RESEARCH ARTICLE
Polymorphism in the Serotonin Receptor 2a (HTR2A) Gene as Possible Predisposal Factor for Aggressive Traits Zsofia Banlaki1, Zsuzsanna Elek1, Tibor Nanasi1, Anna Szekely2, Zsofia Nemoda1, Maria Sasvari-Szekely1, Zsolt Ronai1* 1 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary, 2 Institute of Psychology, Eotvos Lorand University, Budapest, Hungary *
[email protected]
Abstract
OPEN ACCESS Citation: Banlaki Z, Elek Z, Nanasi T, Szekely A, Nemoda Z, Sasvari-Szekely M, et al. (2015) Polymorphism in the Serotonin Receptor 2a (HTR2A) Gene as Possible Predisposal Factor for Aggressive Traits. PLoS ONE 10(2): e0117792. doi:10.1371/ journal.pone.0117792 Academic Editor: Allan Siegel, University of Medicine & Dentistry of NJ—New Jersey Medical School, UNITED STATES Received: July 22, 2014 Accepted: December 31, 2014 Published: February 6, 2015 Copyright: © 2015 Banlaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Aggressive manifestations and their consequences are a major issue of mankind, highlighting the need for understanding the contributory factors. Still, aggression-related genetic analyses have so far mainly been conducted on small population subsets such as individuals suffering from a certain psychiatric disorder or a narrow-range age cohort, but no data on the general population is yet available. In the present study, our aim was to identify polymorphisms in genes affecting neurobiological processes that might explain some of the inter-individual variation between aggression levels in the non-clinical Caucasian adult population. 55 single nucleotide polymorphisms (SNP) were simultaneously determined in 887 subjects who also filled out the self-report Buss-Perry Aggression Questionnaire (BPAQ). Single marker association analyses between genotypes and aggression scores indicated a significant role of rs7322347 located in the HTR2A gene encoding serotonin receptor 2a following Bonferroni correction for multiple testing (p = 0.0007) both for males and females. Taking the four BPAQ subscales individually, scores for Hostility, Anger and Physical Aggression showed significant association with rs7322347 T allele in themselves, while no association was found with Verbal Aggression. Of the subscales, relationship with rs7322347 was strongest in the case of Hostility, where statistical significance virtually equaled that observed with the whole BPAQ. In conclusion, this is the first study to our knowledge analyzing SNPs in a wide variety of genes in terms of aggression in a large sample-size non-clinical adult population, also describing a novel candidate polymorphism as predisposal to aggressive traits.
Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the NIH R03 TW007656 Fogarty International Research grant to Maria Sasvari-Szekely and the following Hungarian Scientific Research Funds: OTKA K100845 and K83766. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction Aggression, defined as any behavior intended to be destructive, lies at the root of numerous major ills of humanity ranging from verbal abuse through both interpersonal and self-directed violence to mass criminal acts. Consequences of aggression-driven acts pose an enormous burden on society and economics, rendering it important to understand the biological basis behind [1,2].
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Serotonin Receptor 2A Polymorphisms and Aggression
Competing Interests: The authors have declared that no competing interests exist.
