Abstract
Objective
The study was designed to compare the expression levels of IL-6 mRNA, long non-coding RNA (lncRNA) NRON (non-coding repressor of the nuclear factor of activated T cells [NFAT]) and TMEVPG1 (Theiler’s murine encephalomyelitis virus persistence candidate gene 1) which play critical roles in the regulation of immune function, and to investigate relationship between expression levels and symptom type and the cognitive functions in patients with schizophrenia and schizoaffective disorder.
Methods
The study included 84 participants (27 patients with schizophrenia, 27 with schizoaffective disorder, and 30 healthy subjects). The lncRNA (TMEVPG1 and NRON) and IL-6 mRNA expression analysis was measured with the real-time PCR method. The Wisconsin Card Sorting Test, the Stroop Test, Clinical Global Impression Scale, Positive and Negative Symptoms Scale, Young Mania Rating Scale and the Hamilton Depression Rating Scale were applied.
Results
The lncRNA TMEVPG1 expression level was determined to be higher in the patient groups than controls. The TMEVPG1 was able to differentiate schizophrenia cases from the controls. In the schizoaffective group, a positive correlation was determined between NRON expression and positive symptomatology, and an increase in NRON expression was determined to make a moderate contribution to cognitive dysfunction. NRON expression was decreased as the dose of antipsychotic drug increased in the schizophrenia group.
Conclusion
The results of this study demonstrated that there are significant differences between schizophrenia and schizoaffective disorder in terms of inflammatory markers and their relationship between the symptom type or cognitive functions.
Keywords: Schizophrenia, Schizoaffective disorder, Inflammation, Non-coding RNA
INTRODUCTION
Schizophrenia is a chronic disease seen with positive and negative symptoms and disruptions in cognitive areas [1]. Despite important studies in neuroscientific areas, the diagnosis of schizoaffective disorder continues to be a matter of debate. There are differences of opinion about whether the disease represents a different clinical entity, whether the emergence of two different mental disorders, or whether it represents a condition that has expanded within the psychotic process from bipolar disorder to schizophrenia [2].
Studies have shown evidence of increased inflammation in schizophrenia patients [3]. While interleukin (IL)-6 may have several protective properties in infection, it can play a role on maintaining chronic inflammation in various neuropsychological diseases when it is over-expressed in the central nervous system [3,4]. Relationship was also determined between cognitive impairment and elevated IL-6 levels in schizophrenia patients [5].
The application of new generation sequencing technology to transcriptome analysis has completely changed the view of genomes, and the section that was previously accepted as “junk DNA” because of non-coding protein has now emerged as formed of thousands of non-coding RNA transcripts which have active regulatory roles. It is estimated that >80% of the human genome is actively transcribed, but as only 2 to 3% are changed to proteins, this means that almost all the genome transcription output is monopolised by non-coding RNA (ncRNAs). Long ncRNAs (lncRNAs) are the most abundant ncRNAs in the human genome. Noticeably, while the protein encoding genes and corresponding number of sequences remain stable during the evolution of eukaryotic organisms, the increasing development of lncRNAs significantly increases together with organ complexity [6]. LncRNAs play critical roles in the regulation of immune function. It has been shown that both TMEVPG1 ([Theiler’s murine encephalomyelitis virus persistence candidate gene 1], also named Ifng-AS1 [Ifng antisense RNA 1] or nettoie Salmonella pas Theiler’s) and NRON (non-coding repressor of the nuclear factor of activated T cells [NFAT]) lncRNAs play a role in the regulation of proinflammatory cytokine expression [7]. In addition, lncRNA TMEVPG1 increases interferon gamma (IFN-γ) expression, and lncRNA NRON regulates NFAT (nuclear factor of activated T-cells) transcription factor, which controls a series of cytokine expressions, including IL-6 and IFN-γ [8,9]. There are extremely few studies that have examined lncRNAs in psychiatric diseases [10,11]. To the best of our knowledge, there is no study in literature that has investigated lncRNAs in schizoaffective disorder.
The aim of this study was to investigate the relationship between cognitive functions and the change according to type of predominant symptom of the disease in lncRNAs, which have a regulatory role in IL-6 and inflammation, in schizophrenia and schizoaffective patient groups. It was aimed for this study to contribute to the knowledge of the similarities and differences between these two diseases which are in the same spectrum.
