Abstract
Objectives: To evaluate the genetic polymorphisms in IL-2 and IL-2RA genes in schizophrenia (SCZ) patients by comparing them with healthy controls.
Methods: A sample of 127 patients with SCZ and 100 healthy volunteers were included in the case-control study. These individuals were consecutively selected from the Malazgirt State Hospital Psychiatry Outpatient Clinic in Mus, Turkey, over the three months from October 2020 to December 2020. The Structured Clinical Interview for DSM-5 Disorders, Clinician Version (SCID-5-CV) was used to confirm the diagnosis according to the DSM-5 criteria. In addition, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine gene polymorphisms from DNA material.
Results: Our findings indicated significant differences in the IL-2 genotype and allele frequencies between SCZ patients and the healthy control group. Specifically, the frequency of the homozygous GG genotype was notably higher in SCZ patients compared to the control group. Conversely, when comparing the IL-2RA genotype and allele frequencies of SCZ patients with the control group, no statistically significant differences were observed between the 2 groups. When compared to individuals with other genotypes, interaction analysis indicated that carriers of the GG/AG (IL-2/IL-2RA) genotype demonstrated a significantly increased risk of SCZ.
Conclusion: In light of the analyses, our study indicates that while the IL-2 genotype polymorphism may be considered a risk factor for developing SCZ, the IL-2RA variant was not associated with SCZ among Turkish patients.
Schizophrenia (SCZ) is a persistent and severe mental disorder affecting approximately 1% of the general population. While the complete pathophysiology of SCZ remains unclear, an escalating number of studies are now concentrating on the involvement of the immune system in this disorder.1-3 Recent studies suggest that uncontrolled activation of microglia and heightened inflammation due to proinflammatory cytokines may contribute to the development of SCZ, particularly in individuals with a genetic predisposition.4 Moreover, infections experienced during embryonic and early childhood stages can hinder fetal brain development, while excessive synaptic pruning during adolescence has been identified as another potential risk factor for psychosis.5 Numerous linkage and association studies have demonstrated the involvement of multiple genes in the development of SCZ.6-8 A thorough examination of 118 meta-analyses revealed an association between SCZ and a set of 24 genetic variants situated in 16 different genes (HP, DRD4, SLC6A4, TPH1, IL1B, DRD2, MTHFR, TP53, GABRB2, DAO, DRD1, APOE, DTNBP1, COMT, PLXNA2, and GRIN2B).9 Among the potential candidate genes for SCZ, those encoding cytokines appear promising. Cytokines play crucial roles in the central nervous system (CNS), acting as essential mediators in the communication between the brain and immune system and participating in neuroinflammatory processes.10-12
Interleukin-2 (IL-2) is a growth factor that regulates the autocrine function of T-cells, specifically regulatory T-cells (Treg), and plays a crucial role in the immune response.13 The IL-2 gene is located on chromosome 4q26-q27, and its various variants can lead to functional differences in activation.14 Several linkage studies have suggested the possibility of a susceptibility gene for SCZ in this chromosomal region, such as 4q22–23 or 4q31,15,16 although conflicting data exist.17 Notably, 2 variants in the IL-2 gene, -330 T/G (rs2069762) and +114 G/T (rs2069763) have been identified as having an impact on IL-2 levels.18 While prior investigations have explored the association between the IL-2-330 T/G (rs2069762) polymorphism and SCZ in German and Polish populations, no similar research has been identified concerning the Turkish population diagnosed with SCZ and its relationship with this IL-2 polymorphism.19,20
The IL-2RA gene, or interleukin 2 alpha-receptor gene, is located on chromosome 10p15.1. It encodes CD25, a protein recognized as the alpha-receptor chain for interleukin 2, constituting the high-affinity alpha subunit of the interleukin receptor. The CD25 is vital for the T lymphocyte response to IL-2, the primary growth factor for these cells. CD25 expression is crucial for fostering proliferation, extending lifespan, and ensuring optimal T-cell function.21,22 Upon evaluating 265 Irish families with SCZ alongside genome scan results, the findings indicate the potential presence of a vulnerability locus for SCZ within region 10p15-p11 in these specific Irish families.23 However, there has been no prior investigation into the genetic polymorphism of IL-2RA genes in SCZ patients by comparing them with healthy controls. Moreover, association studies examining polymorphisms of IL-2 genes in SCZ have been limited. Considering the potential association of immune-related genes with SCZ susceptibility, we hypothesized that polymorphisms in the IL-2 (rs2069762) and IL-2RA (rs2104286) genes might show notable distinctions in Turkish individuals with SCZ compared to healthy individuals. Furthermore, the exploration of gene-gene interactions between IL-2 and IL-2RA gene polymorphisms could offer valuable insights into the progression of this disorder. Hence, our objective was to examine genetic polymorphisms in IL-2 and IL-2RA genes among SCZ patients, drawing comparisons with a healthy control group.