Increased levels of aggression are characteristic to patients with a variety of neurodegenerative and psychiatric disorders as well as to alcoholics and drug addicts [3–7], but can also often be observed among the normal human population, even conferring certain privileges to the aggressor under certain circumstances e.g. by means of social dominance [8,9]. From the evolutionary point of view, some degree of aggression is indeed necessary for gaining adequate fitness (through an improved access of food supplies and other resources) and reproductive success; however, these benefits are compensated for by an increased risk of injury and social isolation. Hence, optimal levels of aggression are presumably shaped by a fine balance between effects of positive and negative selection pressure, implying a strong genetic background next to the role of environment [10,11]. This assumption is further underpinned by the fact that aggression proved to be heritable in several twin studies, with an estimated genetic contribution to the risk of aggressiveness of above 40% [12–17]. Experimental evidence suggest that aggressive manifestations and the accompanying emotions (anger, anxiety, fear) can be strongly related to highly conserved brain regions, chiefly to the amygdala and its linked neural circuits, but also to the anterior cingulated cortex and the prefrontal cortex [18,19]. In terms of biochemistry, it is principally the monoaminergic neurotransmitter systems (e.g. dopamine, noradrenaline and serotonin pathways) that are believed to play a major role in aggressive behavior, though possible effects of sexual hormones, the hypothalamic-pituitary-adrenal (HPA) axis and blood sugar levels have also been implicated [20,21]. Great efforts have been made to decipher the possible genetic background behind predisposition to aggression, describing novel polymorphisms in a variety of genes with a role in neuropsychiatry, and also identifying promising candidates for aggressive behavior and the related mental states (impulsivity, hostility). However, most of these association studies were carried out in small samples, raising the possibility of committing statistical errors (Pavlov 2012). Besides, the vast majority of aggression-related genetic investigations either were based on comparisons between healthy individuals and patients suffering from personality disorders etc., or concentrated on restricted samples not representative of the general population (e.g. [22–28]). These factors render data evaluation challenging, and often lead to controversial results. Our aim was to simultaneously examine the effect of a set of putatively functional single nucleotide polymorphisms (SNP) on aggressive tendencies of the general Hungarian adult population using a microarray system, with a principal focus on monoaminergic pathways and its close interactors. Selected SNPs are located in genes encoding monoaminergic neurotransmitter transporters and receptors, their associated proteins and other signal transduction molecules, enzymes involved in the biosynthesis or degradation of neurotransmitters, neurotrophic factors and regulators of circadian rhythm as well as of neuronal death, all with an implicated role in emotional responses and behavioral traits [20,29–32].
Materials and Methods Individuals involved Non-related individuals of Caucasian Hungarian origin without any known psychiatric disorder were recruited for this study on a voluntary basis at the Institute of Psychology, Eotvos Lorand University (Budapest). Buccal samples and self-filled out aggression questionnaires were obtained from 887 subjects (45.8% males and 54.2% females). The sample comprised of 495 psychology and law enforcement students studying in the Budapest area and 392 random volunteers recruited at academic institutions and events popularizing this survey. All participants belonged to the middle socioeconomic status. Mean age was 23.2 (±7.55) years within the range from 18 to 75 years. All participants gave written informed consent and the study was
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approved by the Scientific and Research Ethics Committee of the Medical Research Council (“ETT TUKEB”—Ministry of Health, Medical Research Council, Budapest, H-1051 Hungary).
Phenotypic measure The original 29-item version of the self-report Buss-Perry Aggression Questionnaire (BPAQ) [33] was used to assess aggressive tendencies. This instrument comprises four subscales: Verbal Aggression (5 items), Physical Aggression (9 items), Anger (7 items) and Hostility (8 items). Individual items are rated from one (‘extremely uncharacteristic of me’) to five (‘extremely characteristic of me’). Total score for aggression was calculated as the sum of ratings for all the items, with a possible range between 29 and 145. Hungarian version of the original English language questionnaire was obtained by the “forward-backward” translation method and was pilot tested prior to the present study [34].
Sample collection Buccal cells were collected by gently scraping the inner cheek with cotton-tipped collection swabs. Genomic DNA preparation was performed by a traditional, salting-out procedure [35]. Briefly, collection swabs were incubated overnight in 450 μl cell lysis buffer (0.2 g/l Proteinase K, 0.1 M NaCl, 0.5% SDS, 0.01 M Tris buffer pH = 8.0) at 56°C, followed by RNase treatment at room temperature. Proteins were precipitated with saturated NaCl (6 M) and removed by centrifugation. DNA was precipitated with isopropanol, purified with 70% ethanol and resuspended in 100 μl of Tris-EDTA pH = 8.0 (containing 0.5 M EDTA). DNA concentrations were measured by a fluorometry based intercalation assay (AccuBlue Broad Range dsDNA Quantification Kit, Biotium). Concentration of samples analyzed in this study ranged between 15 and 200 ng/μl. Isolated DNA samples were kept at −20°C until used.
Marker selection Common SNPs with a higher than 5% minor allele frequency (MAF) were selected from the dbSNP database of NCBI [36]. Priority was given to polymorphisms referred to in various association studies in connection with personality or mood disorders as well as aggression or impulsivity in psychiatric disorders, and to putative functional variants, either causing an amino acid change or with an implicated gene regulatory role.
