Balkan Journal of Medical Genetics

INVESTIGATION OF CIRCULATING SERUM microRNA-328-3p AND microRNA-3135a EXPRESSION AS PROMISING NOVEL BIOMARKERS FOR AUTISM SPECTRUM DISORDER
Popov NT, Minchev DS, Naydenov MM, Minkov IN, Vachev TI
*Corresponding Author: Assistant Professor Tihomir I. Vachev, Ph.D., Department of Plant Phisyology and Molecular Biology, University of Plovdiv “Paisii Hilendarski,” 24 Tzar Assen Str., Plovdiv, Bulgaria. E-mail: tiho9@abv.bg
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MATERIALS AND METHODS

Patients. A total of 30 subjects (24 males and six females) with ASD aged 3 to 11 years, and 30 TDC sex- and age-matched to the ASD group were included in this study. All participants were randomly selected from the family practices in the Plovdiv region. Probands were evaluated by certified psychiatrists and the diagnosis of ASD was made by clinical examination, Gilliam autism rating scale (GARS), childhood autism rating scale (CARS) and autism diagnostic interview (ADI-R), adhering to the diagnostic and statistical manual of mental disorders (DSM V) criteria. The GARS norm-referenced screening instrument was used for ASD symptom assessment. To help differentiate subjects with ASD from those with other developmental delays, we used CARS. The ADR-R is a structured interview performed with the parents of the patients. This is the golden standard for assessment of ASD patients. The DSM-V is the standard classification of mental disorders used by mental health professionals and physicians in the USA and most research teams worldwide. The control group representing TDC was assigned with the aim to match by sex and age to the ASD group. Inspection of all children in the healthy group for absence of autistic features was done by clinical examination and CARS. Children with known infectious, oncological, metabolic or genetic conditions were excluded from the study. No children were receiving any drug therapy when they were recruited. Statement of Ethics. The Institutional Review Board of the Ethics Committee of the Medical University of Plovdiv approved the methodology of the study and the written informed consent forms. Blood Processing and RNA Extraction. Blood was drawn by venipuncture from a peripheral vein while the participants were fasting (>3 hours without a meal). Separation into blood cells and serum was done by centrifuging at 1600 g for 10 min. RNA was extracted from the 200 μL serum aliquots, with the addition of 5 μL (100 nM) spikein cel-miR-39 control, added for internal standardization. Extraction of serum RNA was done using the PAXgene blood miRNA kit (PreAnalytiX GmbH, Hombrechtikon, Switzerland), following the manual purification of total RNA, including miRNA protocol, recommended by the manufacturer. Quantification of Serum MicroRNAs. The Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) were used for miRNA specific cDNA synthesis. MicroRNA-specific primer sequences used in this study are shown in Table 1. MicroRNA specific cDNA (5 μL) were subjected to pre amplification with peqGOLD Taq DNA Polymerase (VWR, Radnor, PA, USA) prior to the reverse transcription-(RT-PCR) step in order to enhance sensitivity of the test. The qRT-PCR was carried out using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and the ABI PRISM® 7500 system (Applied Biosystems, Foster City, CA, USA). All the experiments were performed in duplicate. Each sample was normalized using spiked-in control and relative quantification of miRNAs were calculated applying the 2–ΔΔCt method. Statistical analyses were made by the Statistical Package for the Social Sciences (SPSS) software, version 20.0 (SPSS®; IBM Inc., Armonk, NY, USA). The analysis of variance (ANOVA) t-test on the data of Ct values was used for investigation of dysregulation of the analyzed miRNAs between TDC and ASD groups. MedCalc statistical (https://medcale.org/) software was used to perform receiver operating characteristic (ROC) analysis. Pathways Prediction Analysis of Differentially Expressed miR-328-3p and miR-3135a Serum. First, we managed to obtain every validated target gene available in the miRWalk 2.0 database (zmf.umm.uni-heidelberg. de/ apps/zmf/mirwalk2/) for both of the serum miRNAs we studied. The miRWalk database offers a convenient direct search option, but only for those pathways in which putative target genes take part. However, such a direct search option is not available for validated target genes. We developed our own script that uses a list of validated target genes as a query and automatically explores the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp.kegg) for pathways in which our validated target genes participate. This made it possible to gather information for all of the validated targets without searching the whole KEGG database by hand. Afterwards, the number of target genes was plotted against their respective pathways on separate diagrams for each miRNA we investigated.

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