-violent method suicide victims (p = 0.049, 95 % CI: 0.131
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- 1.106, OR: 0.386); control group without alcohol dependence and suicide victims (p = 0.037, 95 % CI: 0.316 - 0.990, OR: 0.565) and control group without alcohol dependence
and suicide victims without alcohol dependence (p = 0.043, 95 % CI: 0.306 - 0.990, OR:
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0.556).
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Statistical analysis of MAOA-uVNTR polymorphism genotype and allele frequencies
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shown no statistically significant differences in any of the investigated female groups
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(supplementary table S2 and S3). The ratio of homozygous to heterozygous genotypes
4. Discussion
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was 1.8 to 1 (32 to 18) for control group and 1.4 to 1 (38 to 28) for suicide victims.
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The aim of our study was to genotype MAOA-uVNTR polymorphism in Slovenian
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subjects. Our results suggest involvement of MAOA-uVNTR 3R allele in suicidal behaviour of men but not women. 3R allele was more often present in male victims of suicide (regardless of the chosen method of suicide), male suicide victims who have
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chosen a non-violent method; and in male suicide victims without alcohol dependency when compared to control group. Suicide is a complex phenomenon which is determined by many different risk factors, including genetic ones. Slovenia is ranked as one of the leading countries regarding suicide rate in the world, despite numerous protective factors such as warm climate, stable
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economy and a high percentage of Catholics [1]. As the genetic component for suicidal behaviour could be strong for Slovenian population, we questioned whether MAOAuVNTR polymorphism, a 30 bp long sequence that affects gene transcription activity [9, 21] could affect the rate of suicide. Namely, several studies of the serotonergic system on
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Slovenian population showed association with completed suicide so far [22-25]. In the present study we included 266 suicide victims (200 male, 66 female) and 191
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control subjects (141 male, 50 female). Genotype frequency distributions for females were within Hardy-Weinberg equilibrium (supplementary table S1). Among suicide victims there is a higher proportion of men to women (global ratio around 4 to 1,
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Slovenian 3 to 1) [1], which is similar to ratio of subjects of our study. In our study group
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the most commonly used methods was hanging, in both men (46%) and women (39%),
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and more suicide victims chose violent than non-violent methods. Both is in accordance
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with published epidemiological data for Slovenian population and many countries worldwide [19, 26].
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All statistical analysis was done separated by gender due to X-chromosomal location of
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the MAOA gene. First we compared MAOA-uVNTR polymorphism frequencies between suicide victims and control group. As has been shown by other studies MAOA alleles have
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been previously associated with aggression, impulsivity and alcohol abuse [11, 14] so we stratified our data further, which resulted in a smaller number of subjects in stratified groups. From the control group, we selected a control group without alcohol dependency.
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From the suicide victims we selected two groups depending on the use of violent or nonviolent method of suicide victims and two groups depending on alcohol dependency. In males, comparison of allele frequency distribution showed some statistically significant differences. When comparing suicide victims and control group we failed to
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detect statistical significance (chosen as p < 0.05), although the observed p-value of 0.088 is suggestive towards 3R allele being more frequent in suicide victims. Statistical significance was observed comparing three pairs of subgroups. First occurred when comparing control group and non-violent method suicide victims (p = 0.049). 3R
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allele was more often present in male suicide victims who have chosen a non-violent method than in control group. Our results are conclusive with a study conducted on
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healthy volunteers, associating 3R and 5R alleles with lower degree of aggression and
impulsivity in male subjects. Similarly, 3.5R and 4R alleles were found to be associated with a higher degree of aggression and impulsivity [10]. Another conclusive study found
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a link between the attempted suicide by a violent method and the increased frequency of
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3.5R and 4R allele compared to a non-violent method suicide attempt [27]. However, the
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link between the MAOA-u VNTR polymorphism and suicide is not fully established.
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Many studies failed to link MAOA-uVNTR polymorphism genotypes with suicidal behaviour [28-32]. Our result is contradictory to multiple studies of aggression on non-
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suicidal subjects. Incarcerated offenders show higher frequency of low expression alleles
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[11, 33]. We could conclude that low expression alleles probably don’t predispose aggression [34, 35] but when accompanied with childhood adversary associate with
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violent behaviour and impulsivity [36, 37]. Similarly, one study found no association between MAOA and suicide attempts but found an association between lower expression alleles, history of abuse and male subject with high impulsivity [13]. We would also like
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to point out that the average age of violent and non-violent suicides were not equivalent in our sample (mean age 47.32 years ±16.38 and 37.35 years ±15.74 respectively, p = 0.013). While older suicide victims tend to use more premeditated and violent methods we do not have data regarding average age of violent and non-violent suicide victims in
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Slovenia so we cannot completely exclude potential confounds in relation to agedependent differences in impulsivity. Second statistically significant difference occurred in the comparison of control group without alcohol dependency and suicide victims (p = 0.037). 3R allele occurred more
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frequently in male victims of suicide, regardless of the chosen method of suicide, as in control group without alcohol dependency. Third statistically significant difference was
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observed in comparison of control group without alcohol dependency and suicide victims
without alcohol dependency (p = 0.043). 3R allele occurred more frequently in male suicide victims without alcohol dependency than in control group without alcohol
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dependency.
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Majority of studies found no association between MAOA-uVNTR polymorphism and
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alcohol abuse [38]. Alcohol dependant subjects with strong antisocial tendencies showed
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a higher frequency of 3R allele when compared to controls and non-antisocial alcoholics [39, 40]. A study on alcohol dependent males showed a weak link between risk of alcohol
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dependency development and 3R and 5R allele [14]. Similarly male rhesus macaques
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animal model associated lower expression of MAOA in blood with higher alcohol consumption [41]. Our analysis failed to detect association between alcohol abuse and
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suicide. While 3R allele was more frequent in suicide victims, statistical significance was also observed in group of suicide victims without alcohol dependency. Although we did notice a higher frequency of 3R allele in suicide victims with alcohol dependency
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compared to control group without alcohol dependency, the results were not statistically significant.
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Comparison of genotype and allele frequency distribution showed no statistically significant differences between any of the female studied groups (supplementary tables S2 and S3). As a major limitation of our study we would like to emphasize small sample size after
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sample stratification, resulting in lower power of study. Our starting sample size is sufficient but as we stratify subjects in subgroups, the sample size gets noticeably smaller.
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With around 2 million inhabitants, Slovenia is a low populated country which makes
sample collection harder. Data collection would therefore take many more years. We also do not have access to data about potential early childhood adversities, socio-demographic
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data, clinical data and comorbid mental disorders besides alcohol dependency. MAOA
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gene contains many other polymorphisms that could influence suicidal behaviour
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independently, with other genes or through the gene – environment interaction. Studies
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also observed changes in MAOA methylation pattern in depression, panic disorder and antisocial personality disorder [42-44].
