Genetic polymorphism of IL-17F rs763780 contributes to the susceptibility to bipolar disorder but not to schizophrenia in the Turkish population

Abstract This study aims to investigate the genetic polymorphism in the interleukin-17F (IL-17F) (rs763780, 7488 A/G) gene in bipolar disorder (BD) and schizophrenia (SCZ) patients by comparing it with healthy controls considering clinical parameters. A sample of 107 patients with BD, 129 patients with SCZ, and 100 healthy volunteers were included. SCID-I was used to confirm the diagnosis according to DSM-IV-TR criteria. The Young Mania Rating Scale (YMRS) and the Hamilton Depression Rating Scale (HAM-D) were administered to BD patients. The Positive and Negative Symptoms Scale (PANSS) was applied to the patients with SCZ. PCR-RFLP was used to determine IL-17F gene polymorphism. Our results demonstrated that the distributions of the IL-17F genotype and the allele frequencies of BD patients were statistically significantly different from the control group. The AA genotype (OR: 0.283; 95% Cl: 0.140–0.573; p<.001) and A allele (OR: 0.333; 95% Cl: 0.171–0.646; p=.001) frequencies were significantly higher in the control group than in the BD group. The IL-17F genotype and the allele frequency distributions of SCZ patients were not statistically significantly different from the control group. When comparing scale scores due to the IL-17F genotype distributions in patients with BD or SCZ, there was no statistically significant difference between the groups of IL-17F genotypes. In summary, whereas the IL-17F polymorphism may be associated with BD, this polymorphism was not related to SCZ.


Introduction
Schizophrenia (SCZ) and bipolar disorders (BD) are debilitating mental disorders with prevalence rates of approximately 1% of the worldwide population. [1] Biological mechanisms are still poorly understood, where the interaction of genetic and environmental risk factors seems to play a notable role in their etiology. [2,3] Recent findings have demonstrated that many rare copy number susceptibility loci affect genes involved in the neurodevelopmental pathways, cell plasticity, and neurotransmitter systems, evidencing the genetic factor of these conditions. [4][5][6] In addition, although not adequately described, it is known that environmental influences, including psychological events, immune activation, lifestyle, nutrition, and damaging chemical exposure, are important triggers to epigenetic inheritance in SCZ and BD. [7,8] Changes in the methylation pattern of genes related to glutamatergic, GABAergic, dopaminergic, and inflammation-associated systems are seen as critical epigenetic mechanisms (DNA methylation, histone covalent modifications (acetylation, methylation, and phosphorylation), and non-coding RNAs (ncRNAs)) involved in SCZ and BD. [9][10][11] Genome-wide association studies show a great understanding of the genetic overlap between SCZ and BD. [12] Moreover, growing evidence has shown that defects in immune-related genes present SCZ and BD susceptibility. [13][14][15] Therefore, SCZ and BD might be associated with alterations in the systemic immune system, including T-cell activation and low-grade chronic inflammation. [16,17] Earlier studies documented heightened peripheral inflammatory markers in SCZ and BD patients compared to healthy controls. [18,19] The patients with first-episode psychosis and chronic SCZ show increased peripheral levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). In addition, meta-analysis studies reported elevated C-reactive protein (CRP) and IL-6 levels during acute mania and increased IL-6 and TNF-α in the euthymic phase of BD. [19][20][21] Interleukin-17 (IL-17) is produced by T helper 17 (Th17) cells and is involved in numerous immune and inflammatory processes. Th17 cells play a predominant role in mucosal cell integrity and may infiltrate the central nervous system through its cytokine production's direct effects. [22] Six members of the IL-17 family were determined (IL-17A, IL-17B, IL-17C,  IL-17D, IL-17E, and IL-17F). An IL-17F gene is located on chromosome 6p12.3. [23] IL-17F (rs763780, 7488 A/G) polymorphism affects the gene's coding region by inducing Histidine to Arginine (His-to-Arg) substitution at amino acid 161, [24] is located in the third exon of the IL17F gene and was documented to cause a loss in the ability of IL17F to induce expression of specific cytokines and chemokines. Besides, this variant may work as a natural antagonist of the wild-type IL17F since it can bind to its receptor without triggering a signal, blocking the initiation of IL8 expression. [25,26] IL-17A and IL-17F genes are mapped on the same chromosome, and they share the highest degree of homology, about 50%, while the IL-17B, C, D, and E have only 16-30% identity at the primary sequence level, and their genes are localized to different chromosomes. [27] Furthermore, although IL-17A and IL-17F genes are mapped on the same chromosome at position 6p12, IL-17F has been shown to function differently from other members of the IL-17 cytokine family, especially IL-17A. Accordingly, IL-17F is considered a principal mediator of cellular immunity by preserving the expression of essential cytokines that induce pro-inflammatory responses. [28] Copy number variations (CNVs) studies have shown that copy number changes in 6p12 may play a role in SCZ or BD pathophysiology. Genomewide study of CNV determined that copy number changes of 3p22.2, 7p13, 11p15.1, 12p13, and 6p12 are related to SCZ in Han Chinese family trios. [29] Again, for the recessive model, the most notable evidence of linkage occurred at 6p12.3 for BD identified using a combined genomewide linkage and CNV analysis. [30] Several investigations have indicated elevated levels of IL-17 in BD, [31] SCZ, [32] anxiety, and depression, [33] but association studies regarding IL-17 polymorphism in the psychiatric disorder have been lacking. When the literature is reviewed, there is no disease-associated single nucleotide polymorphisms (SNPs) research about the relationship between IL-17F gene polymorphism and patients diagnosed with SCZ or BD considering clinical parameters. We hypothesized that IL-17F gene polymorphism might be related to the etiopathogenesis and clinical parameters of SCZ and BD in Turkish patients. Therefore, this research aims to investigate the connection between the IL-17F gene polymorphism in patients with SCZ or BD by comparing the genotype distribution of the IL-17F gene between patients and the healthy control group regarding clinical characteristics.