Methods
Participant selection
In this case-control investigation, 127 individuals diagnosed with SCZ were included. The psychiatric clinical interview utilized the Structured Clinical Interview for the Diagnostic and Structured Clinical Interview for DSM-5 Disorders, Clinician Version (SCID-5-CV). These patients were consecutively selected from the Malazgirt State Hospital Psychiatry Outpatient Clinic from October 2020 to December 2020 for three months. For comparison, we recruited 100 healthy participants who were age-, sex-, geographic area-, and ethnicity-matched with the patient group. The participants were given details about the study’s objectives, materials, and procedures, and their written informed consent was obtained. The study received approval from the Clinical Research Ethics Committee of the Istanbul Faculty of Medicine, in accordance with the ethical standards for human experimentation outlined in the Declaration of Helsinki (Approval date: 29.01.2021-56267).
DNA analyses
The DNA extraction from blood samples obtained from participants was performed in accordance with the manufacturer’s instructions, utilizing the Quick-DNA Miniprep Plus Kit by Zymo Research. The extracted DNA samples were stored at -20 °C. Genotyping of IL-2RA rs2104286 and IL-2 rs2069762 variants was carried out using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, following established methods. The IL-2RA rs2104286 polymorphism was investigated through PCR with the forward primer 5’-GCAGGTGTCAACGCAAAAAC-3’ and reverse primer 5′-TCCCTGGAATGTCACTGATG-3’. Additionally, IL-2 rs2069762 amplification was performed using the forward primer 5’-ATTCACATGTTCAGTGTAGTTCT-3’ and reverse primer 5’-GTGATAGCTCTAATTCATGC-3’, and the resulting PCR products were examined through gel electrophoresis.24,25
Statistical analyses
Statistical analysis was carried out using IBM SPSS version 21.0 (IBM Corp., released 2012; Armonk, NY, USA). The adherence of genotype distributions in participants to the Hardy-Weinberg Equilibrium (HWE) was assessed. Genotypic distribution of IL-2 (rs2069762) and IL-2RA (rs2104286) in SCZ patients, as well as the potential interaction between IL-2 (rs2069762) and IL-2RA (rs2104286) polymorphisms concerning SCZ risk, were evaluated using the Pearson chi-square test or Fisher’s exact test. Various models, including dominant, over-dominant, recessive, and additive, were employed to assess the relationship between single nucleotide polymorphisms and SCZ, providing 95% confidence intervals (CI) and odds ratios (OR). Statistical significance was considered for a p-value less than 0.05. Power analysis was conducted using “G*power” software (version 3.0.5, http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3/). Additionally, we employed the approach proposed by Lee and Wang to evaluate the potential presence of population stratification bias.26
Results
The sociodemographic characteristics and clinical parameters of SCZ patients were analyzed (Table 1). A total of 227 individuals, including 127 SCZ patients and 100 healthy controls, underwent assessment for the IL-2 rs2069762 and IL-2RA rs2104286 variants. The IL-2 rs2069762 variant’s genotype distribution is detailed in Table 2. Notably, our findings indicated significant statistical differences in IL-2 genotype and allele frequency distribution between SCZ patients and the healthy control group. The homozygous GG genotype frequency was significantly elevated in the SCZ patient group compared to the control group (OR: 0.453; 95% CI: 0.207–0.995; p=0.045). The additive model revealed a significant difference in the frequency distribution of GG and TT genotypes between the 2 groups (OR: 2.191; 95% CI: 0.991–4.846; p=0.049).
- The clinical characteristics of SCZ patients.
- Comparison of frequencies of IL-2 (rs2069762) gene variants between patients with SCZ and healthy controls.
Furthermore, the genotype distribution of the IL-2RA rs2104286 variant is outlined in Table 3. When comparing the distribution of IL-2RA genotype and allele frequency in SCZ patients to the control group, no statistically significant differences were observed between the 2 groups. Compared with other genotypes, interaction analysis indicated that carriers of GG/AG (IL-2/IL-2RA) (OR: 2.236; 95% CI: 0.984–5.079; p=0.050) genotype demonstrated a significantly increased risk of SCZ (Table 4).
- Comparison of frequencies of IL-2RA (rs2104286) gene variants between patients with SCZ and healthy controls.
- Interaction of IL-2 (rs2069762), and IL-2RA (rs2104286) polymorphisms on SCZ risk.