METHODS
Study Design
The study included 27 patients diagnosed with schizophrenia and 27 with schizoaffective disorder according to DSM-5 criteria, and a control group of 30 healthy subjects matched in age and sex. All the patients and control subjects were in the 18 to 60 years age range, were literate, and had normal mental capacity. Olanzapine, risperidone, quetiapine, amisulpride, aripiprazole and paliperidone were the antipsychotics that used in the patient group. Valproic acid, carbamazepine and lamotrigine were the mood stabilizers that used.
The study exclusion criteria were defined as the presence of comorbid disease, autoimmune disease, neurological/physical chronic disease, a known allergy, a history of malignancy, pregnancy, alcohol/substance abuse, a history of surgical intervention within the last 12 months, a history of head trauma, having received electroconvulsive therapy within the last 6 months, the presence of liver transaminase/serum electrolyte/urea-creatine abnormal/mm3 in the full blood count, or use of anti-inflammatory and immunosuppressive drugs (eg., non-steroidal antiinflammatory drugs and steroids).
Blood samples of 12.5 ml were withdrawn from an antecubital vein of all the study participants into 3 tubes; one tube (5 ml) for hemogram, one tube (5 ml) for biochemistry, and one tube (2.5 ml) as an RNA stabilisation tube (for the TMEVPG1 and NRON lncRNAs and IL-6 mRNA expression levels). All the samples were taken as fasting blood samples in the morning from all the study participants.
Approval for the study was granted by the Ethics Committee of University (decision number: 60116787-020/31842, dated: 01.06.2020). Each participant or their legal representative was informed about the study, and written informed consent was obtained in accordance with the Helsinki Declaration.
Clinical Evaluation
A sociodemographic data form was completed by all participants. Positive-Negative Symptoms Scale (PANSS) [12] and Clinical Global Impressions Scale (CGI) [13] were applied in the patient groups. Hamilton Depression Evaluation Scale (HAM-D) [14], and the Young Mania Evaluation Scale [15] were applied to the schizoafffective group.
Neuropsychological Evaluation
Executive functions were evaluated using the Wisconsin Card Sorting Test (WCST) and the Stroop test. These were applied as the Psychology Experiment Building Language (PEBL)-Berg’s WCST [16], which is the short computer version of the WCST [17], and PEBL-Victoria Stroop Test, which is the Victoria form of the Stroop test [18].
Biochemical Analyses
Examination of full blood count and biochemical parameters
The blood samples were analyzed in one biochemistry tube (5 ml) for biochemical analyses and in one EDTA tube (5 ml) for hemogram analyses. The hemogram parameters were examined on a Mindray BC 6800 device, and the biochemical analytes on a Roche/Hitachi c702 device.
Examination of inflammation parameters
A peripheral blood sample of 2.5 ml was withdrawn into Pax Gene RNA tubes for each of the study participants for RNA isolation, and was stored at −80°C until assay.
RNA isolation
The Hybrid-R miRNA (Geneall) kit was used for total RNA isolation. The procedures were applied in sequence according to the kit protocol. The blood sample tubes containing RNA stabiliser were thawed to room temperature, then centrifuged at 3,000 g for 10 minutes. After discarding the supernatant, 4 ml PBS (Capricorn) was added to the pellet and a mild vortex process was applied. The tubes were again centrifuged at 3,000 g for 10 minutes. After discarding the supernatant, 500 μl Riboex (Geneall) was added to the pellet and was left for 5 minutes at room temperature. Then 100 μl chloroform (Sigma) was added, shaken rapidly for 15 seconds, then left at room temperature for 2 minutes. The tubes were then centrifuged at 12,000 g for 15 minutes at 4°C. The upper aqueous phase was transferred to a new tube, to which the same amount of 100% ethanol (Merck) was added. From this a 700 μl sample was transferred to a red-ringed spin column. After centrifugation at 10,000 g for 30 seconds, the filtered part was removed, then the remaining sample was placed in the same column and the last two procedures were repeated. After the addition of 500 μl SW1 to the column, it was centrifuged at 10,000 g for 30 seconds and the filtered part was removed. Then, 500 μl RNW was added to the column, and after centrifugation at 10,000 g for 30 seconds, the filtered part was removed. It was centrifuged at 10,000 g for 1 minute, then 1.5 ml of the column was placed in a new collection tube. Fifty μl ribonuclease (RNase)-free water was added to the column, which was then centrifuged at 10,000 g for 1 minute. The determination of RNA concentrations and purity were made with a Nanodrop 2000 (Thermo Scientific). RNAs that were not converted to complementary DNA (cDNA) were removed and stored at −80°C.