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Suicidal behaviour is s complex behaviour that is influenced by many factors. Thus, it is
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unlikely that the genetic component would be determined only by the influence of an individual gene. Impulsivity and aggression, which may lead to suicidal behaviour, are a
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result of interplay between many genes, environment, development and psychopathology [45]. Our study, conducted on a population with high suicide risk, contributes to existing knowledge of serotonin system and suicidal behaviour by showing a possible effect of
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MAOA-uVNTR polymorphism 3R allele.
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Author’s contributions TZ identified subjects to be included in the study, provided samples and assigned the diagnosis. AVP designed the study. KU preformed PCR and statistical analysis. KU drafted of the manuscript. AVP edited the manuscript. All authors read and approved the
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final manuscript.
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Acknowledgements
The authors would like to thank Sandra Ropret for help with sample preparation and Nejc
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Nadižar for help with statistical analysis.
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Funding
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This work was supported by the Slovenian Research Agency, program grant no. P1-0104
Conflict of interest
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(D), and postdoctoral project no. Z3-2180.
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The authors declare no conflict of interest.
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References
[3]
[4] [5]
[10]
CC E
[11]
PT
[9]
ED
M
[8]
A
[7]
N
U
[6]
IP T
[2]
World Health Organization, 2014. Mental health: suicide prevention. http://who.int/mental_health/suicide-prevention/en/ (accessed 1 Aug 2017). SI-STAT Data Portal, NIJZ, Statistični urad Republike Slovenije. http://pxweb.stat.si/pxweb/Database/Dem_soc/05_prebivalstvo/32_Umrljivos t/15_05L30_umrli-vzrok/15_05L30_umrli-vzrok.asp (accessed 15 Sep 2017). D.J. Statham, A.C. Heath, P.A. Madden, K.K. Bucholz, L. Bierut, S.H. Dinwiddie, W.S. Slutske, M.P. Dunne, N.G. Martin, Suicidal behaviour: an epidemiological and genetic study, Psychol Med 28 (1998) 839-855. D.A. Brent, J.J. Mann, Family genetic studies, suicide, and suicidal behavior, Am J Med Genet C Semin Med Genet 133C (2005) 13-24. Z.Y. Chen, G.S. Hotamisligil, J.K. Huang, L. Wen, D. Ezzeddine, N. AydinMuderrisoglu, J.F. Powell, R.H. Huang, X.O. Breakefield, I. Craig, et al., Structure of the human gene for monoamine oxidase type A, Nucleic Acids Res 19 (1991) 4537-4541. Q.S. Zhu, J. Grimsby, K. Chen, J.C. Shih, Promoter organization and activity of human monoamine oxidase (MAO) A and B genes, J Neurosci 12 (1992) 44374446. G.S. Hotamisligil, X.O. Breakefield, Human monoamine oxidase A gene determines levels of enzyme activity, Am J Hum Genet 49 (1991) 383-392. H.G. Brunner, M.R. Nelen, P. van Zandvoort, N.G. Abeling, A.H. van Gennip, E.C. Wolters, M.A. Kuiper, H.H. Ropers, B.A. van Oost, X-linked borderline mental retardation with prominent behavioral disturbance: phenotype, genetic localization, and evidence for disturbed monoamine metabolism, Am J Hum Genet 52 (1993) 1032-1039. S.Z. Sabol, S. Hu, D. Hamer, A functional polymorphism in the monoamine oxidase A gene promoter, Hum Genet 103 (1998) 273-279. S.B. Manuck, J.D. Flory, R.E. Ferrel, J.J. Mann, M.F. Muldoon, A regulatory polymorphism of the monoamine oxidase-A gene may be associated with variability in aggression, impulsivity, and central nervous system serotonergic responsivity, Psychiatry Research (2000) 9-23. K.M. Beaver, J.P. Wright, B.B. Boutwell, J.C. Barnes, M. DeLisi, M.G. Vaughn, Exploring the association between the 2-repeat allele of the MAOA gene promoter polymorphism and psychopathic personality traits, arrests, incarceration, and lifetime antisocial behavior, Personality and Individual Differences 54 (2013) 164-168. K. Brookes, X. Xu, W. Chen, K. Zhou, B. Neale, N. Lowe, R. Anney, B. Franke, M. Gill, R. Ebstein, J. Buitelaar, P. Sham, D. Campbell, J. Knight, P. Andreou, M. Altink, R. Arnold, F. Boer, C. Buschgens, L. Butler, H. Christiansen, L. Feldman, K. Fleischman, E. Fliers, R. Howe-Forbes, A. Goldfarb, A. Heise, I. Gabriels, I. KornLubetzki, L. Johansson, R. Marco, S. Medad, R. Minderaa, F. Mulas, U. Muller, A. Mulligan, K. Rabin, N. Rommelse, V. Sethna, J. Sorohan, H. Uebel, L. Psychogiou, A. Weeks, R. Barrett, I. Craig, T. Banaschewski, E. Sonuga-Barke, J. Eisenberg, J. Kuntsi, I. Manor, P. McGuffin, A. Miranda, R.D. Oades, R. Plomin, H. Roeyers, A.
SC R
[1]
A
[12]
16
[18]
[19]
[20]
IP T
SC R
U
CC E
[21]
N
[17]
A
[16]
M
[15]
ED
[14]
PT
[13]
Rothenberger, J. Sergeant, H.C. Steinhausen, E. Taylor, M. Thompson, S.V. Faraone, P. Asherson, The analysis of 51 genes in DSM-IV combined type attention deficit hyperactivity disorder: association signals in DRD4, DAT1 and 16 other genes, Mol Psychiatry 11 (2006) 934-953. Y.Y. Huang, S.P. Cate, C. Battistuzzi, M.A. Oquendo, D. Brent, J.J. Mann, An association between a functional polymorphism in the monoamine oxidase a gene promoter, impulsive traits and early abuse experiences, Neuropsychopharmacology 29 (2004) 1498-1505. T. Saito, H.M. Lachman, L. Diaz, T. Hallikainen, J. Kauhanen, J.T. Salonen, O.P. Ryynanen, M.K. Karvonen, E. Syvalahti, T. Pohjalainen, J. Hietala, J. Tiihonen, Analysis of monoamine oxidase A (MAOA) promoter polymorphism in Finnish male alcoholics, Psychiatry Res 109 (2002) 113-119. R. Lu, J. Lee, H. Ko, W. Lin, K. Chen, J.C. Shih, No association of the MAOA gene with alcoholism among Han Chinese males in Taiwan, Progress in NeuroPsychopharmacology & Biological Psychiatry (2002) 457–461. R Core Team, 2017. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available: https://www.R-project.org F. Faul, E. Erdfelder, A.-G. Lang, A. Buchner, G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences, Behavior Research Methods 39 (2007) 175-191. M. Asberg, L. Traskman, P. Thoren, 5-HIAA in the cerebrospinal fluid. A biochemical suicide predictor?, Archives of general psychiatry 33 (1976) 11931197. V. Ajdacic-Gross, Methods of suicide: international suicide patters derived from the WHO mortality database, Bulletin of the World Health Organization 86 (2008) 726-732. P. Räsänen, H. Hakko, J. Jokelainen, J. Tiihonen, Seasonal variation in specific methods of suicide: a national register study of 20 234 Finnish people, Journal of Affective Disorders 71 (2002) 51-59. G. Guo, X.-M. Ou, M. Roettger, J.C. Shih, The VNTR 2 repeat in MAOA and delinquent behavior in adolescence and young adulthood: associations and MAOA promoter activity, European journal of human genetics : EJHG 16 (2008) 626-634. G. Pungercic, A. Videtic, A. Pestotnik, I.Z. Pajnic, T. Zupanc, J. Balazic, M. Tomori, R. Komel, Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms: a study on Slovenian population of suicide victims, Psychiatr Genet 16 (2006) 187-191. A. Videtic, T.T. Peternelj, T. Zupanc, J. Balazic, R. Komel, Promoter and functional polymorphisms of HTR2C and suicide victims, Genes, brain, and behavior 8 (2009) 541-545. A. Videtic, T. Zupanc, P. Pregelj, J. Balazic, M. Tomori, R. Komel, Suicide, stress and serotonin receptor 1A promoter polymorphism -1019C>G in Slovenian suicide victims, Eur Arch Psychiatry Clin Neurosci 259 (2009) 234-238.
