Badanie asocjacji pomiędzy polimorfizmem genów 5-HTT, MAOA i DAT a samobójstwem u mężczyzn z populacji polskiej.pdf

ARCH. MED. SĄD. KRYMINOL., 2010, LX, 112-117 PRACE ORYGINALNE

Katarzyna Linkowska 1 , Patrycja Daca 1 , Marzena Sykutera 2 , Ewa Pufal 2 ,

Elżbieta Bloch-Bogusławska 2 , Tomasz Grzybowski 1

Badanie asocjacji pomiędzy polimorfizmem genów 5-HTT,

MAOA i DAT a samobójstwem u mężczyzn z populacji polskiej

Search for association between suicide and 5-HTT, MAOA and DAT

polymorphism in Polish males

1 Z Zakładu Genetyki Molekularnej i Sądowej Katedry Medycyny Sądowej UMK w Toruniu, Collegium

Medicum im. Ludwika Rydygiera w Bydgoszczy

Kierownik: dr hab. n. med. Tomasz Grzybowski, prof. UMK

2 Z Zakładu Medycyny Sądowej Katedry Medycyny Sądowej UMK w Toruniu, Collegium Medicum

Kierownik Zakładu: prof. dr hab. n. med. Karol śliwka

Kierownik Katedry: dr hab. n. med. Tomasz Grzybowski, prof. UMK

Znalezienie markerów genetycznych umożliwiających

ocenę ryzyka popełnienia samobójstwa miałoby

istotne znaczenie w praktyce klinicznej. Celem prze-

prowadzonych badań było określenie czy istnieje

asocjacja pomiędzy polimorfizmami w genach 5-HTT,

MAOA i DAT a samobójstwem, a także określenie

czy współwystępowanie wariantów alleli tych genów

może predysponować do samobójstwa. Uzyskane

wyniki wykazały brak statystycznie istotnych różnic

w częstości alleli i genotypów w genach 5-HTT, MAOA

i DAT pomiędzy grupą kontrolną a badaną. Analiza

genotypów we wszystkich 4 loci wykazała różnice

w częstości pomiędzy grupą kontrolną a samobój-

cami dla genotypu (3;12-12;S-S;9-10). Genotyp ten

występował tylko w grupie kontrolnej z częstością

8% (p=0,03).

5-HTT, MAOA and DAT genes were investigated in

66 male suicide completers and 51 male control

subjects from the Polish population. There were no

significant differences in the allele and genotype

frequencies between the case and control group.

In the individual genotype tests, examination of the

distribution differences of each genotype showed that

genotype (3;12-12;S-S;9-10) differed between the

suicide victims and control subjects. This genotype

existed only in the control sample and appeared with

the frequency of 8% ( p =0.03).

Key words: suicide, 5-HTT, MAOA, DAT

BACKGROUND

A better understanding of genetic determinants of

suicidal behavior might be very useful in clinical

practice. The objectives of the present study were to

answer the question whether there is an association

between functional polymorphic forms of 5-HTT,

MAOA or DAT and suicidality, and to examine whether

the combination of functional alleles in 5-HTT, MAOA

and DAT genes would predict a predisposition to

suicidal behavior. Functional polymorphisms in

In the last decade, a growing number of mo-

lecular genetic studies have been carried out to

identify candidate genes that may be involved

in pathophysiological mechanisms of suicidal

behavior. Post-mortem studies revealed interest-

ing data on the serotoninergic , noradrenergic

and dopaminergic neurotransmitter systems of

suicide victims. However, most of the attention is

focused on serotoninergic abnormalities, which

are additionally related to a variety of psycho-

* Poszerzona wersja referatu przedstawionego podczas XV Zjazdu Naukowego PTMSiK, Gdańsk 16-18.09.2010 r.

Nr 2-3 113

pathological dimensions such as anxiety, de-

pressed mood, impulsivity and aggression [1].

The crucial role in the regulation of sero-

toninergic transmission by determining the

magnitude and duration of 5-HT synaptic signal

is played by the serotonin transporter (5-HTT)

[2]. The human serotonin transporter is encoded

by a single copy gene located on chromosome

17q11.1-q12. [3]. Two polymorphisms of the

5-HTT gene, which differently modulate tran-

scription, have been identified: a 44-bp insertion-

deletion in the promoter region (5-HTTLPR),

and a variable number of tandem repeats in the

second intron of the gene (VNTR) [4].

