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

RESULTS

Identification of Differentially Expressed HERV Genes in SLI. Using the relative quantification real-time PCR approach described by Livak et al. [19], we measured the expression levels of all five HERV genes we studied: HERV-K (HLM-2) gag, HERV-K env, HERV-W pol, HERV-P env, and HERV-R env. Each of the HERVspecific primer pairs used in the procedure was tested on serial dilutions of pooled genomic DNA samples in order that we check the specificity of amplification. We chose the housekeeping gene GAPDH as an endogenous control based on its negligible overall variance in the samples. Additionally, we checked the Ct values we obtained for each gene in each cDNA sample. Only Ct differences no greater than 0.2 between the technical replicates of every sample were considered acceptable. Calculated mean levels for the five HERV genes are shown in Figure 2. The data we obtained is presented as a relative fold difference (log2) for each gene. Differences in expression between the two groups of individuals were evaluated using the Wilcoxon-Mann-Whitney test. The HERV-K (HML-2) gag and HERV-P env transcript levels appeared to be significantly lower (Mann-Whitney U test p value <0.05) in the group of children with SLI compared with those in the control group (Figure 3). The other three genes studied: HERV-K env, HERV-R env, HERV-W pol, did not show statistically significant changes between the two groups. Statistical significance is defined as p value of <0.05 combined with an absolute fold change of >1.4. In order to determine whether the expression levels of HERV-K (HML-2) gag and HERV-P env can serve as potential discriminative biomarkers, we performed a receiver operating characteristic (ROC) analysis of the data from the qRT-PCR experiment. The ROC analysis is widely used statistical method for assessing the ability of a potential test marker to distinguish between disease carriers and healthy individuals. It provides a statistical model that presents the sensitivity (or the true positive fraction) of the marker as a function of the false positive fraction (defined as 1-specificity) of the same marker at multiple thresholds. Discriminative performance is assessed by the area enclosed between the ROC curve and the X-axis of the graph, and this area is known as area under the curve (AUC). A greater AUC represents a more accurate discrimination between individuals with the disease and no disease. A single point on the ROC curve represents a particular sensitivity value for a given specificity. Since there is a 50.0% chance to predict the health status of an individual (disease or no disease) only by random guessing, a 50.0% discrimination accuracy of a given marker is possible, but statistically meaningless. Thus, the AUC value can never be lower than 0.5, and even the worst test marker can meet a 50.0% predictive accuracy. This random chance is often presented on the ROC curve plot as a dotted diagonal line. The ROC curve was plotted and the AUC values with corresponding 95% confidence intervals (95% CI) were calculated as follows: AUC 0.812 (95% CI: 0.696-0.928) for HERV-K (HML-2) gag and 0.841 (95% CI: 0.736-0.946) for HERV-P env. The ROC analysis was subsequently used to calculate diagnostic sensitivity and specificity. Calculated sensitivity and specificity of HERV-K (HML-2) gag at optimal cutoff were 72.0 and 70.4%, respectively. Moreover, HERV-P env showed higher sensitivity 84.0% and 70.4% specificity (Figure 4). Together, these results indicate that the observed statistically significant differences in expression of HERV-K (HML-2) gag and HERV-P env, can discriminate between SLI cases and healthy controls with considerable accuracy.



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