[22]
A
[23]
[24]
17
[28]
[29]
[30]
M
CC E
[34]
ED
[33]
PT
[32]
A
N
[31]
IP T
[27]
SC R
[26]
T. Zupanc, P. Pregelj, M. Tomori, R. Komel, A.V. Paska, TPH2 polymorphisms and alcohol-related suicide, Neurosci Lett 490 (2011) 78-81. S. Roškar, M. Zorko, A. Podlesek, Suicide in Slovenia Between 1997 and 2010, Crisis 36 (2015) 126-134. P. Courtet, F. Jollant, C. Buresi, D. Castelnau, D. Mouthon, A. Malafosse, The monoamine oxidase A gene may influence the means used in suicide attempts, Psychiatr Genet 15 (2005) 189-193. H.N. Buttenschon, T.J. Flint, L. Foldager, P. Qin, S. Christoffersen, N.F. Hansen, I.B. Kristensen, P.B. Mortensen, A.D. Borglum, O. Mors, An association study of suicide and candidate genes in the serotonergic system, J Affect Disord 148 (2013) 291-298. C.F. Hung, F.W. Lung, T.H. Hung, M.Y. Chong, C.K. Wu, J.K. Wen, P.Y. Lin, Monoamine oxidase A gene polymorphism and suicide: an association study and meta-analysis, J Affect Disord 136 (2012) 643-649. H. Ono, O. Shirakawa, N. Nishiguchi, A. Nishimura, H. Nushida, Y. Ueno, K. Maeda, No evidence of an association between a functional monoamine oxidase a gene polymorphism and completed suicides, Am J Med Genet 114 (2002) 340-342. M. Pompili, G. Gentile, C. Scassellati, C. Bonvicini, M. Innamorati, D. Erbuto, F. Montebovi, G. Ducci, A. Forte, E. De Pisa, S. Ferracuti, G. Serafini, V. De Luca, M. Amore, M. Simmaco, P. Girardi, Genetic association analysis of serotonin and signal transduction pathways in suicide attempters from an Italian sample of psychiatric patients, Neurosci Lett 656 (2017) 94-102. A.L. Rahikainen, S. Majaharju, J. Haukka, J.U. Palo, A. Sajantila, Serotonergic 5HTTLPR/rs25531 s-allele homozygosity associates with violent suicides in male citalopram users, Am J Med Genet B Neuropsychiatr Genet 174 (2017) 691-700. D.A. Stetler, C. Davis, K. Leavitt, I. Schriger, K. Benson, S. Bhakta, L.C. Wang, C. Oben, M. Watters, T. Haghnegahdar, M. Bortolato, Association of low-activity MAOA allelic variants with violent crime in incarcerated offenders, Journal of psychiatric research 58 (2014) 69-75. J.S. Fowler, N. Alia-Klein, A. Kriplani, J. Logan, B. Williams, W. Zhu, I.W. Craig, F. Telang, R. Goldstein, N.D. Volkow, P. Vaska, G.J. Wang, Evidence that brain MAO A activity does not correspond to MAO A genotype in healthy male subjects, Biol Psychiatry 62 (2007) 355-358. P.L. Hurd, K.L. Vaillancourt, N.L. Dinsdale, Aggression, digit ratio and variation in androgen receptor and monoamine oxidase a genes in men, Behavior genetics 41 (2011) 543-556. C. Aslund, N. Nordquist, E. Comasco, J. Leppert, L. Oreland, K.W. Nilsson, Maltreatment, MAOA, and delinquency: sex differences in gene-environment interaction in a large population-based cohort of adolescents, Behavior genetics 41 (2011) 262-272. A. Caspi, J. McClay, T.E. Moffitt, J. Mill, J. Martin, I.W. Craig, A. Taylor, R. Poulton, Role of genotype in the cycle of violence in maltreated children, Science 297 (2002) 851-854.
U
[25]
[35]
A
[36]
[37]
18
[40]
[41]
A
A
CC E
PT
[45]
M
[44]
ED
[43]
N
U
[42]
IP T
[39]
S.A. Nishioka, E.A. Perin, A.S. Sampaio, Q. Cordeiro, C. Cappi, R.S. Mastrorosa, I.A. Morais, V.N.D. Reis, M.C. do Rosario, A.G. Hounie, The role of the VNTR functional polymorphism of the promoter region of the MAOA gene on psychiatric disorders, Rev Psiq Clin-Brazil 38 (2011) 34-42. J. Samochowiec, K.P. Lesch, M. Rottmann, M. Smolka, Y.V. Syagailo, O. Okladnova, H. Rommelspacher, G. Winterer, L.G. Schmidt, T. Sander, Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism, Psychiatry Res 86 (1999) 67-72. L.G. Schmidt, T. Sander, S. Kuhn, M. Smolka, H. Rommelspacher, J. Samochowiec, K.P. Lesch, Different allele distribution of a regulatory MAOA gene promoter polymorphism in antisocial and anxious-depressive alcoholics, J Neural Transm (Vienna) 107 (2000) 681-689. R. Cervera-Juanes, L.J. Wilhem, B. Park, R. Lee, J. Locke, C. Helms, S. Gonzales, G. Wand, S.R. Jones, K.A. Grant, B. Ferguson, MAOA expression predicts vulnerability for alcohol use, Mol Psychiatry 21 (2016) 472-479. D. Checknita, G. Maussion, B. Labonte, S. Comai, R.E. Tremblay, F. Vitaro, N. Turecki, A. Bertazzo, G. Gobbi, G. Cote, G. Turecki, Monoamine oxidase A gene promoter methylation and transcriptional downregulation in an offender population with antisocial personality disorder, Br J Psychiatry 206 (2015) 216222. P.A. Melas, Y. Forsell, Hypomethylation of MAOA's first exon region in depression: a replication study, Psychiatry Res 226 (2015) 389-391. C. Ziegler, J. Richter, M. Mahr, A. Gajewska, M.A. Schiele, A. Gehrmann, B. Schmidt, K.P. Lesch, T. Lang, S. Helbig-Lang, P. Pauli, T. Kircher, A. Reif, W. Rief, A.N. Vossbeck-Elsebusch, V. Arolt, H.U. Wittchen, A.O. Hamm, J. Deckert, K. Domschke, MAOA gene hypomethylation in panic disorder-reversibility of an epigenetic risk pattern by psychotherapy, Translational psychiatry 6 (2016) e773. N. Antypa, A. Serretti, D. Rujescu, Serotonergic genes and suicide: A systematic review, European Neuropsychopharmacology 23 (2013) 1125-1142.