Patient selection
The consecutive sampling procedure was used in this case-control research. We collected a sample of 107 patients with BD and 129 patients with SCZ from the Malazgirt State Hospital Psychiatry Outpatient Clinic for three months; additionally, 100 healthy volunteers were included in the study. The Clinical Research Ethics Committee approved our study of the Istanbul Faculty of Medicine regarding human experimentation standards confirmed by the Helsinki Declaration (2020/1773). [34] We used the Structured Clinical Interview for DSM-IV Axis-I Disorders (SCID-I) to confirm the diagnosis according to DSM-IV-TR criteria. [35,36] While the Positive and Negative Symptoms Scale (PANSS) was applied to patients with SCZ, [37,38] the Hamilton Depression Rating Scale (HAM-D) [39,40] and the Young Mania Rating Scale (YMRS) [41,42] were administered to patients with BD.

Inclusion and exclusion criteria
Based on the SCID-I interview, we involved patients diagnosed with BD or SCZ, of either gender, literate, 18 to 65 years of age, and had no mental retardation, neurodevelopmental disorders, and other systemic/neurological diseases that may affect cognitive functions. Besides, we excluded subjects who refused participation, diagnosed with axis-1 disorder other than BD or SCZ due to SCID-I interview, and BD or SCZ secondary to substance use disorders or general medical conditions.

Blood samples and DNA extraction
We took the blood samples into EDTA tubes and isolated DNAs from the collected blood samples by using the Plus Blood Genomic DNA Purification Kit according to manufacturer recommendations (Gene Mark).

Statistical analyses
We performed the statistical analysis with IBM SPSS version 21.0 (IBM Corp. released 2012; Armonk, NY, USA). Quantitative data (clinical parameters of BD or SCZ patients and IL-17F genotype distributions) were represented as descriptive statistics containing frequency, percentage, mean, and standard deviation. The Pearson chi-square or Fisher's exact test analyzed comparisons of the IL-17F genotype distribution of patients. The suitability of continuous variables to normal distribution was evaluated with the Shapiro-Wilk test. Finally, we performed intergroup comparisons of continuous variables (PANSS pos., PANSS neg., PANSS psycho., PANSS total, HAM-D, and YMRS) with the Mann-Whitney U test since the variables did not have a normal distribution. We had to group the genotypes by considering the presence of mutation allele (AA, AG/GG) to compare scale scores of two groups containing a statistically significant number of patients. The online software platform (https://gene-calc.pl/hardy-weinbergpage) was used to carry out Hardy-Weinberg equilibrium (HWE) tests and all polymorphisms conformed to HWE (χ 2 test, p > 0.05) in schizophrenia, bipolar disorder, and control groups. These quality control measures show that genotyping results of all SNPs are reliable for analyses. We accepted the statistical significance as p <.05 for the results of all analyses. In addition, the odds ratio (OR) and the 95% confidence interval (CI) were also calculated. We conducted the power analysis with the "G*power" software (version 3.0.5, http://www.psycho.uni-duesseldorf.de/ abteilungen/aap/gpower3/), post hoc goodness of fit χ2 test, with an "-error" probability of 0.05. The possible presence of population stratification bias has been gauged, according to Lee and Wang. [43] At the time that we collected the populations and designed this study, the required smallest sample size was calculated as 300 cases (100 in each group) would have at least 80% power (for α = 0.05).