Discussion
According to our findings, the distributions of IL-2 polymorphisms in SCZ patients were significantly different from those in the control group. However, no significant association was found between IL-2RA gene polymorphism and SCZ. Numerous extensive studies have been carried out to investigate alterations in IL-2 levels among patients with SCZ. However, the findings from these studies display significant variation, with some indicating increased IL-2 levels in patients with SCZ,27,28 while others report decreases29,30 or no changes.31 IL-2 appears to exhibit a specific association with the negative and cognitive symptoms observed in SCZ. Asevedo conducted a study that explored potential connections between peripheral IL-2 levels and both negative symptoms and cognitive functioning in individuals with SCZ.29 Again, O’Donnell et al’s23 published research investigated immune activation in SCZ and schizophreniform disorder, specifically the production of IL-2 and sIL-2R from stimulated lymphocytes. Their study revealed increased production of IL-2 in unmedicated patients with SCZ and schizophreniform disorder, but no significant changes in sIL-2R were observed in parallel with our study. Nevertheless, it is important to note that changes in IL-2R and sIL-2R levels in patients with SCZ exhibit a consistent pattern across the majority of studies. Specifically, sIL-2R levels consistently appear higher in patients with SCZ compared to healthy controls across diverse studies, indicating its potential as a diagnostic marker for SCZ.33
In the current study, we found a significant difference in the genotype distribution of IL-2 (rs2069762) between SCZ patients and healthy controls. Notably, the frequency of the homozygous GG genotype was significantly higher in the SCZ patient group compared to the control group. Again, interaction analysis indicated that carriers of the GG/AG (IL-2/IL-2RA) genotype demonstrated a significantly increased risk of SCZ. Upon reviewing the literature, it was evident that several studies investigating the association between IL-2 polymorphism and SCZ had been published. For instance, Schwarz et al20 reported a significant association between the IL-2 –330 TT genotype and the IL-4 –590 CC genotype with SCZ.20 Likewise, Palacz et al34 reported that individuals with GT and GG genotypes have a reduced risk of developing SCZ compared to those with the TT genotype.34 In a recent study, the genotype TT and allele T for IL-2 polymorphism were discovered to be significantly associated with paranoid SCZ in the Polish population.19 Contrary to the results of these studies, our research revealed a significantly elevated vulnerability to the occurrence of SCZ among participants who carried the GG genotype. According to Hoffmann et al., the GG genotype is correlated with elevated levels of IL-2, while the TT and GT genotypes are associated with a decrease in the production of this cytokine.35 As a result, our findings suggest the detrimental consequences of excessive cytokine production in individuals with SCZ.
The uniqueness of our study lies in being the first to investigate the potential connection between IL-2RA polymorphism and the susceptibility to SCZ. Furthermore, our findings were enriched by the fact that both SCZ patients and healthy participants were sourced from the same geographical area, thus minimizing potential environmental influences. Nevertheless, alongside these strengths, our study does exhibit certain limitations. Firstly, the small sample size restricted the statistical power of our analysis. Secondly, the cross-sectional design precluded us from making causal inferences regarding the relationship between IL-2, IL-2RA polymorphisms, and the development of SCZ. Thirdly, due to the timing constraints of our study, we were unable to concurrently measure serum IL-2 and IL-2R levels while investigating IL-2 and IL-2RA gene polymorphisms.
In summary, while the IL-2 rs2069762 polymorphism could potentially exhibit an association with SCZ, the IL-2RA rs2104286 polymorphism did not show any correlation with SCZ. The presence of the IL-2 GG genotype might confer a disadvantage in terms of SCZ diagnosis within the Turkish population. These findings provide insights into the underlying causes of SCZ and underscore inflammation as a crucial factor in the progression of its pathophysiology. Currently, a significant gap remains in the availability of reliable markers for diagnosing and guiding the treatment of SCZ. Exploring drug repurposing, a strategy involving the utilization of existing treatments for new disease contexts, offers a promising avenue to address the limitations of current antipsychotic medications that primarily target dopaminergic pathways.36 The dopamine hypothesis falls short in comprehensively explaining all symptoms, and neuroinflammation is increasingly gaining recognition as an additional contributing factor. The conclusions drawn from ongoing trials imply that supplementary anti-inflammatory strategies could hold potential advantages in alleviating clinical symptoms in individuals with SCZ. Consequently, the use of anti-inflammatory medications (such as aspirin, celecoxib, NAC, minocycline, etc.) could potentially be explored for alleviating psychotic symptoms. Furthermore, there has yet to be an antipsychotic medication developed with IL-2 as its target, opening up a potential avenue for future research in this direction.
Acknowledgement
We would like to acknowledge Cambridge Proofreading LLC for native English editing.
- Received August 16, 2023.
- Accepted November 22, 2023.
- Copyright: © Neurosciences
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