Complementary DNA synthesis
An ABT cDNA synthesis kit was used for cDNA conversion for expression analysis. For the cDNA synthesis, a reaction mixture was prepared (1 μl Reverse Transcriptase (200 U/μl); 1 μl 20X dNTP mix (2.5 mM); 2 μl 10X Reaction Buffer; 2 μl Random hexamer (50 μM); 0.5 μl RNase Inhibitor (40 U/μl); 3.5 μl RNase-free; 10 μl RNA Template; total 20 μl). After preparation of the reaction mixture over ice, the tubes were incubated for 10 minutes at 25°C, then for 120 minutes at 37°C, and finally for 5 minutes at 85°C. The cDNA samples were stored at −20°C until expression analysis.
Expression analysis real-time quantitative polymerase chain reaction
The relative quantitation analyses for TMEVPG1 (target lncRNA), NRON (target lncRNA), 18S RNA (reference lncRNA), IL-6 (target gene) and GAPDH (reference gene) were performed using a real-time PCR system (Rotor Gene, Qiagen). The reaction mixture was prepared using ABT SYBR Green Master Mix and primers specific to the target lncRNA and mRNA (10 μl ABT SYBR green PCR Kit, 2×; 2 μl Forward&Reverse Primer Mix [TMEVPG1, NRON, 18S RNA, IL-6, GAPDH]; 5 μl cDNA; 3 μl dH2O; total 20 μl).
The point at which the fluorescent values pass the threshold value is known as the cycle threshold (CT). The CT value is the time when the system starts to notice an increase in the amount of fluorescence, and the PCR product starts to increase exponentially in the log-linear phase. The target gene expression of each sample was normalised according to the reference gene expression. This normalisation was accomplished with the subtraction of the reference CT from the target CT, and the resulting value was labelled ΔCT. To compare the expression levels of the patient groups with the control group, the mean ΔCT values were calculated for each group. By proportioning the mean ΔCT of the patient and control groups, it was then calculated how many fold difference there was between the expression amounts. To determine the number of fold increase or decrease in each replication cycle, these values were taken as negative power over 2. Thus the changes in expression between the patient groups and the control group were determined with the 2−ΔΔCT method [19,20]. ΔCt = Ct (target) − Ct (reference); ΔΔCt = ΔCt (patient) − ΔCt (control); change = 2−ΔΔCt.
Statistical Analyses
Data obtained in the study were analyzed statistically using SPSS ver. 25.0 software (IBM Co.). Continuous variables were stated as mean ± standard deviation or median, (minimum−maximum) values. The conformity of data to normal distribution was examined with the Shapiro Wilk test. When parametric test assumptions were met, independent group differences were compared using One-Way variance analysis (post hoc: Tukey test) and the significance of the difference between two means test. In the comparisons of differences of independent groups of data not conforming to normal distribution, Kruskal Wallis variance analysis (post hoc: Bonferroni corrected Mann-Whitney Utest) and the Mann-Whitney Utest were used. Covariance analysis was used in the examinations made with corrected body mass index (BMI) values. In the examinations of differences between categorical values, the Chi-square test was used. To determine correlations between quantitative variables, Spearman correlation analysis was used. Receiver operating characteristic (ROC) analysis was applied to determine the success of the expression parameters in differentiating the groups. In all the analyses, a value of p < 0.05 was accepted as statistically significant.
RESULTS
Sociodemographic and Clinical Characteristics
The groups were seen to be similar in respect of age, sex, education level (primary school/high school/university), smoking amount (cigarettes/day), and smoking-alcohol consumption status (Table 1). Patients in the schizo-affective group were determined to have higher BMI values than those in the schizophrenia group and the control group (Table 1).
Table 1.
