Background

Schizophrenia (SCZ) and bipolar disorders (BD) are among the leading causes of disability worldwide, affecting around 1% of the population. However, biological mechanisms are still inadequately understood, where the interaction of environmental and genetic risk factors seems to play a prominent function in their etiology [1,2,3]. Moreover, expanding evidence has demonstrated that defects in immune-related genes present SCZ and BD susceptibility [4,5,6,7,8]. Numerous researchers have documented an imbalance in pro-inflammatory/anti-inflammatory cytokines or the level of their soluble receptors in plasma or cerebrospinal fluid of these patients over the last decade [9, 10]. Th1/Th2 imbalance with decreased levels of Th1-related cytokines and compensatory raised Th2-cytokine levels have been consistently observed in untreated SCZ patients [11]. In addition, IL-6 and TNF-alpha production of manic patients with BD was significantly higher than those of healthy participants, while IL-4 levels of the patients were significantly lower than controls [9].

The interleukin-1 receptor antagonist (IL-1RA) inhibits IL-1 activity by binding to the IL-1 receptors [12]. The IL-1RA encoding gene is found in chromosome 2q14, and the IL-1RA gene has a penta-allelic polymorphic region that contains variable numbers of an 86-bp tandem repeat (VNTR) (rs2234663) in intron 2 [13]. In addition, allele 2 of the IL-1RA VNTR variant is associated with elevated production of the IL-1β in vitro [14]. Plasma levels of IL-1RA and sTNF-R1 have been found to be related to general symptom severity and psychotic features of patients diagnosed with SCZ and BD [15]. Although documented results are contradictory, it has been shown that disease susceptibility in SCZ has been linked to the VNTR polymorphism in intron 2 of IL-1RA [16,17,18]. Besides, some results propose a relationship between BD and VNTR polymorphism in IL-1RA in literature [19].

IL-4 is a prototypic member of Th2 cytokines and has a potent anti-inflammatory feature [20]. It diminishes the production and effect of pro-inflammatory cytokines and concerns the isotype switching from immunoglobulin (Ig)M/IgG to IgE by B lymphocytes [21, 22]. IL-4 encoding gene is found in chromosome 5q31.1, and in its third intron, a 70 bp VNTR polymorphism (rs8179190) could alter the expression level of the IL-4 gene. This VNTR polymorphism includes three alleles: RP1 allele, with three repeats, RP2 allele, with two repeats, and RP3 allele, with four repeats. The frequency of RP2 allele is lower than the RP1 alleles. Again, the RP3 allele is insufficiently detected in a few populations [23]. Besides the VNTRs sequence in the third intron, so far, more than 50 allelic variant polymorphisms have been described for IL-4 gene, including − 33C/T (rs2070874), − 590C/T (rs2243250), 2979G/T (rs2227284), and + 3437C/G (rs2227282) [24]. Although we did not find any research on the relationship between IL-4 VNTR polymorphism and SCZ or BD when we looked at the literature, some studies on the relationship between some peripheral cytokine levels, including IL-4, and these psychiatric disorders, drew our attention. Regarding BD, a meta-analysis and systematic review showed that levels of the soluble IL-2 receptor (sIL-2R), IL-4, sIL-6R, and TNF-α were significantly higher in BD patients than in healthy subjects [25]. Similarly, IL4 and IL-10 levels have been found to be associated with negative symptoms of SCZ [26]. Therefore, these studies might suggest that IL-4-related anti-inflammatory immunological processes may have a role in the SCZ and BD pathophysiology.

Although IL-1RA VNTR polymorphisms have been studied as potential susceptible markers for SCZ and BD, there is no published research about the relationship between IL-4 VNTR polymorphism and SCZ or BD susceptibility. To the best of our knowledge, the present study is the first report investigating the possible association between IL-4 VNTR polymorphism and the risk of SCZ or BD. Therefore, this research aims to investigate the connection between IL-1RA and IL-4 VNTR polymorphisms in SCZ and BD by comparing the genotype distribution of these genes between Turkish patients and the healthy control group.