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[38]
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Table 1: Distribution of MAOA-uVNTR polymorphism allele frequencies in men Allele, n (%) 3R 4R Control group 45 (31.9) 96 (68.1) a Suicide victims 83 (41.5) 117 (58.5) Suicide victims (violent method) 72 (40.0) 108 (60.0) b Suicide victims (non-violent method) 11 (55.0) 9 (45.0)
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65 (71.4) 117 (58.5) 97 (58.1) 20 (60.6)
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26 (28.6) 83 (41.5) 70 (41.9) 13 (39.4)
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Control group without alcohol dependency Suicide victimsc Suicide victims without alcohol dependancyd Alcohol dependent suicide victims a p-value = 0.088, 95 % CI: 0.409 - 1.063, OR: 0.662 b p-value = 0.049, 95 % CI: 0.131 - 1.106, OR: 0.386 c p-value = 0.037, 95 % CI: 0.316 - 0.990, OR: 0.565 d p-value = 0.043, 95 % CI: 0.306 - 0.990, OR: 0.556
S0304-3940(18)30152-6 https://doi.org/10.1016/j.neulet.2018.02.063 NSL 33455
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
21-12-2017 6-2-2018 27-2-2018
Please cite this article as: Katarina Urˇsiˇc, Tomaˇz Zupanc, Alja Videtiˇc Paska, Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.02.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population
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Running head: MAOA polymorphism in suicide Katarina Uršiča, Tomaž Zupancb, Alja Videtič Paskaa1
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000
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Ljubljana, Slovenia. b
Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Korytkova ulica
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2, SI-1000 Ljubljana, Slovenia.
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Corresponding author: [email protected]
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Highlights
Suicide is a complex phenomenon influenced by a number of different risk factors. Polymorphism in MAOA promoter region affects the activity of transcription.
We genotyped MAOA polymorphism in a population with one of highest suicide
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rates in the world.
We observed a possible effect of MAOA-uVNTR 3R allele on suicide.
3R allele was associated with a choice of non-violent suicide method.
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Abstract
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Suicide is a well-defined public health problem and is a complex phenomenon influenced by a number of different risk factors, including genetic ones. Numerous studies have
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examined serotonin system genes. Monoamine oxidase A (MAO-A) is an outer mitochondrial membrane enzyme which is involved in the metabolic pathway of
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serotonin degradation. Upstream variable number of tandem repeats (uVNTR) in the promoter region of MAOA gene affects the activity of transcription. In the present study
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we genotyped MAOA-uVNTR polymorphism in 266 suicide victims and 191 control subjects of Slovenian population, which ranks among the European and world
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populations with the highest suicide rate. Genotyping was performed with polymerase chain reaction and agarose gel electrophoresis. Using a separate statistical analysis for female and male subjects we determined the differences in genotype distributions of MAOA-uVNTR polymorphism between the studied groups. Statistical analysis showed a trend towards 3R allele and suicide, and associated 3R allele with non-violent suicide
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method on stratified data (20 suicide victims). This is the first study associating highly suicidal Slovenian population with MAOA-uVNTR polymorphism.
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Keywords: suicidal behaviour, polymorphism, monoamine oxidase A, VNTR
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1. Introduction Suicidal behaviour is a complex public health problem of global proportions. Worldwide close to 800,000 people die as a result of suicide, translating to a life lost every 40 seconds. According to the suicide rate (the number of suicides per 100,000 inhabitants), Slovenia
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has long been ranked above European and global average rates [1]. Despite a decrease in number of suicides for the past decade, in 2016 Slovenian suicide rate was 18 (with
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European average being around 11) [2].
Genetic predisposition of an individual plays an important role in development of suicidal
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behaviour, as shown with adoptees studies and studies of identical (monozygotic) and
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fraternal (dizygotic) twins. The impact of genetics on the onset of suicidal thoughts and
[3, 4].
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1.1. Monoamine oxidase A
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behaviour is supposed to be 45 %, while the rest can be attributed to environmental factors
MAO is an external mitochondrial membrane enzyme that degrades biogenic amines,
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mainly catecholamines and serotonin by oxidative deamination. There are two known isoenzymes, MAO-A and MAO-B, located on chromosome X in the region p11.23-11.4.
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The MAOA gene consists of 16 exons, making the gene approximately 70 kb long. MAOA preferentially degrades serotonin and norepinephrine. In the central nervous system it
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is found in catecholaminergic cell groups [5, 6]. Many polymorphisms of the MAOA gene have been identified and studied, indicating a possible association with various behaviour patterns and mental disorders. Single nucleotide polymorphisms (SNPs) C1460T, which causes the formation of the EcoRV restriction site, and T941G, which causes the formation of the Fnu4HI restriction site,
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may affect the activity of MAO-A, but do not alter the amino acid sequence [7]. A family study of five men showed that the absence of MAO-A activity leads to aggression, impulsive behaviour and borderline intellectual functioning. Due to point mutation C936T in exon 8, which causes early stop codon, MAO-A is no longer active [8].
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Approximately 1.2 kb upstream from exon 1 lies a promoter region polymorphism, a variable number of tandem repeats (uVNTR). The individual tandem sequence consists
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of 30 bp, and can be repeated 3 times, 3.5 times, 4 times, or 5 times. The number of
tandem repeats affects the rate of gene transcription. Transcription rate is higher in 3.5repeat (3.5R) and 4-repeat (4R) alleles, and lower in 3-repeat (3R) or 5-repeat (5R) alleles
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[9]. Changes in serotonergic activity of the central nervous system are associated with
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impulsivity, decreased learning ability and increased aggression [10]. Later two more
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alleles were discovered; lowest transcription activity 2-repeat (2R) allele, showing a link
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with antisocial and violent behaviour and a higher possibility of arrest [11, 12], and a 6repeat allele [13].
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MAOA-uVNTR polymorphism has also been studied regarding alcohol dependence.
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Some studies found a weak association of reduced MAO-A activity and alcohol dependence [14], while others failed to draw the same conclusion [15].
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We hypothesized that MAOA-uVNTR polymorphism would be statistically related to suicidal behaviour. The main aim of our study was therefore comparing MAOA-uVNTR polymorphism allele frequencies between suicide victims and control group and see if
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they correlate with choice of method and alcohol abuse.