Monoamine oxidase-A (MAOA) is a mitochon-

drial enzyme, encoded by a gene located on

chromosome Xp11.23-Xp11.4, which catalyzes

oxidative deamination of biogenic amines such

as noradrenaline, dopamine and serotonin [5].

Sabol et al. identified a common polymorphism

of a variable number of tandem repeats (VNTR)

in the promoter region of the MAOA gene,

which was shown to be associated with MAOA

transcriptional activity. This polymorphism is

located 1.2 kb upstream of the MAOA coding

sequences and consists of a 30-bp repeated

sequence present in 3, 3.5, 4, or 5 copies [6].

The dopamine transporter (DAT) is a plasma

membrane transport protein, encoded by

a gene located on chromosome 5p15.3, which

mediates an uptake of dopamine into presy-

naptic neurons. The 3’ untranslated region of

the dopamine transporter gene contains a 40-

bp variable number of tandem repeat (VNTR),

with two common alleles of 9 and 10 repeat

elements [7]. A number of studies was devoted

to investigation of the functional role played by

DAT VNTR polymorphism, although the results

remain inconclusive.

Over the past few years, several groups have

investigated the possible association between

suicidal behavior and the above-mentioned

polymorphisms, but usually applied to only one

of them. The aim of our study was to answer

the question whether there is an association

between functional polymorphic forms of 5-HTT,

MAOA or DAT and suicidality, as well as to exam-

ine whether the combination of functional alleles

in 5-HTT, MAOA and DAT genes would predict

a predisposition to suicidal behavior.

who were autopsied at the Institute of Forensic

Medicine of Collegium Medicum in Bydgoszcz.

The methods of committing suicide included

hanging ( n =62), jumping from heights ( n =2),

use of firearms ( n =1) and jumping under a train

( n =1) and were classified as violent. Buccal

swabs were obtained from 51 randomly se-

lected unrelated male individuals (mean age

35.3±12.9 years) from the general population

in the Pomerania-Kujawy region of Poland. who

served as controls. We selected both suicide vic-

tims and controls of male gender because of the

gender-specific association with suicidality [3].

The study protocol was approved by the

Ethical Committee of Collegium Medicum in

Bydgoszcz. Human genomic DNA was extracted

from blood or saliva according to the standard

procedures. Quantification of DNA was per-

formed spectrophotometrically. The PCR pro-

cedures for the examined gene polymorphisms

were described elsewhere: serotonin trans-

porter and monoamine oxidase-A [8], dopamine

transporter [9]. The PCR products for MAOA

and DAT were separated by 2.5% agarose gel

electrophoresis followed by ethidium bromide

staining and visualized under UV light. Various

alleles were determined using Gene Ruler 50

bp DNA ladder (Fermentas). The PCR products

for 5-HTT labeled with different fluorescent dyes

(5-HTTLPR –labeled with FAM and 5-HTTVNTR -

labeled with HEX) were separated and detected

by capillary electrophoresis on ABI PRISM

3130xl (Applied Biosystems).

A simultaneous determination of antidepres-

sant drugs (amitriptyline, chlordiazepoxide, car-

bamazepine, chlorpromazine, citalopram, parox-

etine, clomipramine, doxepin, fluoxetine, levome-

promazine, maprotiline, paroxetine, perazine,

mianserine, promazine, sertraline, thioridazine)

in blood samples, hair and nails was performed

using high-performance liquid chromatography

with mass spectrometry (LC/MS).

The statistical significance of differences

between the case and control group distribu-

tion for alleles and genotypes was determined

using the chi-squared tests. The association

analysis was performed using logistic regression

analysis. The Fisher exact test was performed to

compare distributions of the obtained genotypes

between the case and control groups. The sta-

tistical analyses were performed using Statistica

software (version 8). The Arlequin program was

employed to determine departure from Hardy-

Weinberg equilibrium and linkage disequilibrium

between two loci. The significance level for all

MATERIAL AND METHODS

The case sample consisted of 66 male sui-

cide completers (mean age 42.9±17.9 years),

BADANIE ASOCJACJI POMIęDZY POLIMORFIZMEM A SAMOBóJSTWEM

114 Nr 2-3

Katarzyna Linkowska

statistical tests was 0.05. We applied Bonferroni

correction for multiple tests (the level of signifi-

cance was set to α =0.01).

cies in suicide victims were not significantly

different from those in the control group, for

either 5-HTTLPR ( χ 2=0.24, df =1, p =0.62),

or 5-HTTVNTR ( χ 2=1.66, df =2, p =0.44). We

detected no significant linkage disequilibrium

between the 5-HTTLPR and the 5-HTTVNTR

polymorphism in suicide victims ( χ 2=2.48,

p =0.29) and in control subjects ( χ 2=2.15,

p =0.34). There were no significant differences

between the controls and suicide victims in

allele frequencies of the MAOA gene polymor-

phism ( χ 2=3.72, df =2, p =0.16) and DAT gene

polymorphism ( χ 2=0.91, df =1, p =0.34 ). The

result of association analysis obtained by lo-

gistic regression analysis showed no statistical

significant association with suicidality.