Sociodemographic and clinical characteristics of groups
| Control (n = 30) | Schizophrenia (n = 27) | Schizoaffective (n = 27) | p | |
|---|---|---|---|---|
| Age (yr) | 38 (22−55) | 38 (21−58) | 40 (22−54) | 0.884a (F = 0.123) |
| 37.6 ± 8.38 | 37.89 ± 10.88 | 38.85 ± 10.29 | ||
| Body mass index (kg/m2) | 27.26 (20.15−45.26) 27.07 ± 5.21 |
27.39 (19.37−32.71) 26.77 ± 3.64 |
31.14 (23.44−44.46) 32.13 ± 5.96 |
0.0001c,* (kw = 15.892) (1-3, 2-3) |
| Disease onset age (yr) | - - |
20 (14−36) 22.7 ± 6.38 |
23 (14−42) 23.63 ± 6.92 |
0.612d (t = −0.511) |
| Duration of illness (yr) | - - |
11 (1−36) 15.15 ± 12.06 |
15 (3−32) 15.22 ± 7.96 |
0.628e (z = −0.485) |
| Sex | ||||
| Female | 12 (40.0) | 9 (33.3) | 11 (40.7) | 0.825b |
| Male | 18 (60.0) | 18 (66.7) | 16 (59.3) | |
| Education | ||||
| Primary | 15 (50.0) | 13 (48.1) | 17 (63.0) | 0.448b |
| High school | 12 (40.0) | 8 (29.6) | 8 (29.6) | |
| University | 3 (10.0) | 6 (22.2) | 2 (7.4) | |
| Smoking status | ||||
| Yes | 12 (40.0) | 18 (66.7) | 14 (51.9) | 0.132b |
| No | 18 (60.0) | 9 (33.3) | 13 (48.1) | |
| Cigarette/day | ||||
| ≥ 1 package | 9 (75.0) | 14 (77.8) | 11 (78.6) | 0.975b |
| < 1 package | 3 (25.0) | 4 (22.2) | 3 (21.4) | |
| Alcohol | ||||
| Yes | 25 (83.3) | 24 (88.9) | 22 (81.5) | 0.725b |
| No | 5 (16.7) | 3 (11.1) | 5 (18.5) | |
| Application | ||||
| Inpatient | - | 7 (25.9) | 7 (25.9) | > 0.999b |
| Outpatient | - | 20 (74.1) | 20 (74.1) |
Values are presented as number (%), mean ± standard deviation, or median (minimum−maximum).
F, covariance analysis test value; -, not available.
aOne-way ANOVA test; bChi-square test; cKruskal-Wallis test; dtwo sample ttest; eMann-Whitney Utest.
*p < 0.05 significant.
Mood stabilisers were not being used by any of the cases in the schizophrenia group, and by 23 (85.2%) patients in the schizoaffective group. No statistically significant difference was determined between the patient groups in respect of the use of benzodiazepine, biperiden, and antidepressant drugs. Antipsychotic drugs were converted to chlorpromazine equivalents and daily doses were calculated in milligrams [21]. No statistically significant difference was found between the schizophrenia and schizoaffective patients in respect of chlorpromazine equivalent antipsychotic drugs used daily (1,106.42 ± 590.81 and 1,135.65 ± 623.32 mg, respectively) (p > 0.05).
The mean score of the disease severity section of the CGI was 4.86 ± 0.69 in the schizophrenia group and 4.86 ± 0.38 in the schizo-affective group, with no statistically significant difference (p > 0.05). In the schizo-affective group, the mean Young Mania Score was 7.93 ± 8.16, and the mean HAM-D scale score was 5.41 ± 3.66.
Data Related to IL-6 and TMEVPG1 and NRON LncRNA Expression Levels
IL-6 expression was determined to be reduced by −3.11-fold in the schizo-affective group compared to the control group, and by −9.16-fold in the schizophrenia group compared to the control group. TMEVPG1 expression increased by 4.35-fold in the schizoaffective group compared to the control group, and by 4.69-fold in the schizophrenia group compared to the control group. NRON expression increased by 1.36-fold in the schizoaffective group compared to the control group, and by 1.46-fold in the schizophrenia group compared to the control group. Covariance analysis was applied to examine the difference between the groups in respect of the TMEVPG1, NRON, and IL-6 ΔCT values corrected according to BMI. The TMEVPG1 expression was determined to be statistically significantly higher in both patient groups than in the control group. No significant difference was determined between the groups in respect of the NRON and IL-6 values (Table 2).
Table 2.
IL-6 vs. TMEVPG1 vs. NRON lncRNA DCT values corrected according to BMI values
| Control (n = 30) | Schizophrenia (n = 27) | Schizoaffective (n = 27) | p | |
|---|---|---|---|---|
| IL-6 | ||||
| 95% CI | 1.49−5.13 | 4.56−8.40 | 2.06−6.13 | 0.109 (F = 2.080) |
| Mean ± SD | 3.31 ± 0.91 | 6.48 ± 0.96 | 4.10 ± 1.02 | |
| TMEVPG1 | ||||
| 95% CI | 10.6−13.28 | 8.11−10.96 | 7.77−10.77 | 0.018* (F = 3.542) (1-2, 1-3)a |
| Mean ± SD | 11.94 ± 0.67 | 9.53 ± 0.71 | 9.27 ± 0.75 | |
| NRON | ||||
| 95% CI | 15.28−17.63 | 14.48−16.97 | 14.72−17.34 | 0.759 (F = 0.392) |
| Mean ± SD | 16.45 ± 0.58 | 15.73 ± 0.62 | 16.03 ± 0.65 |
IL-6, interleukin 6; TMEVPG1, Theiler’s murine encephalomyelitis virus persistence candidate gene 1; NRON, non-coding repressor of the nuclear factor of activated T cells; lncRNA, long non-coding RNA; CT, cycle threshold; BMI, body mass index; SD, standard deviation; CI, confidence interval; F, covariance analysis test value.
aPost hoc analysis results.