2. Methods
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2.1. Subjects 272 suicide victims (203 male, 69 female) and 193 control subjects (142 male, 51 female) were included in the presented study. Blood samples were taken during the autopsies, performed at the Institute of Forensic Medicine, Faculty of Medicine University of
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Ljubljana. Genomic DNA was isolated from venous blood, using standard methods and stored at +4 °C. Study samples were collected consecutively through the years 2002–2005
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and 2012–2013. Subjects of both groups were matched for age, sex and ethnicity. Mean age ± SD of the suicide victims group and of the control group were 46.3 years ± 16.5, and 53.0 years ± 17.6 for men and 55.3 years ± 18.6, and 56.3 years ± 18.6 for women.
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Male to female ratio in both groups were 3 to 1, and 2.8 to 1, respectively for suicide
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victims and controls. All subjects were of Slovenian origin.
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Subject selection criteria was sufficient body preservation for blood sample collection.
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Subjects in control group were also selected based on the cause of death which excluded
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possible suicide (predominate cause of death being sudden cardiac arrest). Alcohol abuse was confirmed by autopsy reports and histological liver sections, which showed changes
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of the liver that could be attributed exclusively to excessive alcohol consumption (fatty liver disease with >60% of hepatocytes showing macrovesicular hepatocyte fatty change,
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alcohol hepatitis and hepatic cirrhosis). Simultaneously, causal factors for histologic changes of liver were excluded (abnormalities of hepatic tissue due to obesity, diabetes
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and/or congestive heart failure). The study was approved by the Slovenian National Medical Ethics Committee.
2.2. Genotyping
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We genotyped MAOA-uVNTR polymorphism using polymerase chain reaction (PCR) amplification. Estimated PCR product length was minimum 294 to maximum 384 base pairs, from 2R allele to 5R allele. Products were amplified using a primer pair, based on the work of Sabol [9]. Sequences of primers are as following: sense 5'-
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ACAGCCTGACCGTGGAGAAG-3' (position -1369 to -1350) and antisense primer 5'GAACGGACGCTCCATTCGGA-3' (position -1039 to -1038). Reactions were carried
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out in volume of 15 µl, containing 70 ng of DNA, 7.5 µl of PCR Master Mix (50 U/ml
Taq DNA polymerase, 400 μM dNTPs, 3 mM MgCl2 and reaction buffers) (Promega, Madison, USA), 1.2 µl of each 2.5 µmol primer (Sigma-Aldrich, Hamburg, Germany) and
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water. PCR was performed using GeneAmp PCR system 9700 (Applied Biosystems,
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Foster City, ZDA) with conditions: 30 seconds at 95 °C, 30 seconds at 68 °C and 30
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seconds at 72 °C for 35 cycles. PCR products were separated and visualized using 2.8 %
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agarose gels with ethidium bromide and LAS-4000 (Fujifilm, Tokio, Japan).
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2.3. Sanger sequencing
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The sequence of amplified PCR products was confirmed by Sanger sequencing. For four observed MAOA-uVNTR polymorphism alleles one fragment was send to Macrogen
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(Amsterdam, Netherlands), where they were sequenced. 2.4. Statistics
Data was analysed using R version 3.3.3 (R Core Team, 2017) [16]. Statistical analysis
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was done separated by gender as MAOA-uVNTR polymorphism is located on the X chromosome. For age comparison between groups two-tailed t-test and one-way ANOVA were used. For allele and genotype frequency association between groups two-tailed Fisher’s exact test was used. For allele frequencies odds ratio (OR) with 95% confidence
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interval (CI) was calculated. All female groups where within Hardy-Weinberg equilibrium (supplementary table S1). Power analysis using G*Power 3.1 [17] was conducted to determine a priori sample size (parameters effect size = 0.3, significance level = 0.05, power = 0.80). Total desired sample size was 102. Actual total sample size
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was 341 for male subjects and 116 for female subjects, concluding that sample size is
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appropriate to detect significant differences in the two main studied groups.
3. Results
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Eight samples (four male and four female; three suicide victims and one control for both
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gender) were excluded from the statistical analysis after genotyping. Exclusion criteria
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was presence of a low frequency allele (2R, 3.5R, or 5R allele). Remaining 457 subjects
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were grouped as such: 266 suicide victims (200 male, 66 female) and 191 control subjects (141 male, 50 female). Suicide methods of the 266 suicide victims were distributed as
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follows: hanging, n = 130 (48.8%); shooting, n = 38 (14.3%); jumping from height, n = 31 (11.7%); poisoning, n = 24 (9%); drowning, n = 17 (6.4%); traffic accident, n = 12
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(4.5%); cutting wounds, n = 8 (3%); self-burning and electrocution, n = 4 (1.5%), and
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suffocation, n = 2 (0.8%). Based on previous studies, superficial cutting wounds and poisoning with solid, liquid and gaseous substances were regarded as non-violent suicide methods and remaining methods as violent (hanging, shooting, jumping from height,
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drowning, traffic accident, self-burning, electrocution, suffocation) [18-20] Nucleotide sequence of PCR was determined using Sanger sequencing which confirmed that the correct sequence was amplified (supplementary figure S1 - S4).
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Suicide victims and control group 3R and 4R-allele carriers had equivalent mean age (suicide victims p = 0.3047; control group p = 0.4958 for men and suicide victims F = 1.791 p) = 0.175; control group F = 0.11, p = 0.896 for women). Table 1 shows the distribution of MAOA-uVNTR polymorphism allele frequencies in
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male subjects. Statistically significant differences in allele frequencies were observed between control group and non-violent method suicide victims (p = 0.049, 95 % CI: 0.131
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- 1.106, OR: 0.386); control group without alcohol dependence and suicide victims (p = 0.037, 95 % CI: 0.316 - 0.990, OR: 0.565) and control group without alcohol dependence
and suicide victims without alcohol dependence (p = 0.043, 95 % CI: 0.306 - 0.990, OR:
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0.556).
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Statistical analysis of MAOA-uVNTR polymorphism genotype and allele frequencies
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shown no statistically significant differences in any of the investigated female groups
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(supplementary table S2 and S3). The ratio of homozygous to heterozygous genotypes
4. Discussion
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was 1.8 to 1 (32 to 18) for control group and 1.4 to 1 (38 to 28) for suicide victims.
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The aim of our study was to genotype MAOA-uVNTR polymorphism in Slovenian
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subjects. Our results suggest involvement of MAOA-uVNTR 3R allele in suicidal behaviour of men but not women. 3R allele was more often present in male victims of suicide (regardless of the chosen method of suicide), male suicide victims who have
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chosen a non-violent method; and in male suicide victims without alcohol dependency when compared to control group. Suicide is a complex phenomenon which is determined by many different risk factors, including genetic ones. Slovenia is ranked as one of the leading countries regarding suicide rate in the world, despite numerous protective factors such as warm climate, stable
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economy and a high percentage of Catholics [1]. As the genetic component for suicidal behaviour could be strong for Slovenian population, we questioned whether MAOAuVNTR polymorphism, a 30 bp long sequence that affects gene transcription activity [9, 21] could affect the rate of suicide. Namely, several studies of the serotonergic system on
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Slovenian population showed association with completed suicide so far [22-25]. In the present study we included 266 suicide victims (200 male, 66 female) and 191
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control subjects (141 male, 50 female). Genotype frequency distributions for females were within Hardy-Weinberg equilibrium (supplementary table S1). Among suicide victims there is a higher proportion of men to women (global ratio around 4 to 1,
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Slovenian 3 to 1) [1], which is similar to ratio of subjects of our study. In our study group
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the most commonly used methods was hanging, in both men (46%) and women (39%),
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and more suicide victims chose violent than non-violent methods. Both is in accordance
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with published epidemiological data for Slovenian population and many countries worldwide [19, 26].