Methods

Study population

This case–control study included 234 unrelated patients (127 patients with SCZ, 107 patients with BD). Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision (DSM-IV-TR) was used for the psychiatric clinical interview. They were consecutively accepted in Malazgirt State Hospital Psychiatry Outpatient Clinic for three months. The standardized, well-accepted drug regimen was applied to all patient groups. They were in remission and thus did not describe complaints that required hospitalization. Patients were excluded from the research if they had mental retardation, organic disorder, or psychiatric disorders other than SCZ or BD. We recruited age-, sex-, geographic area-, and ethnicity-matched 204 healthy participants who were well-matched with the patients’ group. The Clinical Research Ethics Committee of the Istanbul Faculty of Medicine approved our study under the ethical standard for human experimentation established by the Declaration of Helsinki (29.01.2021-56267) [27].

DNA analyses

We collected peripheral blood into tubes with EDTA and extracted genomic DNA from the samples with the Quick-DNA Miniprep Plus Kit (Zymo Research). DNA samples were stored at − 20 °C. We investigated IL-1RA VNTR polymorphism by the PCR technique using the forward 5′-CTC AGC AAC ACT CCT AT-3′ and reverse 5′-TTC CAC CAC ATG GAA C-3′ primers. PCR products were separated by electrophoresis within a 2% agarose gel and visualized by ethidium bromide staining. Furthermore, we used the forward 5′AGG CTG AAA GGG GGA AAG C-3′ and reverse 5′-CTG TTC ACC TCA ACT GCT CC-3′ primers for IL-4 amplification and examined the PCR products by gel electrophoresis [28, 29].

Statistical analyses

Statistical analysis was performed using IBM SPSS version 21.0 (IBM Corp. released 2012; Armonk, NY, USA). We analyzed genotype distributions in participants due to the Hardy–Weinberg Equilibrium (HWE). Comparisons of genotype and allele frequencies between SCZ/BD patients and controls were performed using the Pearson chi-square or Fisher's exact test. The power analysis was performed with the “G*power” software (version 3.0.5, http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3/). The possible presence of population stratification bias has been calculated, according to Lee and Wang [30]. Because two polymorphisms were included in this study, the significance level was set as 0.025 (0.05/2) after the Bonferroni correction.

Results

IL-1RA genotyping

According to the differences in 86-bp tandem repeat number, three types of alleles can be recognized through the study. IL-1RA allele 1 (4 repeats, 420-bp) and IL-1RA allele 2 (2 repeats, 240-bp) were the most common; meanwhile, IL-1RA allele 4 (3 repeats, 326-bp) was rare. Most of the patients and control subjects carried the IL-1RA allele 1. The participants were evaluated according to clinical characteristics, as shown in Table 1. When the IL-1RA genotype distributions of SCZ patients were compared with the control group, the genotype distributions were not significantly different between the SCZ patients and the control group (1/1 vs. 1/2 + 1/4 + 2/2) (p = 0.231), (1/2 vs. 1/1 + 1/4 + 2/2) (p = 0.845), (1/4 vs. 1/1 + 1/2 + 2/2) (p = 0.673), and (2/2 vs. 1/1 + 1/2 + 1/4) (p = 0.086). Again, the IL-1RA allele frequency distributions were not significantly different between SCZ patients and the control group (1 vs. 2 + 4) (p = 0.078), (2 vs. 1 + 4) (p = 0.097), and (4 vs. 1 + 2) (p = 0.676). IL-1RA allele 2 homozygous genotype and IL-1RA allele 2 frequencies were non-significantly higher among SCZ patients than in controls (Table 2). When the IL-1RA genotype distributions of BD patients were compared with the control group, the genotype distributions were not significantly different between the BD patients and the control group (1/1 vs. 1/2 + 1/4 + 2/2) (p = 0.571), (1/2 vs. 1/1 + 1/4 + 2/2) (p = 0.392), (1/4 vs. 1/1 + 1/2 + 2/2) (p = 1.000), and (2/2 vs. 1/1 + 1/2 + 1/4) (p = 0.676). Additionally, the IL-1RA allele frequency distributions were not significantly different between BD patients and the control group (1 vs. 2 + 4) (p = 0.826), (2 vs. 1 + 4) (p = 0.830), and (4 vs. 1 + 2) (p = 1.000) (Table 3).