*p < 0.05 significant.
Correlations betweeen IL-6, TMEVPG1 and NRON LncRNA ΔCT Values and Cognitive Tests
In the control group, a positive correlation at a weak level was determined between the NRON lncRNA ΔCT values and the number of persevering errors in the WCST (r = 0.363).
In the schizophrenia group, no significant correlation was determined between the TMEVPG1 lncRNA, NRON lncRNA, and IL-6 mRNA ΔCT values and the cognitive tests (p > 0.05).
In the schizoaffective group, the NRON lncRNA was determined to have a moderate level positive correlation with the total number of correct WCST responses (r = 0.435) and with the number of responses at a conceptual level (r = 0.403) and a moderate level negative correlation was determined with the total number of incorrect responses (r = −0.435).
Correlations between IL-6, TMEVPG1 and NRON LncRNA ΔCT Values and PANSS
In the schizoaffective group, a weak negative correlation was determined between the NRON lncRNA ΔCT values and the PANSS P points (p = 0.05, r = 0.381; Table 3). No other significant correlation was determined in all groups.
Table 3.
Relationships between PANSS scores and IL-6 and lncRNA DCT values
| Schizophrenia group | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||||
| PANSS P | PANSS N | PANSS G | PANSS T | ||||||||
|
|
|
|
|
||||||||
| r | p | r | p | r | p | r | p | ||||
| IL-6 | −0.003 | 0.987 | 0.187 | 0.350 | 0.014 | 0.944 | 0.129 | 0.521 | |||
| TMEVPG1 | −0.169 | 0.398 | 0.010 | 0.619 | 0.090 | 0.655 | −0.018 | 0.930 | |||
| NRON | 0.138 | 0.491 | 0.051 | 0.801 | 0.091 | 0.651 | 0.171 | 0.394 | |||
| Schizoaffective group | |||||||||||
|
| |||||||||||
| PANSS P | PANSS N | PANSS G | PANSS T | ||||||||
|
|
|
|
|
||||||||
| r | p | r | p | r | p | r | p | ||||
| IL-6 | 0.116 | 0.564 | 0.074 | 0.714 | −0.085 | 0.675 | 0.032 | 0.874 | |||
| TMEVPG1 | −0.220 | 0.271 | 0.023 | 0.910 | −0.023 | 0.908 | −0.063 | 0.754 | |||
| NRON | −0.381 | 0.050* | −0.122 | 0.546 | −0.020 | 0.920 | −0.235 | 0.237 | |||
PANSS, Positive and Negative Symptoms Scale; IL-6, interleukin 6; TMEVPG1, Theiler’s murine encephalomyelitis virus persistence candidate gene 1; NRON, non-coding repressor of the nuclear factor of activated T cells; lncRNA, long non-coding RNA; CT, cycle threshold; r, Spearman correlation coefficient; PANSS P, PANSS Positive Symptoms; PANSS N, PANSS Negative Symptoms; PANSS G, PANSS General Psychopathology; PANSS T, PANSS Total.
*p < 0.05 significant.
Correlations between IL-6, TMEVPG1 and NRON LncRNA ΔCT Values and the Threshold Drug Dose of Chlorpromazine
In the schizophrenia group, a moderate-level positive correlation was determined between the NRON lncRNA ΔCT values and the chlorpromazine equivalent drug dose (p = 0.019; r = 0.457). No other significant correlation was determined in all groups.
Correlations between IL-6 and LncRNA (TMEVPG1 and NRON)
No significant correlation was found between IL-6 mRNA and gene expressions (p > 0.05). There were no significant correlation between TMEVPG1 lncRNA, NRON lncRNA and IL-6 mRNA in the control group and schizoaffective group. But a low level positive correlation was found between TMEVPG1 and NRON lncRNAs in the schizophrenia group (r = 0.384) (Table 4).
Table 4.