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All statistical analysis was done separated by gender due to X-chromosomal location of
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the MAOA gene. First we compared MAOA-uVNTR polymorphism frequencies between suicide victims and control group. As has been shown by other studies MAOA alleles have
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been previously associated with aggression, impulsivity and alcohol abuse [11, 14] so we stratified our data further, which resulted in a smaller number of subjects in stratified groups. From the control group, we selected a control group without alcohol dependency.
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From the suicide victims we selected two groups depending on the use of violent or nonviolent method of suicide victims and two groups depending on alcohol dependency. In males, comparison of allele frequency distribution showed some statistically significant differences. When comparing suicide victims and control group we failed to
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detect statistical significance (chosen as p < 0.05), although the observed p-value of 0.088 is suggestive towards 3R allele being more frequent in suicide victims. Statistical significance was observed comparing three pairs of subgroups. First occurred when comparing control group and non-violent method suicide victims (p = 0.049). 3R
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allele was more often present in male suicide victims who have chosen a non-violent method than in control group. Our results are conclusive with a study conducted on
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healthy volunteers, associating 3R and 5R alleles with lower degree of aggression and
impulsivity in male subjects. Similarly, 3.5R and 4R alleles were found to be associated with a higher degree of aggression and impulsivity [10]. Another conclusive study found
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a link between the attempted suicide by a violent method and the increased frequency of
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3.5R and 4R allele compared to a non-violent method suicide attempt [27]. However, the
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link between the MAOA-u VNTR polymorphism and suicide is not fully established.
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Many studies failed to link MAOA-uVNTR polymorphism genotypes with suicidal behaviour [28-32]. Our result is contradictory to multiple studies of aggression on non-
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suicidal subjects. Incarcerated offenders show higher frequency of low expression alleles
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[11, 33]. We could conclude that low expression alleles probably don’t predispose aggression [34, 35] but when accompanied with childhood adversary associate with
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violent behaviour and impulsivity [36, 37]. Similarly, one study found no association between MAOA and suicide attempts but found an association between lower expression alleles, history of abuse and male subject with high impulsivity [13]. We would also like
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to point out that the average age of violent and non-violent suicides were not equivalent in our sample (mean age 47.32 years ±16.38 and 37.35 years ±15.74 respectively, p = 0.013). While older suicide victims tend to use more premeditated and violent methods we do not have data regarding average age of violent and non-violent suicide victims in
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Slovenia so we cannot completely exclude potential confounds in relation to agedependent differences in impulsivity. Second statistically significant difference occurred in the comparison of control group without alcohol dependency and suicide victims (p = 0.037). 3R allele occurred more
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frequently in male victims of suicide, regardless of the chosen method of suicide, as in control group without alcohol dependency. Third statistically significant difference was
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observed in comparison of control group without alcohol dependency and suicide victims
without alcohol dependency (p = 0.043). 3R allele occurred more frequently in male suicide victims without alcohol dependency than in control group without alcohol
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dependency.
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Majority of studies found no association between MAOA-uVNTR polymorphism and
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alcohol abuse [38]. Alcohol dependant subjects with strong antisocial tendencies showed
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a higher frequency of 3R allele when compared to controls and non-antisocial alcoholics [39, 40]. A study on alcohol dependent males showed a weak link between risk of alcohol
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dependency development and 3R and 5R allele [14]. Similarly male rhesus macaques
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animal model associated lower expression of MAOA in blood with higher alcohol consumption [41]. Our analysis failed to detect association between alcohol abuse and
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suicide. While 3R allele was more frequent in suicide victims, statistical significance was also observed in group of suicide victims without alcohol dependency. Although we did notice a higher frequency of 3R allele in suicide victims with alcohol dependency
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compared to control group without alcohol dependency, the results were not statistically significant.
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Comparison of genotype and allele frequency distribution showed no statistically significant differences between any of the female studied groups (supplementary tables S2 and S3). As a major limitation of our study we would like to emphasize small sample size after
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sample stratification, resulting in lower power of study. Our starting sample size is sufficient but as we stratify subjects in subgroups, the sample size gets noticeably smaller.
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With around 2 million inhabitants, Slovenia is a low populated country which makes
sample collection harder. Data collection would therefore take many more years. We also do not have access to data about potential early childhood adversities, socio-demographic
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data, clinical data and comorbid mental disorders besides alcohol dependency. MAOA
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gene contains many other polymorphisms that could influence suicidal behaviour
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independently, with other genes or through the gene – environment interaction. Studies
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also observed changes in MAOA methylation pattern in depression, panic disorder and antisocial personality disorder [42-44].
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Suicidal behaviour is s complex behaviour that is influenced by many factors. Thus, it is
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unlikely that the genetic component would be determined only by the influence of an individual gene. Impulsivity and aggression, which may lead to suicidal behaviour, are a
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result of interplay between many genes, environment, development and psychopathology [45]. Our study, conducted on a population with high suicide risk, contributes to existing knowledge of serotonin system and suicidal behaviour by showing a possible effect of
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MAOA-uVNTR polymorphism 3R allele.
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Author’s contributions TZ identified subjects to be included in the study, provided samples and assigned the diagnosis. AVP designed the study. KU preformed PCR and statistical analysis. KU drafted of the manuscript. AVP edited the manuscript. All authors read and approved the
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final manuscript.
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Acknowledgements
The authors would like to thank Sandra Ropret for help with sample preparation and Nejc
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Nadižar for help with statistical analysis.
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Funding
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This work was supported by the Slovenian Research Agency, program grant no. P1-0104
Conflict of interest
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(D), and postdoctoral project no. Z3-2180.
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The authors declare no conflict of interest.
