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Introduction

Aggressive Behavior can cause physical or emotional harm to others. It may range from verbal abuse to physical abuse. It can also involve harming personal property [1]. Aggressive behavior violates social boundaries. It can lead to breakdowns in your relationships. It can be obvious or secretive. Occasional aggressive outbursts are common and even normal in the right circumstances [2].

From psychological and sociological standpoints, aggression is regarded as intentional behavior aimed at inflicting pain and manifested by hostility and attacking behaviors. In contrast, biologists define aggression as a behavior associated with attack or escalation toward attack, omitting any stipulation about intentions and goals [3]. Like many behaviors, aggression can be examined in terms of its ability to help an animal itself survive and reproduce or to risk survival and reproduction. This cost-benefit analysis can be looked at in terms of evolution. However, there are profound differences in the extent of acceptance of a biological or evolutionary basis for human aggression [4]. Thus we can divide aggression behavior in humans into genetic factors and non-genetic factors.

From psychological and sociological standpoints, aggression is regarded as intentional behavior aimed at inflicting pain and manifested by hostility and attacking behaviors. In contrast, biologists define aggression as a behavior associated with attack or escalation toward attack, omitting any stipulation about intentions and goals [3]. Like many behaviors, aggression can be examined in terms of its ability to help an animal itself survive and reproduce or to risk survival and reproduction. This cost-benefit analysis can be looked at in terms of evolution. However, there are profound differences in the extent of acceptance of a biological or evolutionary basis for human aggression [4]. Thus we can divide aggression behavior in humans into genetic factors and non-genetic factors.

Aggressive behavior is an ancient and conserved trait, habitual for most animals to eat, protect themselves, compete for mating and defend their territories. Genetic factors have been shown to play an important role in the development of aggression both in animals and humans [5]. For example, five polymorphisms of a gene known to be linked with impulsivity, violence, and other severe psychiatric illnesses were specified in the perpetrator of murder DNA [6].

Importance of studying aggressive behavior: Violence has probably always been part of the human experience. Its impact can be seen, in various forms, in all aspects of the world. Each year, more than a million people lose their lives, and many more suffer non-fatal injuries due to self-inflicted, interpersonal, or collective violence. Overall, violence is among the leading causes of death worldwide for people aged 15–44 years [7].

MAOA gene

This gene is one of two genes group (MAOA, MAOB). Codes for mitochondrial enzymes (Monoamine Oxidase A). Catalyze the oxidative deamination of amines neurotransmitters like dopamine, norepinephrine, and serotonin [8]. MAOA gene location is on the short arm (p) of the X-chromosome at position 11.3. (Figure 1). The absence of evidence on inactive X chromosomal has made determining the role of a gene in females difficult. The promoter region of the MAOA gene has a repeat polymorphism unit (uVNTR) length of 30 bp, this subject of the most studied genetic variant in aggressive behaviors [9].

The importance of MAOA in the control of impulsive aggressiveness is based on a population of Dutchmen from the same family who had a point mutation in the MAOA gene, rendering the enzyme inactive [10]. Previous research, indicates that the MAOA-uVNTR genotype is related to measures of reactive impulsive experimental aggression in healthy men. In addition, the link between the MAOA-uVNTR genotype and aggressive responses grows in a linear relationship with the degree of provocation. This suggests that MAOA-uVNTR alleles with low functional alleles are linked to higher aggressive responsiveness to provocation rather than increased violent conduct [11]. Environmental variables factors appear to interact with a functional polymorphism for (MAOA) in the promoter region to determine various kinds of violent behavior. However, it is unclear how MAOA influences other variables such as alcohol intake on antisocial behavior [12]. Maltreatment of children was found to be a significant predictor of teenage delinquency. Furthermore, when childhood maltreatment was taken into account, the MAOA genotype exhibited a substantial main impact [13]. Males with low MAOA activity alleles had a higher chance of joining a gang and utilizing a weapon in a fight. Furthermore, compared to male gang members who do not use weapons in a fight, those who utilized weapons in a fight were more likely to have a low MAOA activity allele [14]. According to a study of adolescent health, African-American men with the 2-repeat gene are substantially more likely than other genotypes to be involved in shooting and stabbing activities and report having many shooting and stabbing victims [15]. Brunner syndrome is caused by loss-of-function mutations in the X-linked MAOA gene, which is characterized by impulsivity, maladaptive externalizing behavior, and modest intellectual handicap [16]. In addition, biogenic amine aberration occurs when MAOA activity is impaired in people with Brunner syndrome [17].