Genotyping Genotyping was performed in 384-well plates on an Open Array real-time PCR platform (Applied Biosystems) based on allele-specific, fluorescent (TaqMan) probes and pre-designed, validated primers immobilized to a solid surface obtained from the manufacturer. Approximately 100 ng DNA per sample was used in each measurement. DNA amplification was carried out in the GeneAmp PCR System 9700 (Applied Biosystems) according to the manufacturer’s instructions, using the master mix, containing each dNTP and AmpliTaq Gold DNA-polymerase, provided by the manufacturer. Endpoint detection of signal intensities of allele specific fluorescent dyes was conducted by the OpenArray NT Imager, and genotypes were called by the TaqMan Genotyper v1.2 software. Call rate for individual SNPs is shown in Table 1 (mean: 77.9%).
Statistical analysis Statistical analyses were performed by the SPSS 22.0 (SPSS Inc.) software. Allele and genotype frequency distributions were determined by the χ2 test. Independent samples t-test was used to assess gender differences, and relationship with age was tested by Pearson correlation. Genetic
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Table 1. Genotype distribution of the studied SNPs. SNP
Gene
N
Genotype MM
Mm
HWE*
Call rate
86%
mm
1.
rs1048101
ADRA1A
763
218
28,6%
384
50,3%
161
21,1%
0.945
2.
rs3808585
ADRA1A
722
396
54,8%
277
38,4%
49
6,8%
0.998
81%
3.
rs2236554
ADRA1D
757
293
38,7%
346
45,7%
118
15,6%
0.641
85%
4.
rs553668
ADRA2A
692
519
75,0%
158
22,8%
15
2,2%
0.770
78%
5.
rs11030104
BDNF
702
393
56,0%
264
37,6%
45
6,4%
0.997
79%
6.
rs2049045
BDNF
690
419
60,7%
241
34,9%
30
4,3%
0.820
78%
7.
rs6265
BDNF
601
362
60,2%
212
35,3%
27
4,5%
0.847
68%
8.
rs7103411
BDNF
715
393
55,0%
276
38,6%
46
6,4%
0.966
81%
9.
rs7094179
CDNF
687
305
44,4%
302
44,0%
80
11,6%
0.924
77%
10.
rs7900873
CDNF
696
384
55,2%
273
39,2%
39
5,6%
0.573
78%
11.
rs1051730
CHRNA3
753
320
42,5%
345
45,8%
88
11,7%
0.943
85%
12.
rs16969968
CHRNA5
663
279
42,1%
307
46,3%
77
11,6%
0.866
75%
13.
rs4680
COMT
603
177
29,4%
295
48,9%
131
21,7%
0.927
68%
14.
rs135745
CSNK1E
718
187
26,0%
375
52,2%
156
21,7%
0.460
81%
15.
rs1997644
CSNK1E
688
176
25,6%
364
52,9%
148
21,5%
0.291
78%
16.
rs1611115
DBH
761
443
58,2%
283
37,2%
35
4,6%
0.482
86%
17.
rs6271
DBH
780
657
84,2%
116
14,9%
7
0,9%
0.759
88%
18.
rs4532
DRD1
761
286
37,6%
357
46,9%
118
15,5%
0.931
86%
19.
rs6277
DRD2
579
169
29,2%
284
49,1%
126
21,8%
0.948
65%
20.
rs1800497
DRD2
605
399
66,0%
192
31,7%
14
2,3%
0.261
68%
21.
rs1079597
DRD2
608
443
72,9%
158
26,0%
7
1,2%
0.226
69%
22.
rs1800498
DRD2
595
215
36,1%
280
47,1%
100
16,8%
0.862
67%
23.
rs2134655
DRD3
760
410
53,9%
295
38,8%
55
7,2%
0.981
86%
24.
rs3732790
DRD3
734
243
33,1%
365
49,7%
126
17,2%
0.857
83%
25.
rs6280
DRD3
749
354
47,3%
326
43,5%
69
9,2%
0.887
84%
26.
rs963468
DRD3
736
246
33,4%
364
49,5%
126
17,1%
0.909
83%
27.
rs11246226
DRD4
685
173
25,3%
347
50,7%
165
24,1%
0.941
77%
28.
rs3758653
DRD4
714
486
68,1%
208
29,1%
20
2,8%
0.923
80%
29.
rs916455
DRD4
702
644
91,7%
56
8,0%
2
0,3%
0.803
79%
30.
rs936460
DRD4
697
344
49,4%
284
40,7%
69
9,9%
0.655
79%
31.