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References
[3]
[4] [5]
[10]
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[11]
PT
[9]
ED
M
[8]
A
[7]
N
U
[6]
IP T
[2]
World Health Organization, 2014. Mental health: suicide prevention. http://who.int/mental_health/suicide-prevention/en/ (accessed 1 Aug 2017). SI-STAT Data Portal, NIJZ, Statistični urad Republike Slovenije. http://pxweb.stat.si/pxweb/Database/Dem_soc/05_prebivalstvo/32_Umrljivos t/15_05L30_umrli-vzrok/15_05L30_umrli-vzrok.asp (accessed 15 Sep 2017). D.J. Statham, A.C. Heath, P.A. Madden, K.K. Bucholz, L. Bierut, S.H. Dinwiddie, W.S. Slutske, M.P. Dunne, N.G. Martin, Suicidal behaviour: an epidemiological and genetic study, Psychol Med 28 (1998) 839-855. D.A. Brent, J.J. Mann, Family genetic studies, suicide, and suicidal behavior, Am J Med Genet C Semin Med Genet 133C (2005) 13-24. Z.Y. Chen, G.S. Hotamisligil, J.K. Huang, L. Wen, D. Ezzeddine, N. AydinMuderrisoglu, J.F. Powell, R.H. Huang, X.O. Breakefield, I. Craig, et al., Structure of the human gene for monoamine oxidase type A, Nucleic Acids Res 19 (1991) 4537-4541. Q.S. Zhu, J. Grimsby, K. Chen, J.C. Shih, Promoter organization and activity of human monoamine oxidase (MAO) A and B genes, J Neurosci 12 (1992) 44374446. G.S. Hotamisligil, X.O. Breakefield, Human monoamine oxidase A gene determines levels of enzyme activity, Am J Hum Genet 49 (1991) 383-392. H.G. Brunner, M.R. Nelen, P. van Zandvoort, N.G. Abeling, A.H. van Gennip, E.C. Wolters, M.A. Kuiper, H.H. Ropers, B.A. van Oost, X-linked borderline mental retardation with prominent behavioral disturbance: phenotype, genetic localization, and evidence for disturbed monoamine metabolism, Am J Hum Genet 52 (1993) 1032-1039. S.Z. Sabol, S. Hu, D. Hamer, A functional polymorphism in the monoamine oxidase A gene promoter, Hum Genet 103 (1998) 273-279. S.B. Manuck, J.D. Flory, R.E. Ferrel, J.J. Mann, M.F. Muldoon, A regulatory polymorphism of the monoamine oxidase-A gene may be associated with variability in aggression, impulsivity, and central nervous system serotonergic responsivity, Psychiatry Research (2000) 9-23. K.M. Beaver, J.P. Wright, B.B. Boutwell, J.C. Barnes, M. DeLisi, M.G. Vaughn, Exploring the association between the 2-repeat allele of the MAOA gene promoter polymorphism and psychopathic personality traits, arrests, incarceration, and lifetime antisocial behavior, Personality and Individual Differences 54 (2013) 164-168. K. Brookes, X. Xu, W. Chen, K. Zhou, B. Neale, N. Lowe, R. Anney, B. Franke, M. Gill, R. Ebstein, J. Buitelaar, P. Sham, D. Campbell, J. Knight, P. Andreou, M. Altink, R. Arnold, F. Boer, C. Buschgens, L. Butler, H. Christiansen, L. Feldman, K. Fleischman, E. Fliers, R. Howe-Forbes, A. Goldfarb, A. Heise, I. Gabriels, I. KornLubetzki, L. Johansson, R. Marco, S. Medad, R. Minderaa, F. Mulas, U. Muller, A. Mulligan, K. Rabin, N. Rommelse, V. Sethna, J. Sorohan, H. Uebel, L. Psychogiou, A. Weeks, R. Barrett, I. Craig, T. Banaschewski, E. Sonuga-Barke, J. Eisenberg, J. Kuntsi, I. Manor, P. McGuffin, A. Miranda, R.D. Oades, R. Plomin, H. Roeyers, A.
SC R
[1]
A
[12]
16
[18]
[19]
[20]
IP T
SC R
U
CC E
[21]
N
[17]
A
[16]
M
[15]
ED
[14]
PT
[13]
Rothenberger, J. Sergeant, H.C. Steinhausen, E. Taylor, M. Thompson, S.V. Faraone, P. Asherson, The analysis of 51 genes in DSM-IV combined type attention deficit hyperactivity disorder: association signals in DRD4, DAT1 and 16 other genes, Mol Psychiatry 11 (2006) 934-953. Y.Y. Huang, S.P. Cate, C. Battistuzzi, M.A. Oquendo, D. Brent, J.J. Mann, An association between a functional polymorphism in the monoamine oxidase a gene promoter, impulsive traits and early abuse experiences, Neuropsychopharmacology 29 (2004) 1498-1505. T. Saito, H.M. Lachman, L. Diaz, T. Hallikainen, J. Kauhanen, J.T. Salonen, O.P. Ryynanen, M.K. Karvonen, E. Syvalahti, T. Pohjalainen, J. Hietala, J. Tiihonen, Analysis of monoamine oxidase A (MAOA) promoter polymorphism in Finnish male alcoholics, Psychiatry Res 109 (2002) 113-119. R. Lu, J. Lee, H. Ko, W. Lin, K. Chen, J.C. Shih, No association of the MAOA gene with alcoholism among Han Chinese males in Taiwan, Progress in NeuroPsychopharmacology & Biological Psychiatry (2002) 457–461. R Core Team, 2017. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available: https://www.R-project.org F. Faul, E. Erdfelder, A.-G. Lang, A. Buchner, G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences, Behavior Research Methods 39 (2007) 175-191. M. Asberg, L. Traskman, P. Thoren, 5-HIAA in the cerebrospinal fluid. A biochemical suicide predictor?, Archives of general psychiatry 33 (1976) 11931197. V. Ajdacic-Gross, Methods of suicide: international suicide patters derived from the WHO mortality database, Bulletin of the World Health Organization 86 (2008) 726-732. P. Räsänen, H. Hakko, J. Jokelainen, J. Tiihonen, Seasonal variation in specific methods of suicide: a national register study of 20 234 Finnish people, Journal of Affective Disorders 71 (2002) 51-59. G. Guo, X.-M. Ou, M. Roettger, J.C. Shih, The VNTR 2 repeat in MAOA and delinquent behavior in adolescence and young adulthood: associations and MAOA promoter activity, European journal of human genetics : EJHG 16 (2008) 626-634. G. Pungercic, A. Videtic, A. Pestotnik, I.Z. Pajnic, T. Zupanc, J. Balazic, M. Tomori, R. Komel, Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms: a study on Slovenian population of suicide victims, Psychiatr Genet 16 (2006) 187-191. A. Videtic, T.T. Peternelj, T. Zupanc, J. Balazic, R. Komel, Promoter and functional polymorphisms of HTR2C and suicide victims, Genes, brain, and behavior 8 (2009) 541-545. A. Videtic, T. Zupanc, P. Pregelj, J. Balazic, M. Tomori, R. Komel, Suicide, stress and serotonin receptor 1A promoter polymorphism -1019C>G in Slovenian suicide victims, Eur Arch Psychiatry Clin Neurosci 259 (2009) 234-238.