Correlation between IL-6 and lncRNA (TMEVPG1 and NRON)
| TMEVPG1 | NRON | IL-6 | ||||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
||||||
| r | p | r | p | r | p | |||
| Control | ||||||||
| TMEVPG1 | 1 | - | 0.120 | 0.951 | 0.054 | 0.776 | ||
| NRON | 0.120 | 0.951 | 1 | - | −0.022 | 0.906 | ||
| IL-6 | 0.054 | 0.776 | −0.022 | 0.906 | 1 | - | ||
| Schizophrenia | ||||||||
| TMEVPG1 | 1 | - | 0.384 | 0.048* | −0.105 | 0.602 | ||
| NRON | 0.384 | 0.048* | 1 | - | −0.018 | 0.929 | ||
| IL-6 | −0.105 | 0.602 | −0.018 | 0.929 | 1 | - | ||
| Schizoaffective | ||||||||
| TMEVPG1 | 1 | - | 0.158 | 0.431 | −0.032 | 0.875 | ||
| NRON | 0.158 | 0.431 | 1 | - | −0.071 | 0.726 | ||
| IL-6 | −0.032 | 0.875 | −0.071 | 0.726 | 1 | - | ||
IL-6, interleukin 6; lncRNA, long non-coding RNA; TMEVPG1, Theiler’s murine encephalomyelitis virus persistence candidate gene 1; NRON, non-coding repressor of the nuclear factor of activated T cells; r, Spearman correlation coefficient; -, not available.
*p < 0.05 significant.
Multiple Linear Regression Models for Additional Confounding Variables
Multiple linear regression analysis was performed to detect independent factors affecting IL-6 and TMEVPG1 and NRON LncRNA expression levels including the model also age, gender, duration of disease, smoking and alcohol habits in the patient groups. In the results of multiple linear regression analysis, it was determined that duration of the disease was found to be an independent factor affecting NRON fold value in the schizoaffective disorder group (Adj R2 = 0.069, beta = −0.755, p = 0.032). There was no significant predictive effect of other variables on IL-6 and TMEVPG1 and NRON lncRNA fold values.
The Differentiation of Groups with ROC Analysis of the IL-6 and TMEVPG1 and NRON LncRNA ΔCT Values
The success of the TMEVPG1 lncRNA, NRON lncRNA, and IL-6 mRNA parameters in differeentiating the groups from each other was examined by applying ROC curve analysis. The analysis results showed that the NRON lncRNA and IL-6 mRNA expression levels could not differentiate the groups from each other. The TMEVPG1 lncRNA expression level was determined to be able to statistically differentiate schizophrenia cases from control cases at a weak level (p = 0.042, area under the ROC curve [AUC] = 0.657) (Table 5).
Table 5.
The success of the expressions in differentiating the groups
| Schizophrenia-Schizoaffective | Schizoaffective-Control | Schizophrenia-Control | ||||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
||||||
| AUC | p | AUC | p | AUC | p | |||
| TMEVPG1 | 0.492 | 0.924 | 0.643 | 0.064 | 0.657 | 0.042* | ||
| NRON | 0.599 | 0.21 | 0.523 | 0.767 | 0.616 | 0.133 | ||
| IL-6 | 0.631 | 0.099 | 0.525 | 0.749 | 0.646 | 0.059 | ||
TMEVPG1, Theiler’s murine encephalomyelitis virus persistence candidate gene 1; NRON, non-coding repressor of the nuclear factor of activated T cells; IL-6, interleukin 6; AUC, area under the ROC curve; ROC, receiver operating characteristic.
*p < 0.05 significant.
DISCUSSION
The main findings of our study were that TMEVPG1 expression was higher in the patient groups than in the control group, the TMEVPG1 lncRNA could differentiate schizophrenia from the control cases, but at a weak level, no relationship was seen between the expression levels and cognitive functions in the schizophrenia patients while an increase in NRON expression in schizoaffective patients could contribute to cognitive dysfunction at a moderate level, and there was a positive correlation at a weak level between NRON expression and positive symptomatology in the schizoaffective group.
As the age, sex and the smoking/alcohol use rates which are known to affect peripheral cytokine levels were similar in all the groups, it can be said that confounding factors were minimised in this study. The BMI values were seen to be higher in the schizoaffective group than in the schizophrenia and the controls. These results raise the question of whether patients with schizoaffective disorder are at higher risk of developing metabolic syndrome. Consistent with these results, a previous study of 7,529 male patients reported a 30% greater probability of obesity and a 36% greater probability of a dyslipidemia diagnosis in patients with schizoaffective disorder compared to schizophrenia patients [2]. The high risk of metabolic syndrome is thought to be associated with hereditary factors or the side-effects of widely used antipsychotic drugs, although it is not clear [4]. As both groups were known to be using high dose antipsychotic drugs, the high BMI values were an expected finding.