Table 1 The clinical characteristics of participants
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Table 2 Comparison of frequencies of IL-1RA VNTR polymorphism between SCZ patients and healthy controls
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Table 3 Comparison of frequencies of IL-1RA VNTR polymorphism between BD patients and healthy controls
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IL-4 genotyping

The distribution of intron 3 VNTR IL-4 genotype frequencies, allele frequencies, and risk association were compared between SCZ patients and controls and summarized in Table 4. When the IL-4 genotype distributions of SCZ patients were compared with the control group, the genotype distributions were not significantly different between the SCZ patients and the control group (P1/P1 vs. P1/P2 + P2/P2) (p = 0.346), (P1/P2 vs. P1/P1 + P2/P2) (p = 0.383), and (P2/P2 vs. P1/P1 + P1/P2) (p = 0.198). Likewise, the IL-4 allele frequency distributions were not significantly different between SCZ patients and the control group (P1 vs. P2) (p = 0.129). When the IL-4 genotype distributions of BD patients were compared with the control group, the genotype distributions were not significantly different between the BD patients and the control group (P1/P1 vs. P1/P2 + P2/P2) (p = 0.321), (P1/P2 vs. P1/P1 + P2/P2) (p = 0.476), and (P2/P2 vs. P1/P1 + P1/P2) (p = 0.270). Similarly, the IL-4 allele frequency distributions were not significantly different between BD patients and the control group (P1 vs. P2) (p = 0.182) (Table 5).

Table 4 Comparison of frequencies of the IL-4 VNTR polymorphism between SCZ patients and healthy controls
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Table 5 Comparison of frequencies of the IL-4 VNTR polymorphism between BD patients and healthy controls
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Discussion

Our results demonstrated that the functional VNTR polymorphism distributions of the IL-1RA and IL-4 of SCZ and BD patients were not significantly different from the control group. The functional VNTR polymorphism of the IL-1RA has also been considerably analyzed in the literature. For example, Kim et al. reported that allele 2 was associated with SCZ in a Korean population [16], while Zanardini et al. documented that IL-1RA allele 1 frequency was significantly higher in Italian patients with SCZ than in the control participants [17]. Other studies, including ours, failed to find any difference [31, 32]. When we carefully examined the p values obtained as a result of statistical analyzes, IL-1RA allele 2 homozygous genotype and IL-1RA allele 2 frequencies were non-significantly higher among SCZ patients than in controls. The IL-1RA allele 2 was known to be related to severe clinical outcomes in chronic inflammatory diseases [33, 34]. Furthermore, it was documented that the presence of the IL-1RA allele 2 is associated with enhanced IL-1β production [35]. Therefore, it is likely that diminished IL-1RA production in the SCZ and BD patient's brain cells that carry the IL-1RA allele 2 by dysregulating IL-1β production is related to pro-inflammatory situations in the brain tissue damage [36]. For example, it has been documented that a higher expression of the IL-1RA allele 2 was related to bifrontal-temporal gray matter volume and generalized white matter tissue deficits in allele 2 carrier SCZ patients [37].

