Molecular Analysis of STin2 (Intron 2) Variant of The SLC6A4 Gene in Children and Adolescents With Attention Decit Hyperactivity Disorder

Attention decit hyperactivity disorder (ADHD) is recognized as one of the most familiar childhood psychiatric disorders. Many molecular genetic reviews suggest that genes play a crucial role in susceptibility to ADHD. The serotonin transporter gene (SLC6A4) has polymorphisms that seem to correlate with ADHD development. The association between ADHD and the SLC6A4 gene variants in the Iranian population has not been investigated yet. This study analyzes the STin2 (intron 2) variant of the SLC6A4 gene in Iranian children and adolescents with ADHD .


Abstract
Background Attention de cit hyperactivity disorder (ADHD) is recognized as one of the most familiar childhood psychiatric disorders. Many molecular genetic reviews suggest that genes play a crucial role in susceptibility to ADHD. The serotonin transporter gene (SLC6A4) has polymorphisms that seem to correlate with ADHD development. The association between ADHD and the SLC6A4 gene variants in the Iranian population has not been investigated yet. This study analyzes the STin2 (intron 2) variant of the SLC6A4 gene in Iranian children and adolescents with ADHD .

Materials and Methods
In this retrospective case-control study, 86 ADHD patients and 99 healthy volunteers aged 5 to 14 years old were enrolled as the case group and the control group, respectively. The STin2 (intron2) fragment of the SLC6A4 gene was ampli ed using speci c primers by conventional PCR, and three STin2 alleles of the SLC6A4 gene (STin2.9, STin2.10, and STin2.12) were examined using the acrylamide gel method.

Conclusion
It is concluded that there was no association between the frequency of STin2 variant alleles of the SLC6A4 gene andADHD, but in the study of risk estimation, it was found that allele 10 of this variant is a risk allele in ADHD patients.

Background
Attention de cit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in children, with a worldwide prevalence of 4-8% in school-matured youngsters and may continue during adulthood in 50-80% of cases (1-3). ADHD is described by symptoms of inattention, hyperactivity, and impulsivity and has a wide range of possible clinical presentations(4&5). Considering the importance of ADHD in children and its impact on their health and the fact that its exact etiology is not yet completely understood, evaluating and researching the possible genetic factors involved in it may help us to comprehend, prevent, and possibly cure this disorder better.
To nd better ways to treat and decrease the risks of ADHD, scientists are researching cause(s) and risk factors. Although the etiology and risk factors for ADHD remain unclear, recent evidence suggests that genetics play a key role (6-10). The ADHD patients' genes have been evaluated and statistically, among these genes, DRD4, DRD5, DAT, DBH, SNAP-25, HTR1B, and 5-HTT seem to be involved in the etiology of ADHD (2 & 11-14).
The location of the serotonin transporter gene (SLC6A4; 5HTT) is on chromosome 17q. This gene encodes a carrier protein responsible for retaking serotonin from the synapse and returning it to presynaptic neurons, which has a crucial role in serotonergic activity regulation within the brain. Attention, memory, and voluntary activity are connected to areas of the brain such as the amygdala, hippocampus, thalamus, putamen, and anterior cortex, which are the areas that 5HTT is expressed (12).
Based on the available evidence, the 5HTT gene could play a signi cant role in ADHD. Studies have observed this gene's role in impulsivity's etiology and stimulus responses in hyperactivity (14).
The serotonin transporter gene (5-HTT/SLC6A4) is amongst the most researched genes in psychiatry and has been linked with a wide variety of diseases (11).
A study reported a signi cant association of the polymorphism within the promoter region of 5-HTT with scores on the Wender Utah Rating Scale, which is used to assess a history of ADHD-associated symptoms, indicating a higher frequency of the long variant allele in individuals with high scores (15).
Although some studies have reported the STin2.12 allele (a major allele of STin2 polymorphism) as a transcriptional enhancer, one research showed that STin2.12/STin2.12 homozygotes appear to show fewer serotonin transporters available inside the brain (16-18). In 2002, Zoroğlu et al. noted that the STin2.12/12 variant of VNTR polymorphismappears to be associated with an increased risk of ADHD (19).
In a study that evaluated the association between ADHD and polymorphism of the two regions of the 5-HTT gene [variable number of tandem repeats (VNTR) and 5-HTTLRR] 5-HTTLPR S/S genotype was signi cantly lower in the ADHD group but Homozygous and heterozygous L variant predominated in it. The VNTR STin2.12/12 genotype was found signi cantly less in the ADHD group but there was no signi cant difference between the frequency of the short (S), long(L), 10, and 12 alleles in the two groups. They suggested the lack of an S/S variant of 5-HTTLPR polymorphism or the STin2.12/12 variant of VNTR polymorphism as a risk factor for ADHD (19).
In a study evaluating the serotonin transporter gene in aggressive children with and without ADHD, the 10R allele of the 5HTT VNTR polymorphism was signi cantly less frequent in the study group and there was a signi cant link between 5HTTLPR and ADHD. Aggressive children were statistically more likely to have at least one copy of the long allele than were those without ADHD (20). In a study evaluating the possible role of the 5-HTTLPR polymorphism in childhood disruptive behaviors using the haplotype relative risk design, a signi cant decrease in the short/short 5-HTTLPR genotype was observed in the ADHD type III combined group. Comparing the allele frequencies yielded similar results (21).
A review article reports that when the 5-HTTLPR studies are combined, the pooled OR for the long allele is 1.31 (95% CI 1.09-1.59) (3).
Although there are many studies about ADHD, few have focused on the role of STin2 variants. Therefore, we aimed to analyze the molecular analysis of the STin2 variants of the SLC6A4 in children and adolescent with ADHD.