[22]
A
[23]
[24]
17
[28]
[29]
[30]
M
CC E
[34]
ED
[33]
PT
[32]
A
N
[31]
IP T
[27]
SC R
[26]
T. Zupanc, P. Pregelj, M. Tomori, R. Komel, A.V. Paska, TPH2 polymorphisms and alcohol-related suicide, Neurosci Lett 490 (2011) 78-81. S. Roškar, M. Zorko, A. Podlesek, Suicide in Slovenia Between 1997 and 2010, Crisis 36 (2015) 126-134. P. Courtet, F. Jollant, C. Buresi, D. Castelnau, D. Mouthon, A. Malafosse, The monoamine oxidase A gene may influence the means used in suicide attempts, Psychiatr Genet 15 (2005) 189-193. H.N. Buttenschon, T.J. Flint, L. Foldager, P. Qin, S. Christoffersen, N.F. Hansen, I.B. Kristensen, P.B. Mortensen, A.D. Borglum, O. Mors, An association study of suicide and candidate genes in the serotonergic system, J Affect Disord 148 (2013) 291-298. C.F. Hung, F.W. Lung, T.H. Hung, M.Y. Chong, C.K. Wu, J.K. Wen, P.Y. Lin, Monoamine oxidase A gene polymorphism and suicide: an association study and meta-analysis, J Affect Disord 136 (2012) 643-649. H. Ono, O. Shirakawa, N. Nishiguchi, A. Nishimura, H. Nushida, Y. Ueno, K. Maeda, No evidence of an association between a functional monoamine oxidase a gene polymorphism and completed suicides, Am J Med Genet 114 (2002) 340-342. M. Pompili, G. Gentile, C. Scassellati, C. Bonvicini, M. Innamorati, D. Erbuto, F. Montebovi, G. Ducci, A. Forte, E. De Pisa, S. Ferracuti, G. Serafini, V. De Luca, M. Amore, M. Simmaco, P. Girardi, Genetic association analysis of serotonin and signal transduction pathways in suicide attempters from an Italian sample of psychiatric patients, Neurosci Lett 656 (2017) 94-102. A.L. Rahikainen, S. Majaharju, J. Haukka, J.U. Palo, A. Sajantila, Serotonergic 5HTTLPR/rs25531 s-allele homozygosity associates with violent suicides in male citalopram users, Am J Med Genet B Neuropsychiatr Genet 174 (2017) 691-700. D.A. Stetler, C. Davis, K. Leavitt, I. Schriger, K. Benson, S. Bhakta, L.C. Wang, C. Oben, M. Watters, T. Haghnegahdar, M. Bortolato, Association of low-activity MAOA allelic variants with violent crime in incarcerated offenders, Journal of psychiatric research 58 (2014) 69-75. J.S. Fowler, N. Alia-Klein, A. Kriplani, J. Logan, B. Williams, W. Zhu, I.W. Craig, F. Telang, R. Goldstein, N.D. Volkow, P. Vaska, G.J. Wang, Evidence that brain MAO A activity does not correspond to MAO A genotype in healthy male subjects, Biol Psychiatry 62 (2007) 355-358. P.L. Hurd, K.L. Vaillancourt, N.L. Dinsdale, Aggression, digit ratio and variation in androgen receptor and monoamine oxidase a genes in men, Behavior genetics 41 (2011) 543-556. C. Aslund, N. Nordquist, E. Comasco, J. Leppert, L. Oreland, K.W. Nilsson, Maltreatment, MAOA, and delinquency: sex differences in gene-environment interaction in a large population-based cohort of adolescents, Behavior genetics 41 (2011) 262-272. A. Caspi, J. McClay, T.E. Moffitt, J. Mill, J. Martin, I.W. Craig, A. Taylor, R. Poulton, Role of genotype in the cycle of violence in maltreated children, Science 297 (2002) 851-854.
U
[25]
[35]
A
[36]
[37]
18
[40]
[41]
A
A
CC E
PT
[45]
M
[44]
ED
[43]
N
U
[42]
IP T
[39]
S.A. Nishioka, E.A. Perin, A.S. Sampaio, Q. Cordeiro, C. Cappi, R.S. Mastrorosa, I.A. Morais, V.N.D. Reis, M.C. do Rosario, A.G. Hounie, The role of the VNTR functional polymorphism of the promoter region of the MAOA gene on psychiatric disorders, Rev Psiq Clin-Brazil 38 (2011) 34-42. J. Samochowiec, K.P. Lesch, M. Rottmann, M. Smolka, Y.V. Syagailo, O. Okladnova, H. Rommelspacher, G. Winterer, L.G. Schmidt, T. Sander, Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism, Psychiatry Res 86 (1999) 67-72. L.G. Schmidt, T. Sander, S. Kuhn, M. Smolka, H. Rommelspacher, J. Samochowiec, K.P. Lesch, Different allele distribution of a regulatory MAOA gene promoter polymorphism in antisocial and anxious-depressive alcoholics, J Neural Transm (Vienna) 107 (2000) 681-689. R. Cervera-Juanes, L.J. Wilhem, B. Park, R. Lee, J. Locke, C. Helms, S. Gonzales, G. Wand, S.R. Jones, K.A. Grant, B. Ferguson, MAOA expression predicts vulnerability for alcohol use, Mol Psychiatry 21 (2016) 472-479. D. Checknita, G. Maussion, B. Labonte, S. Comai, R.E. Tremblay, F. Vitaro, N. Turecki, A. Bertazzo, G. Gobbi, G. Cote, G. Turecki, Monoamine oxidase A gene promoter methylation and transcriptional downregulation in an offender population with antisocial personality disorder, Br J Psychiatry 206 (2015) 216222. P.A. Melas, Y. Forsell, Hypomethylation of MAOA's first exon region in depression: a replication study, Psychiatry Res 226 (2015) 389-391. C. Ziegler, J. Richter, M. Mahr, A. Gajewska, M.A. Schiele, A. Gehrmann, B. Schmidt, K.P. Lesch, T. Lang, S. Helbig-Lang, P. Pauli, T. Kircher, A. Reif, W. Rief, A.N. Vossbeck-Elsebusch, V. Arolt, H.U. Wittchen, A.O. Hamm, J. Deckert, K. Domschke, MAOA gene hypomethylation in panic disorder-reversibility of an epigenetic risk pattern by psychotherapy, Translational psychiatry 6 (2016) e773. N. Antypa, A. Serretti, D. Rujescu, Serotonergic genes and suicide: A systematic review, European Neuropsychopharmacology 23 (2013) 1125-1142.
SC R
[38]
19
Table 1: Distribution of MAOA-uVNTR polymorphism allele frequencies in men Allele, n (%) 3R 4R Control group 45 (31.9) 96 (68.1) a Suicide victims 83 (41.5) 117 (58.5) Suicide victims (violent method) 72 (40.0) 108 (60.0) b Suicide victims (non-violent method) 11 (55.0) 9 (45.0)
N A M ED PT CC E A
IP T
65 (71.4) 117 (58.5) 97 (58.1) 20 (60.6)
SC R
26 (28.6) 83 (41.5) 70 (41.9) 13 (39.4)
U
Control group without alcohol dependency Suicide victimsc Suicide victims without alcohol dependancyd Alcohol dependent suicide victims a p-value = 0.088, 95 % CI: 0.409 - 1.063, OR: 0.662 b p-value = 0.049, 95 % CI: 0.131 - 1.106, OR: 0.386 c p-value = 0.037, 95 % CI: 0.316 - 0.990, OR: 0.565 d p-value = 0.043, 95 % CI: 0.306 - 0.990, OR: 0.556