There are minimal data regarding the function of IL-1RA polymorphism in BD, unlike SCZ. Papiol et al. reported a significant excess of the haplotypic combination -511 allele*1/VNTR allele*2 in Spanish BD patients compared with controls [38]. Furthermore, Hosseini et al. documented that the IL1RN*1/2 genotype was more prevalent in BD patients than in controls [19]. In a recent published case–control study, genotyping of IL-1RA (VNTR; rs2234663), IL-1α (rs1800587), IL-1β + 3954 (rs1143634), and IL-1β − 511 (rs16944) loci revealed that three haplotypes including two SNPs of C-T (rs1800587-rs16944), T-C (rs1143634-rs16944), T-A1 (rs16944-rs2234663) and one haplotype including three SNPs of C-C-T (rs1800587-rs1143634-rs16944) were related to the BD [39]. However, Kim et al. documented contradictory reports indicating that VNTR genetic variation in IL-1RA was not related to BD in a Korean population like our study [16]. Papiol et al. analyzed the influence of the IL-1 cluster on brain morphology in BD. They documented that while − 511C/T polymorphism of the IL-1β gene was related to whole-brain gray matter and left dorsolateral prefrontal cortex gray matter deficits in BD patients, the IL-1RA genetic variability did not have any influence on the brain morphology [40].

In the present study, we showed no association between IL-4 VNTR polymorphism and risk for SCZ and BD in the Turkish population. To the best of our knowledge, there is no study on IL-4 gene VNTR polymorphism and SCZ or BD in the literature. There are, however, several studies on IL-4 VNTR gene polymorphism and other autoimmune and inflammatory diseases from Turkey. Their associations with alopecia areata [41], coronary artery disease [42], diabetic peripheral neuropathy [43], knee osteoarthritis [44], multiple sclerosis [45], and recurrent aphthous stomatitis [46] have been studied in the Turkish population previously. Additionally, IL-4 and IL-1RA VNTR gene polymorphisms have been found related to type 2 diabetes mellitus and frailty syndrome together [47, 48]. The IL-4 coding gene is located within the cytokine gene cluster on chromosome 5q31.1, a chromosomal region identified by linkage analyses containing a susceptibility gene for SCZ [49, 50]. Schwarz et al. reported a significant association of the IL-4 –590 CC genotype with SCZ [51]. However, Jun et al. study results showed no significant difference in the genotypic and allelic frequencies of IL-4 Rα + 1902 and IL-4 − 590 gene polymorphism between patients with SCZ and normal controls [52]. Although in the literature, no study on this subject has examined the role of the IL-4 gene polymorphism in BD, a study by Brietzke et al. on the cytokine levels (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) of patients with BD demonstrated that only IL-4 levels were increased in euthymic BD patients compared with healthy controls. This research also showed higher serum levels of IL-2, IL-4, and IL-6 in manic episodes compared to healthy participants [53]. Again, a meta-analysis of 10 studies (428 patients and 397 healthy controls) showed significantly higher IL-4 levels in BD patients than in controls [54]. Since we did not simultaneously investigate blood peripheral cytokine values in our study, we do not know outcomes regarding the relationship between IL-4 VNTR polymorphism and blood IL-4 cytokine levels and the correlation with these studies in the literature.

Our study’s strength is that the first research investigating the possible association between IL-4 VNTR polymorphism and the risk of SCZ or BD, though IL-1RA VNTR polymorphism has been studied as a potential susceptible marker before. Secondly, our findings were more beneficial since SCZ or BD patients and healthy participants were gathered from the same geographic area. However, besides the strengths of the present research, our study has some drawbacks. The first limitation was the small sample size, limiting the statistical power. Secondly, examination of additional clusters of genes and polymorphic sites related to IL-4 and IL-1RA would be needed to establish the contribution of IL-4 and IL-1RA VNTR gene polymorphisms to the etiology of SCZ and BD.

Conclusions

In summary, the etiopathogenesis of SCZ and BD is still an unsolved puzzle of psychiatry. Moreover, the theories that try to enlighten the exact mechanism for chronic inflammation become more complicated every day. Therefore, we investigated anti-inflammatory cytokine VNTR polymorphisms of IL-4 and IL-1RA in the remitted SCZ and BD patients in the present study. Although these biological markers do not seem to be used to diagnose SCZ and BD in the Turkish population, the finding that different inflammatory patterns exist in these psychiatric disorders can provide a hint for further examinations exploring candidate biomarkers for determining these two disorders. In addition, the association observed between cytokines and prognoses may provide some clues for new therapeutic medications and personal strategies in the future.