No significant difference was determined between the groups in respect of NRON expression, while in TMEVPG1 expression increase was observed in both patient groups compared to the control group. Limited studies in literature that determined lower TMEVPG1 expression levels in patients than in control subjects were in contrast to the current study [10,11]. On the other hand; Melbourne et al. [10] did not find any association of atypical antipsychotic chlorpromazine equivalents with TMEVPG1 or NRON. On the other hand; Ghafelehbashi et al. [11] showed that TMEVPG1 expression were decreased in bipolar and schizophrenia patients compared with controls. Considering patients respond to antipsychotic drug treatment, they comment that the anti-inflammatory effect of the drugs may play a role in these decreased inflammatory gene levels indirectly. In the schizophrenia group of the current study, a moderate-level positive correlation was determined between the chlorpromazine equivalent drug doses and the NRON lncRNA ΔCT values. This result shows that as the dose of antipsychotic drug use increased, the NRON lncRNA expression could decrease.
As a result of the ROC analysis in this study, it was determined that TMEVPG1 lncRNA could statistically differentiate schizophrenia from the control cases but this differentiation was at a weak level. A previous study in literature similarly reported that as a result of ROC analysis, IFNG-AS1 (TMEVPG1) could differentiate patients with schizophrenia from healthy control subjects [11]. However, the ability to differentiate was seen to be higher (AUC = 0.79) than in the current study. There is a need for further studies to examine lncRNAs in schizophrenia, schizoaffective disorder, and control subjects.
An increase in IL-6 levels in schizophrenia patients has been shown relatively consistently in previous studies [22]. In a recent meta-analysis, cytokines were investigated in schizophrenia patients at various disease stages and treatment conditions. The results showed that IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 levels were significantly elevated in patients with a first episode of psychosis and in those with acute psychosis recurrence, and the IL-6, IL-1β, and IFN-γ levels decreased with antipsychotic treatment, and therefore, IL-6 could be used as a status marker in schizophrenia patients [23]. In another meta-analysis, the high serum IL-6 levels of schizophrenia patients were determined to decrease after treatment, but even after treatment, the levels were determined to still be higher than those of healthy control subjects [24]. In affective psychosis, there is an increase in inflammatory responses in the manic period and IL-6, TNF-α, IL-1RA levels have been shown to be elevated [25]. But there are studies in the literature that have not reported any change in the IL-6 levels of schizophrenia patients with a first episode of psychosis and acute relapse, similar to the current study [26,27]. In both patient groups in our study, two or more antipsychotic drugs were used at maximum dosage and there was seen to be a response to treatment. That the high IL-6 levels determined in most studies in literature were not seen in the current study could be asssociated with the effect of the antipsychotic drugs used. There is evidence in literature that both typical and atypical antipsychotics suppressed the microglial activation which inhibits inflammatory mediators such as proinflammatory cytokines and nitric oxide, thereby causing an anti-inflammatory effect [28-30]. These results may show that IL-6 could be used as a biological marker to observe the therapeutic effect of psychotropic drugs.
When the relationship was examined in the current study between the IL-6 mRNA and lncRNA (TMEVPG1 and NRON) ΔCT values and the PANSS points, it was seen that there could be a weak positive correlation between an increase in NRON expression and positive symptomatology in the schizoaffective group. As far as could be seen in the literature, no previous study has examined symptom severity in schizophrenia and schizoaffective disorder together with lncRNAs (TMEVPG1 and NRON). Except for a limited number of studies [31,32], the majority of research has not shown a definite positive relationship between the severity of positive symptoms in schizophrenia and levels of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α [33,34].
It has been shown that proinflammatory cytokines significantly impair emotional and social functions, causing anhedonia and social dysfunction, which are linked to the negative symptoms of schizophrenia [35,36]. IL-6, which is related to emotional and social functions, shows an effect on the neurotransmission of catecholamines by increasing the dopaminergic and serotonergic cycles in the hippocampus and frontal cortex, and is known to activate the kynurenine pathway which plays a role in glutamatergic neurotransmission [4]. Increasing proinflammatory events may contribute to central serotonin deficiency. A positive correlation between plasma IL-6 concentrations and negative symptoms, consistent with these mechanisms has been determined in previous studies [33,37]. An important finding of the current study was that inflammatory processes were not correlated with symptomatology in schizophrenia, but a correlation with positive symptomatology was determined in schizoaffective disorder.