Samples
The study group consisted of 86 children from Northwest area of Iran, who were diagnosed with ADHD by the Diagnostic and Statistical Manual (DSM-5) criteri (22). The control group consisted of 99 nonpsychiatric participants with similar demographic features such as mean age and gender and were referred to the children's hospital a liated to Tabriz University of medical sciences for adenotonsillectomy and required routine lab tests. The sampling of target members was subject to the psychiatrist's convenience sampling technique based on inclusion and exclusion criteria. Additionally, participants' parents lled up informed consent.
This study's con rmation is contributed to the Scienti c and Ethics Committee of Tabriz University of Medical Sciences (approval number REC.1396.186.IR.TBZMED) as a thesis for a doctoral degree.

Inclusion Criteria
Inclusion criteria were the diagnosis of ADHD through psychiatrists' clinical interviews based on indicated criteria in DMS-5 and the age range of 4 to 14 years.

Exclusion Criteria
Head trauma and epilepsy history, concurrent psychiatric disorder, Intellectual disability, and other severe medical conditions were considered as the exclusion criteria.

PCR-Gene Ampli cation
The peripheral blood (in the amount of 3-5 mL) was obtained under sterile conditions and was stored under proper storage circumstances. DNA extraction from blood samples of all participants was performed by the proteinase K method. In the following step, the STin2 (intron2) fragment in the SLC6A4 gene was ampli ed through speci c primers, designed using Primer 3 software (version 4), via polymerase chain reaction (PCR), and three types of STin2 alleles of the SLC6A4 gene were examined using the acrylamide gel method. The frequency and distribution of variants were calculated using POPGENE software version 1.32. Then the study's data were coded and analyzed in SPSS software version 26.

Statistical Analysis
The data were entered, coded, and statistically analyzed in SPSS software (version 26.0; IBM Corp, Armonk, NY, USA), and the mean values and standard deviations were computed with this software.The statistical analysis was performed by Pearson's Chi-square test and Fisher's exact test to compare the frequency of different alleles between the two groups to determine possible associations. The p-value < 0.05 was considered statistically signi cant.

Results
A total of 186 children were enrolled in this study. They were divided into two groups, the ADHD group and the control group that contained 86 (46 males and 40 females) and 99 (54 males and 45 females), respectively. All of the participants took part in the study except one of the healthy members who refused the PCR test.
The ndings revealed no signi cant difference between groups in average age (p-value = 0.886) and gender (p-value = 0.982).
A comparison of the analysis of STin 2 (intron 2) variant alleles, which included 9, 10, and 12 alleles, is as follows.
Allele 9: The nine positive alleles frequency were compared between two populations as shown in Table 1.   Allele 10: The ten positive alleles frequency were compared between two populations as follows.
allele 12: The 12 positive alleles frequency were compared between two populations as illustrated in

Discussion
ADHD is a behavioral disorder in which the neurotransmitters and their balance play a crucial role. As there is a failure in behavioral inhibitions in ADHD, serotonin's role in its pathophysiology has been considered in recent years. It has now been suggested in animal studies that serotonin is involved in hyperactivity, inattention, and impulsive behaviors (5&23). Previous reviews demonstrated the association between the SLC6A4, one of the best-studied genes in the psychiatry eld, and a wide range of disorders, and we evaluated the relation between the frequency of STin2 variant alleles of this gene and ADHD.
Statistically, none of the STin2 variant alleles had any essential distinction between the two populations in our study.
To assess the risk, the estimated OR values for alleles 9, 10, and 12 were 0.824, 1.354, and 0.986, respectively, and a positive allele 10 had a higher risk of ADHD development.
Banerjee's study in which the polymorphisms of the STin2 variant in patients with ADHD were thoroughly examined, found that the risk of ADHD development had a relation with the inheritance of allele 12 of this variant (24). To Summarize his results and ours, the association of ADHD with the inheritance of allele 12 of this variant can be concluded.
In 2003, Langley, an examiner of 5-HTT transporter gene polymorphisms,reported no association between the 9, 10, and 12 alleles of the STin2 variant with ADHD (25), which is consistent with our results.
In contrast to our study, Zoroglu in 2002 listed several reasons for the relationship between Homozygosity in Allele 12 (A12 / A12) and an increased risk of ADHD development within the Turkish population, although there was an associationwith Homozygosity in allele 10 (A10 / A10) and a higher risk of ADHD, similar to our results. (19).
Overall the association between the frequency of STin2 variant alleles of SLC6A4 and ADHD seems inconsistent. This may be due to the complex genetic architecture of ADHD and the effects of non-genetic factors on the disorder and also the nature of case-control studies evaluating the association. This in turn necessitates more studies with larger sample sizes and case-control studies evaluating maternal and paternal inheritance of variants of genes involved in serotonin homeostasis in patients with ADHD.

Conclusion
In conclusion, there was no association between the frequency of STin2 variant alleles of the SLC6A4 gene and ADHD, but in the study of risk estimation, it was found that allele 10 of this variant is a risk allele in ADHD patients. However, we recommend further studies with larger sample sizes and among different races in different areas. Also, we suggest case-control studies evaluating maternal and paternal inheritance of variants of genes involved in serotonin homeostasis in patients with ADHD.

Declarations
Ethics approval and consent to participate This study's con rmation is contributed to the Scienti c and Ethics Committee of Tabriz University of Medical Sciences (approval number REC.1396.186.IR.TBZMED) as a thesis for a doctoral degree. Furthermore, Informed consent to participate in this study was obtained from all participant's parent.

Consent for publication
All authors, give their consents for information about this paper to be published in the Journal of Child and Adolescent Psychiatry and Mental Health.