Sociodemographic characteristics and clinical parameters of BD and SCZ patients
The participants were evaluated according to sociodemographic characteristics and clinical parameters, as shown in Table 1. The scale scores of the BD and SCZ patients are shown in Table 2. According to the IL-17F genotype distribution, 65.4% (n = 70) of the patients diagnosed with BD had AA, 34.6% (n = 37) had AG, and 0% (n = 0) had GG genotypes. 85.3% (n = 110) of the patients diagnosed with SCZ had AA, 13.2% (n = 17) had AG, and 1.6% (n = 2) had GG genotypes. Again, 87% (n = 87) of the healthy controls had AA, 13% (n = 13) had AG, and 0% (n = 0) had GG genotypes. The BD patients' mean age was 41.64 ± 11.75, the SCZ patients' mean age was 40.91 ± 10.73, and the mean age of the healthy controls was 31.03 ± 10.50.

IL-17F (rs763780, 7488 A/G) genotyping
When the IL-17F (AA, AG, GG) genotype and the allele frequency (A, G) distributions of BD patients were compared with the control group, the IL-17F genotype and allele frequency distributions of BD were significantly different from the healthy control (

Comparison of the scale scores according to IL-17F genotype distributions (AA, AG/GG) in patients with BD or SCZ
When comparing scale scores (PANSS pos., PANSS neg., PANSS psycho., and PANSS total) due to the IL-17F genotype distributions (AA, AG/GG) in patients with SCZ, there was no statistically significant difference found between the groups of IL-17F genotype (p=.299; p=.294; p=.325; p=.586,  respectively) (data not shown). Again, when we compared the scale scores (HAM-D and YMRS) due to the IL-17F genotype distributions (AA, AG/ GG) in patients with BD, there was no statistically significant difference between the groups of IL-17F genotype (p=.816; p=.158, respectively) (data not shown).