COMT gene

The COMT gene, identified in the 1950s, codes for an enzyme called catechol-O-methyl transferase. The gene produces two forms of this enzyme. The longer form, known as membrane-bound catechol-O-methyl transferase (MB-COMT) and the major brain species, is synthesized mainly by brain nerve cells. However, a shorter variant form of the enzyme called soluble catechol-O-methyl transferase(S-COMT) is a predominant, other tissues product in like the liver, kidneys, and blood [18]. The COMT gene, located on chromosome 22q11, contains a frequent G>A polymorphism that leads to a valine-to-methionine (Val/Met) substitution at codons 108 and 158 S-COMT and MB-COMT, respectively, resulting in a trimodal distribution of COMT activity in human populations [19]. The Val/Met locus is the most common name for the polymorphism, although it is also known by the reference sequence identification code rs4680 (previously rs165688). The Valine (Val) allele is also known as the high activity (H) allele or the G allele [20].

Schematic representation of COMT gene. The six exons of COMT are shown together to indicate the structure of the two transcripts, S-COMT and MB-COMT. The three polymorphisms (Val/Met = rs4680) that have been most widely studied are shown in figure 2 [21].

It methylates catechol-containing substrates, such as the catecholamines adrenaline, norepinephrine, and dopamine. Thereby deactivating them. Thus, Catechol-O-methyl transferase has a role in a wide range of biological processes. Although a study conducted on mice found that the role of COMT in the oxidation of dopamine is less than that of MAOA [22], it still plays an important and influential role in the oxidation of dopamine and other neurotransmitters [20]. According to the research results conducted on a group of males at a Chinese university to assess the effect of Val158Met polymorphism on aggressive behavior, there is a definite correlation between Val158Met and the appearance of aggressive behavior [23]. Lower anxiety and pain sensitivity are common in ValVal genotype carriers [24], leading to increased physical violence. Carriers of the Met allele, which determines reduced enzyme activity, had greater prefrontal cortex activation while retaining limbic system stimulation. As a result, the Met allele increased sensitivity to negative stimuli [25]. The Met- Met genotype is linked to women’s high harm avoidance score, as evaluated by the Cloninger assessment. These people have a fear of risk and the unknown and uneasiness and timidity [26]. Physical violence is the polar opposite of these characteristics.

Materials and Methods

One hundred blood samples were taken from males ranging from 30 to 60 years old in the current study. Fifty blood samples from convicts at Basrah Central Prison (Hamdan Prison) were found guilty of murder. In addition, fifty control samples were obtained randomly from Umm Qasr city, Basra Police Command, Basrah City Center, and Basrah University College of Science. Two milliliters of peripheral blood were collected from each group using a sterilized syringe and stored in sterilized EDTA tubes for DNA extraction. Genomic DNA was extracted from peripheral blood using the Genomic DNA Mini Kit (Geneaid, Taiwan) in the following manner

Genotyping of MAOA uVNTR Polymorphism

Genotyping was performed using conventional PCR technique of the MAOA –uVNTR polymorphism region. The polymorphism was detected using primers F: 5’- ACA GCC TCG CCG TGG AGA AG -3’, R: 5’- GAA CGG ACG CTC CAT TCG GA -3’ [27]. PCR reaction volumes for amplification of the MAOA gene using Bioneer master mix (25 μl) and the PCR condition for amplification of the MAOA gene are shown in tables (1 and 2).

We were able to diagnose the difference repeats in the MAOA gene promoter through gel electrophoresis of PCR product used with a 100 bp. DNA ladder and was analyzed statistically by an online SISA program

Genotyping of COMT gene (rs 4680, rs 165599) Polymorphism

Genotyping was performed using the conventional PCR technique of the COMT gene (rs 4680, rs 165599) polymorphism region. The polymorphism for rs 4680 was detected using primers F: 5´ - AGC CCT CCG TGC TGC TGG AGC TGG - 3´, R: 5´ - CAT GCC CTC CCT GCC CAC AGC CGG - 3´ [28]. The condition of PCR amplification of the COMT rs 4680 gene is listed in table (4). The polymorphism for rs 165599 was detected using primers, F: 5´- GAA GGA GAT GCT TCC ACT CTG T - 3´, R: 5´- ACT TTC AAA GCT CCC CTT GAC- 3´ [28]. PCR condition for amplification of the COMT rs 165599 gene is listed in table (4).

