COMPARATIVE EXPRESSION ANALYSIS OF HUMAN ENDOGENOUS RETROVIRUS ELEMENTS IN PERIPHERAL BLOOD OF CHILDREN WITH SPECIFIC LANGUAGE IMPAIRMENT
Minchev DS1,2,*, Popov NT3, Naimov SI1, Minkov IN4, Vachev TI1
*Corresponding Author: Assistant Professor Danail S. Minchev, Department of Medical Biology, Faculty of Medicine, Medical University-Plovdiv, 4000, Plovdiv, Bulgaria. Tel: +359-896-313-627. E-mail: dante17@abv.bg
page: 49

MATERIALS AND METHODS

Participants. This study included a group of 25 children with SLI and 25 age- and sex-matched healthy controls. All participants were Bulgarians. None of them had received any medications before blood sampling. The SLI patients met the diagnostic criteria according to Diagnostic and Statistical Manual of Mental disorders, 4th edition [17]. Briefly, these criteria include: 1) language performance, evaluated using standard tests, is two standard deviations below the limit for the respective child’s age; 2) verbal skills are at least one standard deviation below the non verbal IQ for the respective child’s age; 3) there are no observable neurological and sensory incapacities or physical impairments that directly affect use of spoken language, nor a pervasive developmental disorder; 4) differential diagnosis is made with respect to the receptive language disorder (comprehensive skills more than two standard deviations below the limit for the child’s age) and expressive language disorder (comprehension is within the two standard deviations from the age level limit); 5) the language impairment affects the academic or occupational achievement or with social communication [17-18]. Children were accessed by a multi disciplinary team of child and adolescent psychiatrists and psychologists. Clinical assessment included a standard interview with the parents, free communication with the child and free play. All children who were clinically positive for language delay, vocabulary reduction and limited sentence structure as child language capacity is significantly below what is expected were referred for standard testing with age appropriate and standardized for the country test (HAWIK-R, Manova- Tomova test, Stanford-Binet Intelligence Scale). Clinical data we obtained from the patients were summarized as follows: mean IQ 85.67 (± 12.669); verbal IQ 85.27 (± 9.376) non verbal IQ 86.93 (± 10.498). All patients who have shown significant difference between verbal and nonverbal development, have had no other developmental disorder signs [autism sepctrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), learning disorder (LD)], were included in the study. Patients with behavior problems such as oppositional, defiant and/or negative behavior, were not excluded. From all the patients referred, four were excluded, two due to an IQ below 70 and two due to no difference between verbal IQ and non-verbal IQ. From all tested patients in 20, HAWIK-R was used, in three Manova-Tomova test and in two Stanford-Binet Intelligence Scale. The control group individuals were not tested with specialized developmental tests, just standard clinical testing. Ethics Statement. Ethical admissibility of the study design and the informed consent form was confirmed by the Ethics Committee of Plovdiv Medical University. The Institutional Review Board approved the use of peripheral blood samples in this study. Written informed consent was obtained from the parents of the children in both study groups, with SLI and the typically developing children of the control group. All parents were informed of the aims and the procedures of this study prior to giving their informed consent. All personal information was kept in strict confidence. Blood Collection, Sample Storage and RNA Isolation. A total amount of 2.5 mL peripheral blood from each participant (SLI or healthy) was drawn directly in a PAXgene blood RNA monovette (PreAnalytiX GmbH, Hombrechtikon Switzerland), according to the manufacturer’s protocol. All samples were kept at –20 °C and thawed at room temperature for 8 hours, prior to RNA extraction. All procedures required for total RNA isolation were performed using the PAXgene blood miRNA kit (PreAnalytiX GmbH, Feldbachstrasse Switzerland), according to the manual purification protocol. RNA quantity (absorption at 260 nm) and purity (A260/A280 ratio) were measured using the Epoch Micro-Volume Spectrophotometer System (BioTek, Winooski, VT, USA). Only A260/A280 ratios higher than 1.8 were considered acceptable. Quantitative RT-PCR Analysis of HERV Expression. We analyzed the expression of five HERV genes: HERV-K (HLM-2) gag, HERV-K env, HERV-W pol, HERV-P env, and HERV-R env, using a relative qRTPCR method. An amount of 1 μg total RNA from each sample was subjected to DNAse I treatment using RQ1 RNase-Free DNase (Promega, Madison, WI, USA) These DNAse treated aliquots were then used for cDNA synthesis using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) and oligo-dT/ random hexamere primers. Reverse transcription reactions were set as follows: 11 μL of total RNA, 4 μL 5 × reaction mix (with the oligo-dT/random hexamere primers), 2 μL Maxima Enzyme Mix (Thermo Scientific), and nuclease free water to a final volume of 20 μL. The qRT-PCR was performed using Maxima SYBR Green qPCR Master Mix (Ňhermo Scientific) in an ABI PRISM® 7500 real-time thermal cycler (Applied Biosystems, Waltham, MA, USA). Each sample was normalized using the GAPDH gene as endogenous control. Experiments were performed in duplicates. Each experiment was completed with a melting curve analysis to confirm the specificity of amplification and the lack of non specific amplification (Figure 1). Expression levels of HERV genes were obtained by relative quantification according to the 2−ΔΔCt method. Primer Sequences. Individual cDNA samples from SLI (25) and healthy controls (25) were analyzed using a qRT-PCR assay. All primer pairs used for real-time PCR reactions are summarized in Table 1. The two separate HERV-K primer pairs produced amplicons located on different genome sites that are not related to each other.



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