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
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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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