Sequencing

From 100 samples, 10 to 18 μl of COMT gene PCR products were sent to Macrogen Company “https://dna.macrogen.com” for sequencing. The tube of each sample was labeled with a number identical to the number of the Excel sheet sent by the company. The sequences were processed and analyzed using Basic Local Alignment Search Tool ‘BLAST’ to search for homologous sequences in the National Center for Biotechnology Information database (NCBI). https://www.blast.ncbi.nlm.nih.gov.

The Results

Analysis of MAOA VNTR polymorphism: In the promoter region of the human MAOA gene, there is a 30 bp. VNTR polymorphism. The number of repeats of this polymorphism varies between 2 and 5 as shown in Figure (3).

The Genotypes distribution of MAOA VNTR polymorphism with allele frequency in control and case groups

The distribution of the observed MAOA uVNTR genotype and allele frequencies in the control and cases groups are shown in table (5). The highest genotype in the control group was 350 bp 3R (41 participants) followed by rare alleles genotype observed at 320 bp 2R, 320/350 bp 2R/3R, and 380 4R with (5, 2, 2) participants respectively. In the case group, tow highest genotypes are 350 bp 3R and 320/350 bp 2R/3R (23 participants) for everyone. 320 bp 2R and 380 bp 4R are rare genotypes observed (2, 1 participant) respectively. The findings revealed that no significant differences existed in 320 bp 2R and 380 bp 4R at the two groups. While there were significant differences between the two groups, in heterozygote 320/350 bp 2R/3R (OR=20.5).

COMT gene rs4680:

Figure (5) showing the bands of COMT gene rs4680 PCR product on agarose gel electrophoresis.

Electropherogram: mutation (G>A) in COMT rs4680 polymorphism sequence.

Reference sequence for COMT gene rs 4680 from NCBI. Indicator in red

By using Jalview program version 2.11.1.7, the aliment of control and case sequences of COMT gene (rs 4680) were done as showing in figure (8).

Distribution of polymorphism with an allele frequency of COMT gene rs4680 and their association in control and cases groups

The distribution of the observed COMT rs4680 genotypes and allele frequencies in the control and cases groups are shown in table (6). The highest genotype in the control group was Val (G) 33 participants 66% flowed by met (A) 44%. However the genotype in the participants of the case was 50% for all (A=G). The results showed there is a significant association (OR=1.9411) for (A) alleles between control and case.

COMT gene rs 165599:

Reverse sequence for COMT gene rs165599 Indicator in red.

Figure 10 represents the sequences aliment of control and case samples sequence.

Electropherogram: Figure (11) showing mutation (G>A in COMT rs 165599 polymorphism sequence

COMT gene rs 165599 distribution

The distribution observed COMT rs 165599 genotypes and allele frequencies in both groups are shown in table (7). The highest genotype in the control group was (C) 29 participants 58% flowed by (T) 21 participants 42%. While the genotype in the participants of the cases was 50% for all (C=T). The results showed there is no significant association (OR=0.75—CI=0.75—P=0.476) or in other words no link (rs165599) polymorphism alleles with aggressive behavior.

The total effect of the polymorphism studied

Individuals in both groups who have at least one MAOA uVNTR (2R/3R) or polymorphism in COMT rs4680 or COMT rs165599 compared to those who do not have any polymorphism. As shown in table (8). The highest percentage of the studied polymorphism appeared in the cases group 80% (40 participants), while the control group was 54% (27 participants). The results showed there is a significant association (OR=3.407- CI 95% =1.401-8.285- P.V=0.00569) between studied polymorphism and aggressive behavior.

MAOA uVNTR 2R/3R, COMT rs4680, and COMT rs 165599 polymorphism are in the same participant

Participants for either group (cases and controls) who had an MAOA promoter uVNTR (2R/3R), a polymorphism in COMT rs165599 and COMT rs 4680 were matched to those who had no polymorphism. The results demonstrated a significant connection (OR=21, CI 95% =2.352 -187.494, P.V=0.00081) between investigated polymorphism and aggressive behavior, as shown in table (9).