Discussion
In our study, while the distributions of the IL-17F genotype and the allele frequencies of the BD patients were significantly different from the control group, there was no statistically significant difference between the IL-17F genotype and the allele frequency distributions of SCZ patients and the healthy control group. When the literature is reviewed, there is no disease-associated SNPs research examining the association between IL-17F polymorphism and SCZ or BD. All studies in the literature were about the linkage between serum IL-17 levels and these psychiatric disorders' etiopathogenesis. IL-17 is an efficient pro-inflammatory cytokine that can induce monocytes and neutrophils. In addition, IL-17 has a function in T-cell-triggered inflammation by stimulating stromal cells to secrete growth factors and different cytokines such as IL-1β, IL-6, and TNF-a, resulting in an inflammatory response via the expression of IL-17 receptors. [24,44] Numerous types of research showed that IL-17 gene polymorphisms are related to various autoimmune diseases such as asthma, [45] psoriasis, [46] and rheumatoid arthritis. [47] On the other hand, in our recently published research examining the catechol-O-methyltransferase (COMT) (rs4680), the cannabinoid receptor type 2 (CNR2) (rs2501432), CNR2 (rs2229579), mitochondrial uncoupling protein-2 (UCP2) (rs659366), and IL-17F (rs763780) gene variants in synthetic cannabinoid use disorder (SCUD) patients have been reported that the IL-17F genotype distribution of SCUD patients was not significantly different from the healthy participants. [48] The present study showed that the participants carrying the IL-17F AA genotype and A allele had a lower risk of BD occurring. We suggest that the IL-17F (rs763780) polymorphism may play a crucial function in the pathogenesis of BD by its direct or indirect effects on the production of IL-17. Since we did not measure the simultaneous plasma IL-17 level, we could not determine how the IL-17F polymorphism affects the IL-17 level in Turkish patients with BD. When the literature was examined, it was noticed that various studies about the connection between IL-17F polymorphism and IL-17 level had been published. Kawaguchi et al. suggested that the IL-17 expression and activity may be suppressed in IL-17F (rs763780) G allele carriers. [26] That was in disagreement with Mahfouz et al.'s study. They reported that a higher frequency of IL-17F GG genotype and G allele was related to an increase in IL17 plasma level ≥ 9.3 pg/ mL (cut-off point). [49] IL-17 contributes to blood-brain barrier (BBB) disruption and the consequent attraction of monocytes and macrophages into the nervous system by recruiting infiltration neutrophils and the activating matrix metalloproteinases. [50] When the Th17 cells disrupt tight junctions and transmigrate across the BBB, it causes the killing of neurons by releasing granzyme B. [51] Therefore, producing of antibodies against neural cell antigens might be the mechanism of potential autoimmunity in BD. When the literature was reviewed, a recent study reported that IL-17 levels significantly increased in BD patients compared with controls. Also, a positive correlation between IL-17 levels and the disorder's duration showed that inflammation is included in the disorder's progression. [31] Li et al. have reported elevated IL-17 levels in BD patients and showed its relationship with the severity of mania symptoms. [52] Again, Chen et al. found an increased level of RORγt (the specific transcription factor of Th17 cell) mRNA expression in blood lymphocytes and higher serum IL-17 concentration in major depressive disorder (MDD) patients compared to healthy controls. [53] On the other hand, Saraykar et al. [54] and Kim et al. [55] did not show proof to support the involvement of IL-17A in MDD.
We found no difference between the IL-17F genotype and allele frequency distributions of SCZ patients and healthy controls. Besides, no disease-associated SNPs have examined the relationship between IL-17F polymorphism and SCZ; the reports about IL-17 levels in SCZ patients are also inconsistent. El Kissi et al. reported that drug-free SCZ patients in the acute phase of the disorder exhibited significantly higher IL-17 than healthy controls. [32] Again, two current studies showed increased IL-17 concentrations in antipsychotic-naïve psychotic patients. [56,57] Contrastly, some researchers showed decreased serum levels of IL-17 in psychotic patients. [58] In a recent meta-analysis study, Fang showed that IL-17 might not be involved in the pathological mechanism of SCZ, similar to our study. [59] Comparing PANSS scale scores due to the IL-17F genotype distributions in patients with SCZ, there was no significant difference between the groups of IL-17F genotypes in our study. Ding et al. published that the PANSS total score correlated positively with the levels of Th17 cells in drug naïve, first episode SCZ. [57] IL-17 is found to be related to PANSS positive, psychopathology, and total scale scores in SCZ patients by Dimitrov et al.. [60] Our results are in line with the El Kissi et al. study, that they also reported no significant associations between IL-17 serum level and PANSS scale scores, although the Scale for the Assessment of Negative Symptoms (SANS) global score was negatively correlated with IL-17 serum level. [32] Our study's strength is the first disease-associated SNPs that showed the interaction between IL-17F gene polymorphism and the pathogenesis of SCZ or BD, considering clinical parameters. Besides this strength of the present research, there are also several limitations. First of all, our study was a single-center study with a small sample, limiting our findings' generalizability. Secondly, the cross-sectional design precludes any causal inferences for the relationship between IL-17F polymorphism and the pathogenesis of BD or SCZ. Thirdly, we could not check the serum IL-17 level at the same time while the IL-17F gene polymorphism was being studied.
In conclusion, while the IL-17F polymorphism might be associated with BD, this polymorphism was not related to SCZ. Having the IL-17F AA genotype and A allele may be advantageous in not being diagnosed with BD in the Turkish population. These findings shed light on the etiology of BD and may promote inflammation as an essential function in the progression of BD pathophysiology; thus, for future studies, we suggest investigating new therapeutic agents, including N-acetylcysteine, non-steroidal anti-inflammatory drugs and GSK3 inhibitors, as a possible co-adjuvant for classic BD treatment. Although this biological marker does not seem to be used to diagnose SCZ, and it is not associated with the clinical variables in the Turkish population in the present study, the findings of this study do not exclude the possibility that other variations of the IL-17F gene may be involved in the etiology of SCZ. Therefore, further studies with larger populations and assessing other critical SNPs should be performed to evaluate better the relationship between immune-related gene polymorphisms and these psychiatric disorders. the study. SP and YO are the responsible provisions of study materials and laboratory samples. HMA drafted the manuscript. All authors critically revised the manuscript. SP and MP supervised the study.

Data availability Statement
The authors confirm that all relevant data are included in the article, which does not contain any supplementary material.

Disclosure statement
No potential conflict of interest was reported by the authors.

Ethical standards
The authors declare that all methods contributing to this work comply with the relevant national and institutional committees' ethical standards on human experimentation and the Helsinki Declaration of 1975, as revised in 2013 [34]

Informed consent
The written informed consent has been taken from the patient to publish her case details.

Funding
The author(s) reported there is no funding associated with the work featured in this article.