RESULTS

The sample of 66 suicide victims and 51 con-

trol subjects was genotyped for 5-HTTLPR and

intron 2 polymorphism of 5HT transporter gene

and two polymorphisms of a variable number

tandem repeat: one in the promoter region of

the MAOA gene and the other in 3’ untrans-

lated region of the dopamine transporter gene

(DAT). Distribution of genotype frequencies

in all loci was in accord with Hardy-Weinberg

equilibrium in both groups. The allele frequen-

Table 1. Polymorphism of 5-HTT, MAOA and DAT gene in suicide victims and control population.

Tabela 1. Polimorfizm genów 5-HTT, MAOA i DAT w grupie badanej i w grupie kontrolnej.

Locus

Allel

Grupa badana

(n=66)

Grupa kontrolna

(n=51)

HTTLPR

78 (59,1)

54 (40,9)

57 (55,9)

45 (44,1)

0,62

HTTVNTR

2 (1,5)

51 (38,6)

70 (59,8)

4 (3,9)

35 (34,3)

63 (61,8)

0,44

MAOA

22 (33,3)

44 (66,7)

0 (0,0)

21 (41,2)

28 (54,9)

2 (3,9)

0,16

hDAT

30 (22,7)

102 (77,3)

18 (17,6)

84 (82,4)

0,34

We obtained 41 genotypes from both the sui-

cide victims and control subjects. We observed

sixteen genotypes (genotypes with frequencies

> 3% in the suicide group), which accounted

for 75% and 56% of all the observed genotypes

combinations in the suicide victims and the

control sample, respectively. In the individual

genotype tests, examination of the distribution

differences of each genotype showed that only

one genotype, existing only in the control sam-

ple with the frequency of 8% (tab. 2), differed

significantly the between suicide victims and

control subjects ( p =0.03), but the significance

was lost when applying Bonferroni correction.

All the suicide victims underwent a toxicologi-

cal screening of blood, hair and nails. Antide-

pressant drugs were found in 25 case subjects

(38%). Positive screening results included SSRI

( n =10) and other antidepressant drugs ( n =15).

A total of 22 individuals (33%) were under the

influence of antidepressant drugs at the moment

of death. In 15 of the cases, the traces of drugs

were also found in hair and nails, which means

that they were taking drugs for a prolonged pe-

riod. Three individuals took antidepressants in

the past but were not under their influence at the

time of death (one had promazine in nails, the

second had fluoxetine in nails and the third had

amitryptiline in both hair and nails). Alcohol was

detected in blood of 27 suicide victims (41%); 22

individuals had blood alcohol level above 1‰.

Table 2. The genotype observed in the control sample

only.

Tabela 2. Genotyp występujący tylko w grupie kon-

trolnej.

Genotyp

DISCUSSION

In the last decade, several studies concerning

the genetics of suicidal behaviour were carried

out. Since there is convincing evidence that the

MAOA 5-HTTVNTR 5-HTTLPR DAT

12,12

S,S

9,10 0,03

Nr 2-3 115

serotoninergic system is involved in suscepti-

bility to suicide, it is reasonable that molecular

genetic studies focused primarily on the genes

of the serotonin pathway. Since the serotonin

transporter acts as a key regulator of the 5-HT

transmission, polymorphisms of the 5-HTT gene

became an attractive target for association stud-

ies in suicide and were investigated extensively;

in particular polymorphism in the promoter re-

gion of the gene. However, the results of these

studies were inconsistent: some reported an

association between the S-allele and suicidality

[10, 11], while other found that the L-allele was

more frequent in suicide victims [12]. In contrast,

Fitch et al. (2001) and Mann et al. (2000) could

not find any association between the 5-HTTLPR

genotype and suicidality [13,14]. In addition,

no association was found between suicide and

VNTR polymorphism in the intron 2 of the 5-HTT

gene. However, a combined analysis of the

5-HTTLPR and 5-HTTVNTR showed a tendency

toward an increase of the 5-HTTLPR allele L and

5-HTTVNTR allele 10 in the suicide victims [15].