Discussion

MAOA-uVNTR: a functional variable number of tandem repeats (VNTR)-polymorphism in the promoter region of the MAOA gene (encodes for MAO-A), is one frequently researched genetic component in this context [11]. This VNTR, which consists of a 30-bp repeated sequence, has been found in human subjects in quantities of 2, 3, 3.5, 4, or 5 copies [9]. The uVNTR prominent in the control samples is 3R. Present in 41 participants (82 %). Not appear to us in the control samples, 3R/4R uVNTR, as stated in the results. The results also revealed two participants who had the genotype 4R (380 bp) and two participants who had the genotype 2R/3R. By looking at the results of the studied cases, we see new repeat has a high frequency beside 3R uVNTR is heterozygote uVNTR 2R/3R in 23 samples for each one. The resulting release one sample has 410 bp, in addition to 480 bp and 480/450 bp in one sample for each.

The theory that aggressive behavior is caused by an excess of adrenaline, norepinephrine, and serotonin neurotransmission suggests that enzymes in the catecholamine pathway are likely to be involved.

This leads us to believe that the 2R/3R polymorphisms uVNTR produces a low-efficiency enzyme, which results in a rise in amino neurotransmitter concentration. People with 2R/3R are more sensitive to unpleasant environmental stimuli as a result. The statistical comparison of the examined cases with the control samples release high significance between 2R/3R repeat and aggressive behavior (OR=19.166- CI=2.508-146.499 -- P.V=0.001). There was a strong link between uVNTR number and violent criminal activity, as evidenced by the commission of a murder crime as a phenotype.

However, several researches, have failed to identify any MAOA-VNTR primary impact [30], [31] and [32]. The results of this study confirmed meta-analytic research that indicated a relationship between MAOA and behavioral disorders [10] and another new study in Pakistan [33].

In Iraqis convicted of murder, MAOA repetitions have not been investigated. So they represent a unique ethnic group. Repeating the tests on a larger number of cases is also recommended, with the Scones focusing on the genetic regions that showed a statistical difference. The findings of this study should not be used to prejudice others.

COMT rs4680: Reuptake is the primary method by which dopamine synaptic action is ended, followed by metabolic breakdown. The principal mammalian enzyme involved in the metabolic breakdown of released dopamine, catechol O-methyl transferase (COMT).

COMT is responsible for more than 60% of the metabolic degradation of dopamine in the frontal cortex [34]. This indicates an impact on cognition via effects on dopaminergic function.

Dopamine, also known as the “happy hormone,” is an important neurotransmitter. It’s made in response to joyous occasions. The Val genotype of the COMT gene was previously known to create an enzyme that was more efficient at breaking down dopamine. As a result, normal dopamine levels are maintained. A mutation in the COMT rs4680 gene causes a change in the enzyme that makes it. Because the resultant enzyme is less efficient at getting rid of dopamine, there is an excess of it, making persons with this mutation more susceptible to neurological shock, which may lead to aggressive behavior.

COMT rs165599: Several studies have looked into this COMT polymorphism. There was suspicion about the function of this mutation (G to A) in the development of schizophrenia. Comprehensive research of Ashkenazi Jews was successful in demonstrating a strong link between this mutation and schizophrenia G is a risk factor [35]. Other investigations supported these results. A study that reviewed the results of a group of research on this subject, revealed a discrepancy in the results about the role of COMT rs165599 in causing schizophrenia [36]. From our point of view, the evidence for the contradiction is weak.

Although there seems to be a difference between schizophrenia and violence, there is a connection. People who have schizophrenia are misunderstood by others. This puts additional pressure on them that may lead to violent behavior.

However, in the current study, a weak association was observed between this genetic variation and aggressive behavior OR=0.75. Although the results are trustful, the sample size in this study is relatively small, and it is important to replicate it with a bigger group of people.

The total effect of the polymorphism studied

The statistical calculations show that the person who have three genetic polymorphisms (MAOA 2R/3R + COMT rs165599 +COMT rs4680) is in high risk of violent behavior (OR=21, CI=2.352 -187.494, P.V= 0.00081).

The results showed a higher significant connection between participants who hold at least one of the genetic polymorphisms (MAOA 2R/3R or COMT rs165599 or COMT rs4680) and those who carry no genetic variants. This demonstrates how these particular genetic variations contribute to the establishment of aggressive behavior.

With an increase in the number of genes with polymorphisms, the influence of genetic polymorphisms on violent behavior tends to grow. This is due to the presence of more neurotransmitters in the synapse region for longer periods of time, resulting in a longer period of nervous stimulation, which leads to aggressive behavior toward others or the same person. The loss or absence of enzymes that break down neurotransmitters causes a rise in neurotransmitters.