An increase in IL-6 has been associated with sustained attention impairment and psychomotor retardation [38,39]. Higher IL-6 levels in schizophrenia patients have also been found to be associated with cognitive impairment [5]. In the current study, the association of inflammation and cognitive impairment could not be shown in patients with schizophrenia. This may have been due to the inclusion of patients at different clinical stages and that the cognitive functions before and independently of the disease were not known. Very little is known about the relationship between stress at different stages of the disease and cytokine concentrations. Therefore, to be able to understand the relationship between disease prodrome, progression, and response to treatment, and cytokine concentrations, there is a need for further research. However, an important finding of the current study was that in the schizoaffective group, a moderate-level positive correlation was determined between the lncRNA NRON ΔCT values and the WCST total number of correct responses and the number of responses at the conceptual level, and a moderate level negative correlation was determined with the total number of incorrect responses. These results suggest that an increase in NRON expression could contribute to impairment in executive functions in schizoaffective disorder. There is an NFAT regulatory function of lncRNA NRON, and it has been previously reported that NFAT controls a series of cytokine expressions, including IL-6 and IFN-g [10]. This relationship determined in the current study schizoaffective group could be important for new studies to be made examining the relationship between lncRNA NRON, inflammation and cognition.
Non-coding RNAs are thought to play a role in the pathophysiology of diseases, and are seen as both targets and tools in new treatment approaches. It is thought that by elucidating the functions of all non-coding RNAs in the human genome, new treatment approaches can be developed and they can also be used as markers in the early diagnosis of disease. It is also stated in the literature that many pharmaceutical companies are trying to develop ncRNA-based strategies against cancer, cardiovascular, neurological and muscle diseases and to ensure the use of treatments in the clinic in the near future [40]. On the other hand, studies have shown that cognitive functions are an essential determinant of psychosocial functioning in patients with schizophrenia [41]. And, individuals with schizophrenia, the severity of the disease seems to be related to the impairment of cognitive functions [42,43]. So, the results of the current study could contribute to the development of diagnostic tools or therapeutic strategies in schizophrenia spectrum disorders.
There were some limitations to this study. Although there are longitudinal studies in literatüre [44,45], the majority of studies measuring inflammatory biomarkers in psychosis have been cross-sectional. Therefore, there is the limitation that the time relationship between inflammation and psychosis cannot be directly deduced [3]. The cross-sectional design of this study could have affected the results. A longitudinal study design would be valuable to track changes in lncRNA expression over time and their association with symptom progression or treatment outcomes. When it is considered that some studies have shown that cytokine plasma levels and soluble cytokine receptor levels are affected by lithium and benzodiazepines [46], another limitation of this study could be said to be that the potential effect of drugs could not be fully discounted. Although it is difficult to find medication naive patients with schizoaffective disorders by its descriptive nature; to eliminate the effects of medications, the results of studies conducted with drug naive schizophrenia patients will be more instructive. In addition, the lack of a mechanism for measuring genotoxic variables such as nutritional habits and the small sample group in terms of influencing the strength of statistical dependence can also be said tonbe limitations of this study. Future studies should include larger sample sizes to increase statistical power and allow for subgroup analyses, such as comparisons based on medication status.
In conclusion, the results of this study demonstrated that TMEVPG1 expression was greater in the patient groups than in the control group. An increase in NRON expression in schizoaffective patients was positively correlated at a weak level with positive symptomatology, and an increase in NRON expression in the schizoaffective group could contribute to impairment in cognitive functions. Thus, there were seen to be significant differences between schizophrenia and schizoaffective disorder in respect of inflammatory markers. Although it was not strong, a correlation was determined between the inflammation parameters measured and symptom type and cognitive functions.
ACKNOWLEDGMENTS
We thank all patients and volunteers who participated in the study as well as the referring specialists. We would like to thank Pamukkale University Scientific Research Projects Coordination Unit for their support to the study (Project number: 2020TIPF014).
Footnotes
Funding
This study was supported by the decision of Pamukkale University Scientific Research Projects Coordination Unit (Project number: 2020TIPF014).
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Author Contributions
Conceptualization: Kadriye Cansu Suguler, Osman Ozdel, Ibrahim Acikbas. Data curation: all authors. Formal analysis: Ibrahim Acikbas, Aysen Buket Er Urganci. Investigation: Kadriye Cansu Suguler, Osman Zulkif Topak, Osman Ozdel. Methodology: Kadriye Cansu Suguler, Osman Ozdel, Osman Zulkif Topak. Resources: all authors. Writing—original draft: Kadriye Cansu Suguler. Writing—review & editing: all authors.
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