The main cause of these conflicting observa-

tions can be either a high genetic heterogenity of

European populations, inadequate sample sizes

or differences in sample compositions. Different

diagnostic groups, as well as different diagnostic

distributions between samples, could lead to

different results if the 5-HTT polymorphisms are

not associated with suicidal behavior but rather

with one of the psychiatric diagnoses which are

prevalent among suicides. In addition, studies

on different ethnic groups could lead to differ-

ent outcomes because allele frequencies of

both promoter and intron polymorphisms vary

among the subjects of different ethnicities and

races [16]. The role of DAT VNTR polymorphism

in the etiology of neuropsychiatric disorders

also seems unresolved. A number of studies

investigated the possible association between

this polymorphism and bipolar disorder, schizo-

phrenia, alcoholism, also with mixed result [17,

18]. No association has been found between

the MAOA gene VNTR polymorphism in the

promoter region and vulnerability to a suicidal

act. However, the MAOA gene variants may influ-

ence the methods used in suicide attempts [19].

The presence of the above-mentioned differ-

ences shows that the question how the genetic

factors contribute to suicide is still open. We

assume that it is unlikely that the few genes

alone are conferring risk of suicidal behavior.

Thus, in our study, the 5-HTT, MAOA and DAT

data were analyzed together to find out whether

the simultaneous coexistence of functional poly-

morphic forms of the 5-HTT gene, MAOA gene

and DAT gene may predispose to suicide. The

study was conducted on a group of 117 males

from Poland. The case sample consisted of 66

male subjects who committed suicide classified

as violent. Because of the gender-specific as-

sociation with suicidality we studied only males

[3]. Despite the previous reports [10, 11], we

were unable to show any association between

the 5-HTT gene polymorphism and suicidality.

In our study, there are no significant differences

in allele and genotype frequencies between the

case and control group. Therefore, we may ar-

gue that none of these genes alone predisposes

to suicide. We checked the influence of all of the

functional polymorphic forms on the occurrence

of suicide by the logistic regression analysis. The

result of this analysis did not show any associa-

tion between the investigated polymorphic forms

and suicidality. We determined all of the geno-

types in 4 loci, but the individual genotype tests

for all 4 loci did not show any associations with

suicidality. However, one genotype (3;12-12;S-

S;9-10) differed significantly between the suicide

victims and control subjects. This genotype ex-

isted only in the control sample and appeared

with frequency of 8%. Probably, this genotype

includes variants of genes that may provide

some protection against suicidal behavior. The

“protective” genotype contains 9 and 10 allele

in the DAT gene and low-activity allele in the

MAOA gene. However, the high-activity alleles

in the MAOA gene have been reported to show

a significantly higher frequency in men who had

attempted suicide by violent means [19]. The

“protective” genotype also includes variants

SS in 5-HTTLPR and 1212 in 5-HTTVNTR. In

agreement with our results, an increase of the

5-HTTLPR allele L and the 5-HTTVNTR allele 10

was observed in the suicide victim group [15].

However, it is worth noting that after Bonferroni

correction for multiple testing, the observed

difference in frequency is no longer significant.

The results of our study should be considered

with caution, for two reasons. First, the study

sample was relatively small, and a larger sample

would be more appropriate to detect genetic ef-

fect in association study of suicide. Second, in

our study we had no detailed information about

psychiatric diagnoses either in case subjects or

control subjects. After toxicological screening of

case subjects we only found that 38% of them

had taken antidepressants in the past and 41%

had alcohol in blood. This allows for suggest-

BADANIE ASOCJACJI POMIęDZY POLIMORFIZMEM A SAMOBóJSTWEM

116 Nr 2-3

Katarzyna Linkowska

ing that a portion of the subjects might suffer

from psychiatric disorders leading ultimately to

suicide.

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CONCLUSIONS

In conclusion, we did not find any association

between the polymorphisms in the 5-HTT, MAOA

and DAT genes and completed suicide in Polish

population. Probably, the risk of suicide is a re-

sult of an action of a greater number of genes,

each contributing a small part to the overall risk,

while numerous non-genetic factors might also

influence this genetic base of the susceptibility

to suicide. A better understanding of the genetic

determination of suicide needs further investi-

gation into the interaction of genes involved in

synthesis, release, uptake and receptor function

for a variety of neurotransmitters.

ACKNOWLEDGEMENTS

This study was financed by the UMK 42/2008 grant.

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