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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RESULTS

Identification of MicroRNA Deregulation Signature in Autism Spectrum Disorder. We analyzed the expression of two serum miRNAs using the stem-loop qRT-PCR assay. Amplicon-specific PCR amplification corresponding to miR-3135a and miR-328-3p was confirmed by melting curve analysis (Figure 1). Our data indicate changes in relative expression levels of miR-3135a and miR-328-3p, which were markedly lower in ASD patients than those in TDC, and the relative serum levels of these miRNAs could distinguish ASD from healthy control patients (Figure 2). To assess the ability of the analyzed serum miRNAs as promising biomarkers for ASD, we performed a ROC analysis, and the area under curve (AUC) was measured (Figure 3). Our relative qRT-PCR data suggests that miR-3135a and miR-328-3p are significantly downregulated in ASD patients (Figure 4). The t-test defined the expression changes of both miRNAs as statistically significant (with fold change ≤2 and p <0.05). The obtained data was subsequently used to assess the diagnostic specificity and sensitivity of analyzed serum miRNAs. Diagnostic sensitivities of miR-3135a and miR-328-3p for ASD were 76.3 and 78.9%, respectively. The corresponding specificities were 88.9 and 88.9%, and AUC 95% confidence interval (95% CI) were 0.828 (0.715-0.911%), and 0.858 (0.749-0.932%), respectively. In addition, a combined ROC analysis was done. The combined ROC curve analysis showed a better diagnostic value than individual miRNAs in ASD. Area under the ROC AUC (95% CI) 0.858. Confidence interval (0.749-0.932). Together, these results indicate that the identified serum miRNAs, alone or in combination, can discriminate between ASD cases and TDC with high accuracy. Identification of MicroRNAs Associated Biological Pathways. In all the 162 genes denoted as unique, validated targets in miRWalk, 71 appeared to participate in biological pathways described in the KEGG database. (Figures 5 and 6). One of the target genes we obtained, the amyloid β precursor protein (APP), is involved in synaptic pathways. This gene encodes a membrane protein that undergoes proteolytic processing. In serotonergic synapses, the soluble APP α fragment interacts with the cyclic adenosine monophosphate (cAMP) signal transduction protein exchange factor directly activated by cAMP (EPAC) to promote neuroprotection. Another gene, solute carrier family eight member A1 (SLC8A1), participates in a specific exteroceptive transduction pathway. The SLC8 A1 protein is an Na+/Ca2+ (K+) antiporter. In olfactory neurons, it has a role in membrane repolarization and annihilates the consequences of a previously occurred action potential. Another six different target genes showed involvement in neurodegenerative diseases: Huntington’s, Parkinson’s or Alzheimer’s. Two genes: APP and BACE1 directly involved in pathogenesis of Alzheimer’s disease. The amyloid precursor protein encoded by APP is cleaved by β-secretase (encoded by BACE1) that leaves a particular peptide responsible for amyloid plaque formation. Two proteins (DNAL1 and POLR2I) are described to take part in Huntington’s disease pathogenesis in an indirect manner, while interacting with or being influenced by, the product of the Huntingtin (HTT) gene. As a result nonspecific changes in cytoskeleton organization or gene expression occur. Two other genes whose protein products take part in the mitochondrial respiratory chain NADH:ubiquinone oxidoreductase subunit A1 (NDUFA1) and NADH: ubiquinone oxidoreductase subunit V3 (NDUFV3) are described to be important not only for Alzheimer’s but also for Huntington’s and Parkinson’s diseases. A correlation between the Alzheimer’s syndrome and the reduced expression of energy metabolism genes has been well established [9]. However, the precise mechanisms in which NDUFA1 and NDUFV3 contribute to a specific neuro-development condition and their regulatory roles is yet to be clarified.

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