INVESTIGATION OF FASCICULATION AND ELONGATION PROTEIN ζ-1 (FEZ1) IN PERIPHERAL BLOOD REVEALS DIFFERENCES IN GENE EXPRESSION IN PATIENTS WITH SCHIZOPHRENIA
Vachev TI1, Stoyanova VK, Ivanov HY, Minkov IN, Popov NT
*Corresponding Author: Associate Professor Vili K. Stoyanova, M.D., Ph.D., Department of Pediatrics and Medical Genetics, Medical University ‒ Plovdiv, 15A Vasil Aprilov St., 4000 Plovdiv, Bulgaria. Tel: +359-32- 602-431; Fax: +359-32-602-593. E-mail: vi1sto@abv.bg
page: 31

MATERIALS AND METHODS

Ethics Statement. This study and the informed consent forms were approved by the Medical University of Plovdiv Ethics Committee. Participants. Written informed consent was obtained from 29 patients recruited at the State Psychiatry Hospital Pazardjik, Pazardzk, Pazardzk, Bulgaria and 24 healthy volunteers. Routine psychiatric examination, wide medical hystory and the Mini- International Neuropsychiatric Interviews were done by a certified psychiatrist to evaluate the diagnosis of paranoid SZ only, on Diagnostic and Statistical Manual of Mental Disorders (IVth edition) criteria and to exclude any mental disorder in the controls. Important inclusion criteria were that the participants had not received any medication (even psychotropic) 1 month before blood sampling and they had a standard breakfast, so they were assessed in a state of exacerbation. Persons with other chronic medical and current acute somatic/neurologic illness, alcohol or drug abuse/dependancy were also excluded. The sample population included 15 males/14 females diagnosed with SZ and 24 age- and gender-matched general population controls (12 males/12 females) with no evidence for any psychiatric or neurological disorder in first-grade relatives (Table 1). Blood Collection and RNA Isolation. Blood samples from the patients and control groups were collected in PAXgene Blood RNA collection tubes (PreAnalyticX GmbH, Hombrechtikon, Switzerland) that contain a reagent that lyses blood cells and immediately stabilizes intracellular RNA to preserve the gene expression profile. To reduce any potential bias in gene expression due to diurnal variation, blood was drawn in the morning, from all of the subjects. We used the PAXgene Blood miRNA Kit (PreAnalyticX) to extract total RNA from the blood samples [14]. Total RNA was then quantified by absorbance at A260 nm using Epoch Micro-Volume Spectrophotometer System (BioTek, Winooski, VT, USA) and the purity was estimated by the ratio A260/A280 nm. The absorbance ratio of 260 nm and 280 nm (A260/A280) was between 1.93 and 2.1 for all samples included for further analysis. The RNA integrity was confirmed by non denaturing agarose gel electrophoresis, which was then stored at ‒80 °C until further analysis. The resulting RNA was treated with RNase-free DNase I (Promega BioSciences, San Luis Obispo, CA, USA) according to the manufacturer’s protocol and checked for DNA contaminations prior to copy DNA synthesis step.PAGE Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) Analysis of FEZ1 mRNA Level. The qRT-PCR analyses were performed in at least three physically separate rooms in to order to reduce the chance for contamination. Copy DNA for the protein coding gene was synthesized from total RNA with oligo (dT)18 primer using RevertAid First Strand cDNA Synthesis Kit according to the assay protocol (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription reactions contained 1 μg of total RNA samples, 1 μL oligo (dT)18 primer and nuclease free water to a final volume of 12 μL, after incubation at 65 °C for 5 min., we added 4 μL 5X RT buffer, 1 μL RiboLock RNase Inhibitor (20 U/μL) (Thermo Fisher Scientific), 2 μL 10 mM dNTP Mix and 1 μL RevertAid MMuLV Reverse Transcriptase (200 U/μL) (Thermo Fisher Scientific), to final volume of 20 μL. A relative gene expression method was employed to determine gene expression levels. The reactions were set up in duplicate in a 96-well format using the 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and Maxima SYBER Green/Rox qRT-PCR Kit (Thermo Fisher Scientific). Amplification of the single amplicon coresponding to the FEZ1 sequence was confirmed by monitoring the dissociation curve (melting curve analysis) and by agarose gel electrophoresis. The qRT-PCR forward and reverse primers for FEZ1 used in this study were 5’-GGG ACT GCA TGA GAC CAT GT-3’ and 5’-TTG AGG GCT GTA GCC AGA CT-3’, respectively [15]. B-actin (ACTB) was used as an internal control for normalization. ACTB-specific qRT-PCR primers were as follows: forward 5’-AGT GTG ACG TGG ACA TCC GCA-3’ and reverse 5’- GCC AGG GCA GTG ATC TCC TTC T-3’. After performing the qRT-PCR analysis, the Ct values were measured, different methods could be used to determine the expression level of the target gene in the test sample relative to the calibrator sample. Here, we used the Livak and Schmittgen method [16], also known as the comparative 2‒Ct method. All the analyzed SZ samples showed mean Ct values range from 26.7 to 34.9 and mean control Ct values ranged from 28.0 to 31.3. The mean Ct values of the reference ACTB gene for SZ samples ranged from 16.6 to 21.7 and the range for control samples from 16.3 to 18.0, respectively. First, for the normalization of the Ct values of the target genes to that of the reference gene, for both the test sample and the calibrator samples was made using the equation: Ct(test) = Ct(target, test)‒Ct(reference, test) Ct(calibrtator) = Ct(target, calibrator)‒Ct(reference, calibrator) Second, normalization of the Ct of the test sample to the Ct of the calibrator was made using the equation: Ct = Ct(test)‒Ct(calibrator) Finally, calculation of the expression ratio with the following equation: 2‒Ct = normalized expression ratio. The result obtained is the fold increase (or decrease) of the target gene in the test samples relative to the calibrator sample. Statistical Analyses. All statistical calculations were performed using the Statistical Package for the Social Sciences (SPSS) software, version 20.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) t-test of Ct was used to examine differences in expression levels of FEZ1 mRNA across healthy controls and SZ subjects. A Spearman correlation analysis was also done for searching relation between expression levels and demographic characteristics of the tested individuals. To investigate the characteristics of FEZ1 gene expression as potential diagnostic biomarkers in SZ patients, a ROC (receiver operating characteristic) curve was done and AUC (area under the ROC curve) was calculated. Statistical tests were two-sided with a p value of 0.05. The STRING Pathway Analysis of FEZ1 Protein Interactions. The FEZ1 interaction data for each validated protein was obtained using the STRING database (version 9.0; http://string-db.org/) (Figure 1).



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