rs3733829
EGLN2
683
263
38,5%
321
47,0%
99
14,5%
0.998
77%
32.
rs222843
GABARAP
683
307
44,9%
293
42,9%
83
12,2%
0.601
77%
33.
rs11111
GDNF
719
540
75,1%
160
22,3%
19
2,6%
0.241
81%
34.
rs1549250
GDNF
710
231
32,5%
353
49,7%
126
17,7%
0.907
80%
35.
rs1981844
GDNF
576
320
55,6%
223
38,7%
33
5,7%
0.771
65%
36.
rs2910702
GDNF
705
387
54,9%
269
38,2%
49
7,0%
0.971
79%
37.
rs2973041
GDNF
695
492
70,8%
182
26,2%
21
3,0%
0.710
78%
38.
rs2973050
GDNF
582
242
41,6%
275
47,3%
65
11,2%
0.608
66%
39.
rs3096140
GDNF
671
320
47,7%
287
42,8%
64
9,5%
1.000
76%
40.
rs3812047
GDNF
679
521
76,7%
144
21,2%
14
2,1%
0.559
77%
41.
rs6925
HTR1A
607
167
27,5%
289
47,6%
151
24,9%
0.510
68%
42.
rs1228814
HTR1B
599
432
72,1%
153
25,5%
14
2,3%
0.995
68%
43.
rs130058
HTR1B
595
330
55,5%
232
39,0%
33
5,5%
0.642
67%
44.
rs13212041
HTR1B
606
376
62,0%
209
34,5%
21
3,5%
0.467
68%
45.
rs11568817
HTR1B
600
187
31,2%
292
48,7%
121
20,2%
0.937
68% (Continued)
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Serotonin Receptor 2A Polymorphisms and Aggression
Table 1. (Continued) SNP
Gene
N
Genotype MM
HWE*
Mm
Call rate
mm
46.
rs6296
HTR1B
607
325
53,5%
233
38,4%
49
8,1%
0.730
68%
47.
rs6311
HTR2A
777
243
31,3%
391
50,3%
143
18,4%
0.809
88%
48.
rs6313
HTR2A
769
240
31,2%
385
50,1%
144
18,7%
0.893
87%
49.
rs6314
HTR2A
773
640
82,8%
130
16,8%
3
0,4%
0.409
87%
50.
rs7322347
HTR2A
765
242
31,6%
370
48,4%
153
20,0%
0.866
86%
51.
rs7984966
HTR2A
758
411
54,2%
293
38,7%
54
7,1%
0.984
85%
52.
rs3813929
HTR2C
744
555
74,6%
117
15,7%
72
9,7%
0.975
84%
53.
rs518147
HTR2C
717
379
52,9%
166
23,2%
172
24,0%
0.237
81%
54.
rs6318
HTR2C
769
570
74,1%
127
16,5%
72
9,4%
0.737
87%
55.
rs907094
PPP1R1B
705
409
58,0%
246
34,9%
50
7,1%
0.308
79%
M: major allele, m: minor allele *Hardy Weinberg Equilibrium. doi:10.1371/journal.pone.0117792.t001
associations were tested by one way analysis of covariance (ANCOVA) assuming a dominant model of inheritance with sex and age as covariates. Bonferroni correction for multiple testing was applied for the total number of SNPs in this study when assessing relationship between BPAQ scores and individual SNPs (the corrected level of significance was p = 0.05 / 55 = 0.0009). In all other cases, p < 0.05 values were regarded as significant. Effect of prior associations in males and females was analyzed by two-way ANCOVA with age as covariate. All tests were two-tailed. Lewontin’s D’ and r2 values of linkage disequilibrium were calculated using HaploView 4.2. [37]. Haplotypes were determined by the PHASE software [38,39].
Results Reliability of the markers analyzed Internal consistency of the self-report BPAQ was assessed by Chronbach’s alpha, which had a value of 0.895 for total scores ensuring reliability of the study. Coefficients for Verbal Aggression, Physical Aggression, Anger and Hostility were 0.640, 0.842, 0.831 and 0.792, respectively. Alleles of all the SNPs studied were in Hardy-Weinberg equilibrium (Table 1).
Potential confounders Gender differences on the BPAQ scale were evaluated by Independent samples t-test. Males presented significantly higher scores than females (68.52±17.14 compared to 64.49±15.09; p
